InteractiveFly: GeneBrief

Stem-loop binding protein: Biological Overview | References

BIOLOGICAL OVERVIEW

The early embryos of many animals including flies, fish, and frogs have unusually rapid cell cycles and delayed onset of transcription. These divisions are dependent on maternally supplied RNAs and proteins including histones. Previous work suggests that the pool size of maternally provided histones can alter the timing of zygotic genome activation (ZGA) in frogs and fish. This study examine the effects of under and overexpression of maternal histones in Drosophila embryogenesis. To understand the effects of histone concentration on the MBT maternally supplied histones were reduced by downregulating the gene encoding a crucial histone regulator, Stem-Loop Binding Protein (Slbp) via maternally driven RNAi. Decreasing histone concentration advances zygotic transcription, cell cycle elongation, Chk1 activation, and gastrulation. Conversely, increasing histone concentration delays transcription and results in an additional nuclear cycle before gastrulation. Numerous zygotic transcripts are sensitive to histone concentration, and the promoters of histone sensitive genes are associated with specific chromatin features linked to increased histone turnover. These include enrichment of the pioneer transcription factor Zelda and lack of SIN3A and associated histone deacetylases. These findings uncover a critical regulatory role for histone concentrations in ZGA of Drosophila (Wilky, 2019).

Downregulating Slbp via maternally driven RNAi, histone H2B was reduced by ~50% and H3 by ~60% at the MBT. Approximately 50% of embryos laid by Slbp RNAi mothers (henceforth Slbp embryos) that form a successful blastoderm do not undergo the final division and attempt gastrulation in NC13. Another ~30% exhibit an intermediate phenotype of partial arrest, with only part of the embryo entering NC14. A minority of Slbp embryos begin gastrulation with all nuclei in NC14. NC12 duration was predictive of NC13 arrest, with NC12 being an average of ~5min longer in Slbp embryos that went on to arrest compared with those that did not arrest (Wilky, 2019).

Cellularization was first detected in wild-type (WT) embryos ~20 min into NC14. Partially arrested Slbp embryos also began cellularization ~20 min into NC14, with nuclei that arrested in NC13 waiting until the remainder of the embryo had entered NC14 to cellularize. Fully arrested embryos began cellularization ~20min into NC13, initiating cellularization one cycle early and ~20min earlier in overall developmental time than WT. Despite their reduced cell number, these embryos form mitotic domains and gastrulate without obvious defects, however they die before hatching (Wilky, 2019).

To examine the effects of increased histone concentration on developmental timing cell cycle progression was monitored in embryos from abnormal oocyte (abo) mutant mothers (henceforth abo embryos). abo is a histone locus-specific transcription factor, the knockdown of which increases the production of replication-coupled histones, particularly H2A and H2B (Berloco, 2001). abo increased H2B by ~90%, whereas total (combined replication-coupled and replication-independent) H3 was not affected in NC14 embryos. Approximately 60% of abo embryos displayed fertilization defects or catastrophic early nuclear divisions. Of abo embryos that formed a functioning blastoderm, ~6% underwent a complete extra nuclear division before gastrulating in NC15, whereas ~4% underwent a partial extra nuclear division. Embryos from abo mothers that completed total extra divisions had faster NC14s in which they did not cellularize and spent 40-60 min in NC15 before gastrulating. This suggests an alteration of the normal transcription-dependent developmental program. In some cases, the cell cycle program and transcriptional program may be decoupled, evidenced by the fact that some abo embryos attempted to gastrulate while still in the process of division. abo embryos that underwent extra divisions exhibited a range of gastrulation defects including expanded mitotic domains and ectopic furrow formation (Wilky, 2019).

Since alterations in histone levels can both decrease and increase the number of divisions before cell cycle slowing, it was reasoned that histone levels might affect activation of checkpoint kinase 1 (Chk1, also known as grp), which is required for cell cycle slowing at the MBT. To test this, a fluorescent biosensor of Chk1 activity was crosses into the Slbp background. Even in Slbp embryos that did not undergo early gastrulation, Chk1 activity was higher than in WT, consistent with the lengthened cell cycle. This result indicates that the observed cell cycle phenotypes in the histone-manipulated embryos are likely mediated through changes in Chk1 activity (Wilky, 2019).

As cellularization and gastrulation require zygotic transcription, it was suspected that embryos with altered development likely have altered gene expression. Single-embryo RNA-seq was performed on staged Slbp embryos that remained in NC13 for more than 30 min. These were compared with either NC-matched (NC13) or time-matched (NC14) WT embryos. To control for maternal effects of Slbp RNAi, pre-blastoderm stage WT and Slbp embryos were compared. The Slbp embryos underwent ZGA one NC earlier than WT. ~5000 genes were identified that were differentially expressed between Slbp and WT NC13, with ~60% being upregulated. The upregulated genes have largely previously been identified as new zygotic transcripts, including cell cycle regulators such as fruhstart (frs, also known as Z600) and signaling molecules such as four-jointed (fj), whereas the downregulated genes are enriched for maternally degraded transcripts. This is thought to represent a coherent change in ZGA timing instead of global transcription dysregulation, as 98% of the genes that are overexpressed in Slbp are expressed before the end of NC14 in the control or previously published datasets. Indeed, the transcriptomes of histone-depleted embryos that stopped in NC13 are more similar to WT NC14 than WT NC13, which suggests a role for cell cycle elongation in ZGA. Nonetheless, ~1500 genes are differentially expressed between Slbp NC13 and WT NC14 without accounting for differences in ploidy. Of these, the majority of the ~1000 overexpressed genes are again associated with zygotic transcription, and downregulated genes associated with maternal products. Thus, ZGA is even further accelerated in the histone knockdown than can be explained by purely time alone (Wilky, 2019).

As ZGA is accelerated by histone depletion, it was asked whether ZGA would be delayed in the histone overexpression mutant. RNA-seq was performed on pools of abo and WT embryos collected 15-30 min into NC14. More than 1000 genes were identified that were differentially expressed between abo and WT, with approximately equal numbers of genes up- and down-regulated. As expected, the downregulated genes in abo were enriched for previously identified zygotically expressed transcripts, and upregulated transcripts were enriched for maternally deposited genes. Thus, histone overexpression delays the onset of ZGA (Wilky, 2019).

Zygotic genes, the transcription of which is upregulated by histone depletion and downregulated by histone overexpression, contain many important developmental and cell cycle regulators including: frs, hairy (h), fushi tarazu (ftz) and odd-skipped (odd). Conversely, the maternally degraded transcripts that are destabilized by histone depletion and stabilized by histone overexpression include several cell cycle regulators such as Cyclin B (CycB), string (stg, also known as Cdc25string) and Myt1. Therefore, histone concentration can modulate the expression and stability of specific cell cycle regulators, which may contribute to the onset of MBT (Wilky, 2019).

Since histone concentration has previously been implicated in sensing the nuclear-cytoplasmic (N/C) ratio (Amodeo, 2015), this study compared the genes that are changed in both the histone under- and overexpression embryos with those that had previously been found to be dependent on either the N/C ratio or developmental time (Lu, 2009). Both previously identified N/C ratio-dependent and time-dependent genes (Lu, 2009) followed the same general trends as the total zygotic gene sets, indicating that histone availability cannot explain these previous classifications (Wilky, 2019).

Next, attempts were made to disentangle the effects of cell cycle length from transcription in the histone overexpression mutant. Single-embryo time-course RNA-seq was performed on abo and WT embryos collected 3 min before mitosis of NC10-NC13 and 3 min into NC14. In addition, unfertilized embryos (henceforth NC0) of both genotypes were collected to control for differences in maternal contribution. Even with a stringent selection process that accounted for cell cycle time and embryo health, a small set of robustly upregulated (179) and downregulated (260) genes was detected across NC10-NC14. Of the newly transcribed genes, 111 genes were detected with delayed transcription, including frs and only 37 that are upregulated. These results were confirmed using qPCR. When compared with previous datasets, zygotic genes tend to be underexpressed, as was the case for the pooled abo dataset; however, the majority of these enrichments are not statistically significant. Nonetheless the majority of these underexpressed genes are expressed during NC14 in WT. This geneset, in combination with the time-matched Slbp comparison, enables further examination of the chromatin features that underlie histone sensitivity for transcription independent of cell cycle changes (Wilky, 2019).

To identify chromatin features associated with histone sensitivity, the presence was compared of 143 modENCODE chromatin signals near the transcriptional start site (TSS±500 bp) of genes whose expression was altered by changes in histone concentration independent of cell cycle time. A clear pattern was found of unique chromatin features for the histone-sensitive genes, compared with all newly transcribed genes, that was highly similar between the histone over- and underexpression experiments. The pioneer transcription factor Zld, known to be important for nucleosome eviction during ZGA, was enriched in the promoters of histone-sensitive genes. Insulator proteins such as BEAF-32 and CP190 were depleted in histone-sensitive genes. Promoters of histone-sensitive genes also show a strong reduction for SIN3A, a transcriptional repressor associated with cell cycle regulation. SIN3A is known to recruit HDACs to TSSs, and almost all HDACs also show significant de-enrichment at the TSSs of histone-sensitive genes. Taken together, these marks make up a unique chromatin signature that may sensitize a locus to changes in histone concentration, as is likely for pioneer factors such as Zld. Other aspects of this signature may indicate that these genes are subsequently subject to later developmental regulation, as indicated by H3K4me3 and H3K27me3 (Wilky, 2019).

This study has demonstrated that histone concentration regulates the timing of the MBT in Drosophila, resulting in both early gastrulation and extra pre-MBT divisions from histone reduction and increase, respectively. Histone concentration also regulates ZGA. Thousands of genes are prematurely transcribed in histone-depleted embryos and hundreds of genes are delayed in histone-overexpressing embryos. The majority of these genes appear to be downstream of changes in cell cycle duration, suggesting a model in which histones directly regulate cell cycle progression. In other cell types, histone loss halts the cell cycle via accumulation of DNA damage and stalled replication forks. In the early embryo, changes in histone availability may similarly create replication stress to directly or indirectly activate Chk1 as this study has shown. In turn, Chk1 would inhibit Stg and/or Twine to slow the cell cycle. This mechanism is supported by previous observations that loss of zygotic histones causes the downregulation of Stg in the first post-MBT cell cycle. In this case, the observed transcriptional changes would be independent or downstream of the altered cell cycle (Wilky, 2019).

Alternatively, direct changes in transcription downstream of histone availability may feed into the cell cycle. In bulk, histone-sensitive transcripts might underlie the replication stress that has been previously proposed to slow the cell cycle at the MBT. Consistent with this, the cell cycle lengthening and partial arrest phenotypes observed in mutant RNA Pol II embryos occur at a similar frequency to those observed as the result of histone depletion. Another possibility is that specific histone-sensitive transcripts are responsible for cell cycle elongation. One promising candidate for a histone-sensitive cell cycle regulator is the N/C ratio-sensitive CDK inhibitor frs, as zygotic transcription of frs plays a crucial role in stopping the cell cycle at the MBT. In contrast, tribbles, an N/C ratio-dependent inhibitor of Twine that has also been implicated in cell cycle slowing, does not show a consistent response between histone perturbations. In this previously proposed model, maternal histone stores may compete with pioneer transcription factors to set the timing of transcription initiation. Indeed, the central Drosophila pioneer transcription factor Zld is enriched at the promoters of histone-sensitive genes. Moreover, this study has identified a broader set of chromatin features that may sensitize individual loci to changes in histone concentrations. These include less obvious candidates for global early transcriptional regulators, such as SIN3A, HDACs and class I insulator proteins that may protect transcripts from changes in histone concentrations. This work highlights the importance of histone concentration in regulating the timing of MBT and provides evidence that promoters of histone-sensitive genes possess a unique chromatin signature. However, future studies will be required to isolate the specific downstream effectors that respond to changes in histone concentrations in the early embryo (Wilky, 2019).

Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker

3' end cleavage of metazoan replication-dependent histone pre-mRNAs requires the multi-subunit holo-U7 snRNP and the stem-loop binding protein (SLBP). The exact composition of the U7 snRNP and details of SLBP function in processing remain unclear. To identify components of the U7 snRNP in an unbiased manner, a novel approach was developed for purifying processing complexes from Drosophila and mouse nuclear extracts. In this method, catalytically active processing complexes are assembled in vitro on a cleavage-resistant histone pre-mRNA containing biotin and a photo-sensitive linker, and eluted from streptavidin beads by UV irradiation for direct analysis by mass spectrometry. In the purified processing complexes, Drosophila and mouse U7 snRNP have a remarkably similar composition, always being associated with CPSF73, CPSF100, symplekin and CstF64. Many other proteins previously implicated in the U7-dependent processing are not present. Drosophila U7 snRNP bound to histone pre-mRNA in the absence of SLBP contains the same subset of polyadenylation factors but is catalytically inactive and addition of recombinant SLBP is sufficient to trigger cleavage. This result suggests that Drosophila SLBP promotes a structural rearrangement of the processing complex, resulting in juxtaposition of the CPSF73 endonuclease with the cleavage site in the pre-mRNA substrate (Skrajna, 2018).

In metazoans, 3' end processing of replication-dependent histone pre-mRNAs occurs through a single endonucleolytic cleavage, generating mature histone mRNAs that lack a poly(A) tail. This specialized 3' end processing reaction depends on the U7 snRNP, the core of which consists of a ∼60-nt U7 snRNA and a unique heptameric Sm ring. In the ring, the spliceosomal subunits SmD1 and SmD2 are replaced by the related Lsm10 and Lsm11 proteins, whereas the remaining subunits (SmB, SmD3, SmE, SmF and SmG) are shared with the spliceosomal snRNPs (Skrajna, 2018).

Lsm11 contains an extended N-terminal region that interacts with the N-terminal region of the 220 kDa protein FLASH. Together, they recruit a specific subset of the proteins that participate in 3' end processing of canonical pre-mRNAs by cleavage and polyadenylation, resulting in formation of the holo-U7 snRNP (Skrajna, 2014). This subset of polyadenylation factors is referred to as the histone pre-mRNA cleavage complex (HCC) and in mammalian nuclear extracts includes symplekin, all subunits of CPSF (CPSF160, WDR33, CPSF100, CPSF73, Fip1 and CPSF30) and CstF64 as the only CstF subunit. The remaining components of the cleavage and polyadenylation machinery, including CstF50 and CstF77, the two CF Im subunits of 68 and 25 kDa, and the two subunits of CF IIm (Clp1 and Pcf11) were consistently absent in the HCC (Yang, 2013). A similar subset of polyadenylation factors is associated with the Drosophila holo-U7 snRNP (Sabath, 2013; Skrajna, 2018 and references therein).

The substrate specificity in the processing reaction is provided by the U7 snRNA, which through its 5' terminal region base pairs with the histone downstream element (HDE), a sequence in histone pre-mRNA located downstream of the cleavage site. This interaction is assisted by the stem-loop binding protein (SLBP), which binds the highly conserved stem-loop structure located upstream of the cleavage site (Wang, 1996; Martin, 1997; Tan, 2013) and stabilizes the complex of U7 snRNP with histone pre-mRNA (Dominski, 1999), likely by contacting FLASH and Lsm11 (Skrajna, 2017). In mammalian nuclear extracts, histone pre-mRNAs that form a strong duplex with the U7 snRNA are cleaved efficiently in the absence of SLBP. In contrast, Drosophila nuclear extracts lacking SLBP are inactive in cleaving histone pre-mRNAs, suggesting that Drosophila SLBP plays an essential role in processing in addition to stabilizing binding of the U7 snRNP to histone pre-mRNA (Skrajna, 2017, Sabath, 2013, Dominski, 2003; Lanzotti, 2002; Dominski, 2005; Skrajna, 2018 and references therein).

Within the HCC, CPSF73 is the endonuclease, acting in a close partnership with its catalytically inactive homolog, CPSF100, and the heat-labile scaffolding protein symplekin (Kolev, 2005). RNAi-mediated depletion of these three HCC subunits in Drosophila cultured cells results in generation of polyadenylated histone mRNAs, an indication of their essential role in the U7-dependent processing. Depletion of the remaining components of the HCC had no effect on the 3' end of histone mRNAs and their function in the U7 snRNP, if any, is less clear. Previous in vivo studies implicated multiple other proteins, in addition to SLBP and components of the U7 snRNP, in generation of correctly processed histone pre-mRNAs. These proteins include ZFP100, CDC73/parafibromin, NELF E, Ars2, CDK9, CF Im68 and RNA-binding protein FUS/TLS (Fused in Sarcoma/Translocated in Sarcoma). ZFP100, CF Im68 and FUS were shown to interact with Lsm11, whereas Ars2 was shown to interact with FLASH, raising the possibility that they may be essential components of the cleavage machinery (Skrajna, 2018).

To determine which factors are required for the cleavage reaction, a novel method for purification of in vitro assembled Drosophila and mouse processing complexes was developed. In this method, histone pre-mRNAs containing biotin and a photo-cleavable linker in either cis or trans are incubated with a nuclear extract and the assembled processing complexes are immobilized on streptavidin beads, washed and released into solution by irradiation with long wave UV. This approach yielded remarkably pure processing complexes that were suitable for direct and unbiased analysis by mass spectrometry, providing a complete view of the holo-U7 snRNP and other proteins that associate with histone pre-mRNA for 3' end processing (Skrajna, 2018).

In this method, processing complexes were assembled in a nuclear extract on a synthetic histone pre-mRNA containing biotin and a photo-cleavable linker at the 5' end. The major cleavage site and the two neighboring nucleotides on each side were modified with a 2'O-methyl group, hence preventing endonucleolytic cleavage of the pre-mRNA and increasing the efficiency of capturing intact processing complexes. Following immobilization on streptavidin beads, the pre-mRNA and the bound proteins were washed and released to solution by irradiation with long wave UV. This UV-elution step, by eliminating all background proteins non-specifically bound to streptavidin beads, resulted in isolation of remarkably pure processing complexes that were suitable for direct analysis by mass spectrometry. This is the first successful use of the photo-cleavable linker and the UV-elution step for purification of an in vitro assembled RNA/protein complex. Parallel experiments with pre-mRNA substrates lacking 2'O-methyl nucleotides at the cleavage site demonstrated that the immobilized processing complexes retain catalytic activity. Thus, the mass spectrometry analysis of the UV-eluted material is likely to provide a global and unbiased view of all essential proteins that associate with histone pre-mRNA for 3' end processing (Skrajna, 2018).

Since chemical synthesis of RNAs containing covalently attached biotin and the photo-cleavable linker (cis configuration) is both expensive and limited to sequences not exceeding 60-70 nt, longer histone pre-mRNAs generated by T7 transcription were tested. Biotin and the photo-cleavable linker can be attached to the 3' end of these pre-mRNAs in trans via a short complementary oligonucleotide. This modification makes the UV-elution method more cost effective and potentially applicable for purification of RNA-protein complexes that require longer RNA binding targets, including spliceosomes and complexes involved in cleavage and polyadenylation (Skrajna, 2018).

In the UV-eluted mouse and Drosophila processing complexes, mass spectrometry identified SLBP and all known subunits of the U7-specific Sm ring, including Lsm10 and Lsm11. Readily detectable in mouse and Drosophila processing complexes were also FLASH and subunits of the HCC. The HCC is remarkably similar in composition between the two species, with symplekin, CPSF100, CPSF73 and CstF64 being most abundant and present in close to stoichiometric amounts, as determined by both silver staining and emPAI value analysis. The remaining CPSF subunits (CPSF160, WDR33, Fip1 and CPSF30) are present in lower amounts, suggesting that they are substoichiometric, being stably associated only with a fraction of the U7 snRNP (Skrajna, 2018).

In both mouse and Drosophila experiments, SLBP and the components of the U7 snRNP were the only proteins that consistently failed to bind histone pre-mRNAs in the presence of processing competitors: SL RNA and αU7 oligonucleotide. Other proteins were detected both in the samples containing processing complexes and in the matching negative controls, where formation of processing complexes was blocked. Among them, the most prevalent were non-specific RNA binding proteins, including hnRNP Q in mouse nuclear extracts, and IGF2BP1 in Drosophila nuclear extracts. All these proteins likely bind to sites in histone pre-mRNAs unoccupied by SLBP and U7 snRNP, and play no essential role in processing (Skrajna, 2018).

CstF50 and CstF77 were not detected in the UV-eluted mouse processing complexes and were present only in some Drosophila complexes, always with low scores, consistent with a previous conclusion that of the three CstF subunits only CstF64 stably associates with the U7 snRNP (Yang, 2013). No peptides were detected for CF Im (68 and 25 kDa) and CF IIm (Clp1 and Pcf11) in any of the mouse experiments, suggesting that these factors are also uniquely involved in cleavage and polyadenylation. Mass spectrometry identified the orthologues of the 68 and 25 kDa subunits in some Drosophila experiments, but they were clearly contaminants, persisting in the presence of the SL RNA and αU7 oligonucleotide. CF Im68 was previously reported to interact with Lsm11 and to co-purify with U7 snRNP. Based on this analysis, this subunit is unlikely to interact with Lsm11 in the processing complex (Skrajna, 2018).

Catalytically active mouse processing complexes also lacked ZFP100 (ZN473), a zinc finger protein that co-localizes with Lsm11 and stimulates expression of a reporter gene containing U7-dependent processing signals. ZFP100 was initially identified by the yeast two-hybrid system as a protein interacting with SLBP bound to the SL RNA and suggested to function as a bridging factor in the SLBP-mediated recruitment of the U7 snRNP to histone pre-mRNA. However, the absence of ZFP100 in the UV-eluted mouse processing complexes containing both SLBP and U7 snRNP strongly argues against this function. ZFP100 may instead participate in a different aspect of histone gene expression in vivo, perhaps acting as a coupling factor that integrates transcription of histone genes with 3' end processing of the nascent histone pre-mRNAs (Skrajna, 2018).

A similar role in vivo may be played by the multi-functional protein FUS and other proteins previously linked to 3' end processing of histone pre-mRNAs in mammalian cells, including Ars2, CDC73/parafibromin, NELF E and CDK9. These factors were never specifically detected in the UV-eluted mouse processing complexes, suggesting that they have no direct role in processing in vitro. Their downregulation by RNAi results in production of a small amount of polyadenylated histone mRNAs, which may be due to a defect in coupling of histone gene transcription with processing and/or cell-cycle progression (Skrajna, 2018).

Although this study identified several polyadenylation subunits in a stable association with the U7 snRNP, the experiments do not directly address which of them are essential for processing of histone pre-mRNAs. In Drosophila cultured cells, RNAi-mediated depletion of each of only three U7-associated polyadenylation subunits, symplekin, CPSF100 and CPSF73, consistently resulted in accumulation of histone mRNAs terminated with a poly(A) tail, an indication of a defect in the U7-dependent processing mechanism. Depletion of the remaining HCC subunits had no effect, suggesting that their association with the U7 snRNP is not essential for 3' end processing of histone pre-mRNAs. Symplekin, CPSF100 and CPSF73 are present in Drosophila cells as a stable sub-complex (Sullivan, 2009) and likely act together as an autonomous cleavage module recruited for processing to either histone or canonical pre-mRNAs by specialized RNA recognition sub-complexes. For canonical pre-mRNAs, this role is played by the remaining CPSF subunits, CPSF160, WDR33, Fip1 and CPSF30, recently shown to co-operate in recognizing the AAUAAA signal during the polyadenylation step. In 3' end processing of histone pre-mRNAs, the recruitment of the cleavage sub-complex is mediated by the U7 snRNA, which recognizes the substrate by the base pairing interaction, further arguing that CPSF160, WDR33, Fip1 and CPSF30 are likely non-essential bystanders in the U7 snRNP (Skrajna, 2018).

A less clear role in 3' end processing of histone pre-mRNAs is played by CstF64, which in spite of being relatively abundant in Drosophila U7 snRNP can be depleted from Drosophila cells without causing a detectable misprocessing of histone pre-mRNAs. A defect in the U7-dependent processing was however observed in human cells partially depleted of CstF64, suggesting that in mammalian cells this subunit may play a more critical role, perhaps helping to stabilize the three essential subunits of the HCC on the FLASH/Lsm11 complex. Clearly, determining which subunits are essential for cleavage will require reconstitution of a catalytically active processing complex from recombinant components (Skrajna, 2018).

This study brings a new perspective on the essential role of Drosophila SLBP in processing. It was recently demonstrated that Drosophila SLBP, like its mammalian counterpart, enhances the recruitment of U7 snRNP to histone pre-mRNA (Skrajna, 2017). A small amount of U7 snRNP binds to histone pre-mRNA in the absence of Drosophila SLBP but the bound U7 snRNP in spite of containing all major HCC subunits is catalytically inactive. This study now shows that processing complexes assembled in the absence of SLBP can be activated for cleavage by simply adding recombinant WT SLBP, providing evidence that SLBP is the only missing factor in the assembled complexes. A mutant Drosophila SLBP that is deficient in recruiting U7 snRNP to histone pre-mRNA is also unable to activate the assembled complex for cleavage. Based on these results, it is proposed that the interaction of Drosophila SLBP with the U7 snRNP promotes an essential structural rearrangement of the entire processing complexes that juxtaposes the catalytic site of CPSF73 with the pre-mRNA (see A hypothetical model explaining essential role of Drosophila in processing). It is possible that higher metazoans developed an additional positioning mechanism for the CPSF73 endonuclease, resulting in efficient cleavage in the absence of SLBP (Skrajna, 2018).

U7 snRNP is recruited to histone pre-mRNA in a FLASH-dependent manner by two separate regions of the stem-loop binding protein

Cleavage of histone pre-mRNAs at the 3' end requires stem-loop binding protein (SLBP) and U7 snRNP that consists of U7 snRNA and a unique Sm ring containing two U7-specific proteins: Lsm10 and Lsm11. Lsm11 interacts with FLASH and together they bring a subset of polyadenylation factors to U7 snRNP, including the CPSF73 endonuclease that cleaves histone pre-mRNA. SLBP binds to a conserved stem-loop structure upstream of the cleavage site and acts by promoting an interaction between the U7 snRNP and a sequence element located downstream from the cleavage site. This study shows that both human and Drosophila SLBPs stabilize U7 snRNP on histone pre-mRNA via two regions that are not directly involved in recognizing the stem-loop structure: helix B of the RNA binding domain and the C-terminal region that follows the RNA binding domain. Stabilization of U7 snRNP binding to histone pre-mRNA by SLBP requires FLASH but not the polyadenylation factors. Thus, FLASH plays two roles in 3' end processing of histone pre-mRNAs: It interacts with Lsm11 to form a docking platform for the polyadenylation factors, and it cooperates with SLBP to recruit U7 snRNP to histone pre-mRNA (Skrajna, 2017).

In spite of passing 20 years since the discovery of SLBP as a factor that binds the conserved stem-loop structure in histone pre-mRNA (Wang, 1996; Martin, 1997), its precise role in processing and interactions with the rest of the processing machinery are incompletely understood. Initial studies in human nuclear extracts demonstrated that human SLBP promotes binding of U7 snRNP to histone pre-mRNA and that the in vitro requirement for SLBP could be bypassed by increasing the extent of complementarity between the 5' end of the U7 snRNA and the histone downstream element (HDE). In contrast, Drosophila SLBP is essential for cleavage of all five Drosophila histone pre-mRNAs in vitro (Dominski, 2005), but whether it acts in the same manner as mammalian SLBP and/or has other functions in processing has not been unambiguously determined (Skrajna, 2017).

Determining the role of Drosophila SLBP in processing proved challenging, in part due to the rapid cleavage of histone pre-mRNAs and hence disruption of the processing complexes containing U7 snRNP during short incubation in Drosophila nuclear extracts. This study used a modified approach for the assembly and purification of Drosophila processing complexes and clearly demonstrates that Drosophila SLBP functionally resembles mammalian SLBP and acts by stabilizing the interaction between the U7 snRNP and histone pre-mRNA. By using a substrate containing biotin at the 3' end, it was shown that the U7 snRNP becomes partially destabilized on the HDE after cleavage, when SLBP is no longer part of the complex, further confirming the role of Drosophila SLBP in promoting stable association of U7 snRNP with histone pre-mRNA. Interestingly, the U7 snRNP that remains associated with the HDE following endonucleolytic cleavage contains FLASH and all subunits of the HCC. This result suggests that holo-U7 snRNP does not undergo a major remodeling during the course of processing reaction (Skrajna, 2017).

Consistent with a recent studies (Sabath, 2013), a detectable amount of Drosophila U7 snRNP binds to histone pre-mRNA in the absence of SLBP, possibly as a result of base-pairing between the 5' end of the U7 snRNA and the HDE. The bound U7 snRNP contains all the key subunits of the HCC, including the CPSF73 endonuclease, but remains functionally inert. This is in sharp contrast to mammalian holo-U7 snRNP, which when bound to the HDE can result in cleavage of histone pre-mRNA even in the absence of SLBP. The reason for this difference between the two systems is unknown (Skrajna, 2017).

Drosophila and human SLBP use the same regions to recruit U7 snRNP to histone pre-mRNA The results indicate that two regions in mammalian and Drosophila SLBPs are critical for the recruitment of the U7 snRNP to histone pre-mRNA: parts of the RBD that do not directly contact the SL RNA and 15-20 amino acids of the C-terminal region located immediately downstream from the RBD. SLBP mutants altered in these regions retain the ability to bind the SL RNA but are partially or completely impaired in supporting cleavage of histone pre-mRNAs. This study shows that these mutants are also impaired in recruiting U7 snRNP to histone pre-mRNA, providing a likely molecular basis for their reduced activity in processing (Skrajna, 2017).

Within the RBD, the most critical role is played by helix B, including the highly conserved D(E)/R motif. Mutating this motif itself is sufficient to strongly reduce processing activity of human SLBP (Dominski, 2001) and the recruitment of U7 snRNP by Drosophila SLBP. An important role in recruiting U7 snRNP may also be played by other amino acids of the RBD, including evolutionarily variable residues in the loop that connects helices B and C. Despite overall conservation, human and Drosophila RBDs are not functionally interchangeable, yielding chimeric proteins that are inactive in both the U7 snRNP recruitment and cleavage of histone pre-mRNAs. It is possible that these variable amino acids are largely responsible for the observed incompatibility between the two RBDs (Skrajna, 2017).

In Drosophila SLBP, the C-terminal region consists of only 17 amino acids and is highly acidic, containing four stoichiometrically phosphorylated serines alternating with four aspartic acids. In addition to these four SD motifs, the C-terminal region contains one TD motif, and the current study indicates that the threonine residue in this motif may be phosphorylated sub-stoichiometrically. The high density of negative charge in this region is critical for the activity of Drosophila SLBP in promoting stable recruitment of the U7 snRNP to histone pre-mRNA and in supporting the cleavage reaction. Interestingly, in the absence of SL RNA the acidic C terminus of Drosophila SLBP associates with helices A and C, bringing them together and preparing for maximum strength interaction with the SL RNA (Zhang, 2014). Upon binding to the RNA target, the phosphorylated C-terminal tail is repelled from the RBD (Zhang, 2014) and may become available for the independent function in the recruitment of the U7 snRNP (Skrajna, 2017).

The C-terminal region of human SLBP lacks the repeated SD motif present in the Drosophila SLBP. Interestingly, six out of 20 residues that immediately follow the RBD in human SLBP are acidic, suggesting that the overall negative charge of this segment may also be important for activity of this protein (Zhang, 2014). However, the C-terminal regions of human and Drosophila SLBP are not functionally interchangeable, indicating that they co-evolved with other component(s) of their respective processing machineries, resembling the divergent evolution of the two RBDs (Skrajna, 2017).

SLBP tightly bound to the upstream stem-loop promotes stable recruitment of U7 snRNP to histone pre-mRNA likely by directly or indirectly interacting with a subunit(s) of the U7 snRNP. In both Drosophila and mouse nuclear extracts, SLBP is active in recruiting U7 snRNP that lacks the HCC. Thus, SLBP is unlikely to interact with any of the polyadenylation factors of the holo-U7 snRNP. In contrast, removal of FLASH from Drosophila U7 snRNP by RNAi abolishes the ability of SLBP to recruit U7 snRNP to histone pre-mRNA and this ability can be restored by addition of the N-terminal fragment of Drosophila FLASH. Mouse nuclear extracts contain primarily core U7 snRNP (FLASH is limiting), and addition of human N-terminal fragments of human FLASH to a mammalian extract stimulates the activity of SLBP in recruiting the U7 snRNP to histone pre-mRNA (Skrajna, 2017).

The N-terminal region of human and Drosophila FLASH was initially characterized as a protein that interacts with Lsm11, hence forming a docking platform for the HCC. This study now identified a new role for the N-terminal FLASH in processing by showing that it is also essential for the SLBP-mediated 'loading' of the U7 snRNP on histone pre-mRNA. This dual FLASH function may be important for the fidelity of 3' end processing of histone pre-mRNAs in vivo. Clearly, the FLASH-bound U7 snRNP that readily associates with the HCC forming holo-U7 snRNP has an advantage in binding to histone pre-mRNA over functionally incompetent core U7 snRNP, likely preventing misprocessing at downstream cryptic sites by cleavage and polyadenylation (Skrajna, 2017).

Previous studies on processing in human cells suggested that the recruitment of U7 snRNP by SLBP might be mediated by ZFP100, a zinc finger protein of 100 kDa that binds both human SLBP and Lsm11. Mrultiple attempts to detect ZFP100 in the mouse processing complexes by silver staining and mass spectrometry failed, although CPSF100 of the same molecular weight was readily identified, arguing against its involvement in the SLBP-mediated recruitment of the U7 snRNP. ZFP100 is a component of histone locus bodies and may play a role in vivo in coupling transcription of histone genes with 3' end processing of the nascent histone pre-mRNAs (Skrajna, 2017).

It is unclear whether SLBP is completely unable to act on the core U7 snRNP and whether its interaction with FLASH is direct or indirect. A model is favored in which SLBP interacts with the FLASH/Lsm11 complex rather than with FLASH alone. Alternatively, FLASH, upon binding Lsm11, induces a structural shift in part of Lsm11, making it competent to interact with SLBP. The interaction may also include Lsm10. Clearly, further studies are required to identify interactions that span across the cleavage site and bring SLBP and U7 snRNP together into a tight processing complex (Skrajna, 2017).

This study has developed a method to identify regions in human and Drosophila SLBP that are essential for the recruitment of U7 snRNP to histone-pre-mRNA. In both proteins, this activity is mediated by helix B and likely other amino acids of the RBD that do not directly contact the SL RNA, and by ∼20 C-terminal amino acids that follow the RBD. The activity of SLBP in promoting stable recruitment of U7 snRNP to histone pre-mRNA depends on FLASH but not the HCC. Thus, FLASH has two functions in processing: First, it is essential for bringing the HCC to U7 snRNP and second, it cooperates with SLBP in facilitating the interaction between U7 snRNP and histone pre-mRNA. The fact that Drosophila and human SLBP recruit U7 snRNP to histone pre-mRNA through the same regions, and that FLASH but not the HCC is essential for this activity of SLBP, suggests that both processing machineries utilize a conserved network of interactions spanning across the cleavage site (Skrajna, 2017).

Biochemical fractionation of time-resolved Drosophila embryos reveals similar transcriptomic alterations in replication checkpoint and histone mRNA processing mutants

In higher eukaryotes, maternally provided gene products drive the initial stages of embryogenesis until the zygotic transcriptional program takes over, a developmental process called the midblastula transition (MBT). In addition to zygotic genome activation, the MBT involves alterations in cell-cycle length and the implementation of DNA damage/replication checkpoints that serve to monitor genome integrity. Previous work has shown that mutations affecting histone mRNA metabolism or DNA replication checkpoint factors severely impact developmental progression through the MBT, prompting this study to characterize and contrast the transcriptomic impact of these genetic perturbations. This study defined gene expression profiles that mark early embryogenesis in Drosophila through transcriptomic analyses of developmentally staged (early syncytial vs late blastoderm) and biochemically fractionated (nuclear vs cytoplasmic) wild type embryos. The transcriptomic profiles were compared of loss-of-function mutants of the Chk1/Grapes replication checkpoint kinase and the Stem Loop Binding Protein (SLBP), a key regulator of replication-dependent histone mRNAs. This analysis of RNA spatial and temporal distribution during embryogenesis offers new insights into the dynamics of early embryogenesis. In addition, it was found that grp and Slbp mutant embryos display profound and highly similar defects in gene expression, most strikingly in zygotic gene expression, compromising the transition from a maternal to a zygotic regulation of development (Lefebvre, 2017).

In vivo characterization of the Drosophila mRNA 3' end processing core cleavage complex

A core cleavage complex (CCC) consisting of CPSF73, CPSF100, and Symplekin is required for cotranscriptional 3' end processing of all metazoan pre-mRNAs, yet little is known about the in vivo molecular interactions within this complex. The CCC is a component of two distinct complexes, the cleavage/polyadenylation complex and the complex that processes nonpolyadenylated histone pre-mRNAs. RNAi-depletion of CCC factors in Drosophila culture cells causes reduction of CCC processing activity on histone mRNAs, resulting in read through transcription. In contrast, RNAi-depletion of factors only required for histone mRNA processing allows use of downstream cryptic polyadenylation signals to produce polyadenylated histone mRNAs. This study used Dmel-2 tissue culture cells stably expressing tagged CCC components to determine that amino acids 272-1080 of Symplekin and the C-terminal approximately 200 amino acids of both CPSF73 and CPSF100 are required for efficient CCC formation in vivo. Additional experiments reveal that the C-terminal 241 amino acids of CPSF100 are sufficient for histone mRNA processing indicating that the first 524 amino acids of CPSF100 are dispensable for both CCC formation and histone mRNA 3' end processing. CCCs containing deletions of Symplekin lacking the first 271 amino acids resulted in dramatic increased use of downstream polyadenylation sites for histone mRNA 3' end processing similar to RNAi-depletion of histone-specific 3' end processing factors FLASH, SLBP, and U7 snRNA. A model is proposed in which CCC formation is mediated by CPSF73, CPSF100, and Symplekin C-termini, and the N-terminal region of Symplekin facilitates cotranscriptional 3' end processing of histone mRNAs (Michalski, 2015).

Developmentally regulated Elimination of damaged nuclei involves a Chk2-dependent mechanism of mRNA nuclear retention

The faithful execution of embryogenesis relies on the ability of organisms to respond to genotoxic stress and to eliminate defective cells that could otherwise compromise viability. In syncytial-stage Drosophila embryos, nuclei with excessive DNA damage undergo programmed elimination through an as-yet poorly understood process of nuclear fallout at the midblastula transition. This study shows that this involves a Chk2-dependent mechanism of mRNA nuclear retention that is induced by DNA damage and prevents the translation of specific zygotic mRNAs encoding key mitotic, cytoskeletal, and nuclear proteins required to maintain nuclear viability. For histone messages, this study shows that nuclear retention involves Chk2-mediated inactivation of the Drosophila Stem loop binding protein (SLBP), the levels of which are specifically depleted in damaged nuclei following Chk2 phosphorylation, an event that contributes to nuclear fallout. These results reveal a layer of regulation within the DNA damage surveillance systems that safeguard genome integrity in eukaryotes (Iampietro, 2014).

Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation

Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop-binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. This study shows that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine were used to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA (Zhang, 2014).

3'-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors

3'-End cleavage of animal replication-dependent histone pre-mRNAs is controlled by the U7 snRNP. Lsm11, the largest component of the U7-specific Sm ring, interacts with FLASH, and in mammalian nuclear extracts these two proteins form a platform that recruits the CPSF73 endonuclease and other polyadenylation factors to the U7 snRNP. FLASH is limiting, and the majority of the U7 snRNP in mammalian extracts exists as a core particle consisting of the U7 snRNA and the Sm ring. In this study the U7 snRNP was purified from Drosophila nuclear extracts and its composition was characterized by mass spectrometry. In contrast to the mammalian U7 snRNP, a significant fraction of the Drosophila U7 snRNP contains endogenous FLASH and at least six subunits of the polyadenylation machinery: symplekin, CPSF73, CPSF100, CPSF160, WDR33, and CstF64. The same composite U7 snRNP is recruited to histone pre-mRNA for 3'-end processing. A motif was identified in Drosophila FLASH that is essential for the recruitment of the polyadenylation complex to the U7 snRNP and analyzed the role of other factors, including SLBP and Ars2, in 3'-end processing of Drosophila histone pre-mRNAs. SLBP that binds the upstream stem-loop structure likely recruits a yet-unidentified essential component(s) to the processing machinery. In contrast, Ars2, a protein previously shown to interact with FLASH in mammalian cells, is dispensable for processing in Drosophila. These studies also demonstrate that Drosophila Symplekin and three factors involved in cleavage and polyadenylation-CPSF, CstF, and CF Im-are present in Drosophila nuclear extracts in a stable supercomplex (Sabath, 2013).

U7 snRNA mutations in Drosophila block histone pre-mRNA processing and disrupt oogenesis

Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem–loop structure generated by an endonucleolytic cleavage involving the U7 snRNP, which interacts with histone pre-mRNAs through base-pairing between U7 snRNA and a purine-rich sequence in the pre-mRNA located downstream of the cleavage site. Null mutations of the single Drosophila U7 gene were generated and U7 snRNA was demonstrated to be required in vivo for processing all replication-associated histone pre-mRNAs. Mutation of U7 results in the production of poly A⁺ histone mRNA in both proliferating and endocycling cells because of read-through to cryptic polyadenylation sites found downstream of each Drosophila histone gene. A similar molecular phenotype also results from mutation of Slbp, which encodes the protein that binds the histone mRNA 3' stem–loop. U7 null mutants develop into sterile males and females, and these females display defects during oogenesis similar to germ line clones of Slbp null cells. In contrast to U7 mutants, Slbp null mutations cause lethality. This may reflect a later onset of the histone pre-mRNA processing defect in U7 mutants compared to Slbp mutants, due to maternal stores of U7 snRNA. A double mutant combination of a viable, hypomorphic Slbp allele and a viable U7 null allele is lethal, and these double mutants express polyadenylated histone mRNAs earlier in development than either single mutant. These data suggest that SLBP and U7 snRNP cooperate in the production of histone mRNA in vivo, and that disruption of histone pre-mRNA processing is detrimental to development (Godfrey, 2006).

Chromosome duplication during the cell cycle requires the production of histones during S phase to package newly replicated DNA into chromatin. Bulk histone production during S phase is achieved through the biosynthesis of replication-dependent histone mRNAs, which are cell-cycle regulated and accumulate only in S phase. In animal cells these histone mRNAs are unique: The 3' end terminates in a conserved 26-nt sequence that forms a stem-loop rather than in a poly A⁺ tail. Since histone genes lack introns, the only processing step required for mature histone mRNA production is endonucleolytic cleavage of the pre-mRNA to form the 3' end of the mRNA. Much of the cell-cycle regulation of histone mRNAs is post-transcriptional and is mediated by the 3' end of the mRNA. Thus, a complete understanding of cell-cycle-regulated histone mRNA production requires a full understanding of the factors required for histone pre-mRNA processing (Godfrey, 2006).

The processing of histone pre-mRNAs requires two cis elements and a number of trans-acting factors. The cis elements are the stem-loop at the 3' end of histone mRNA and a purine-rich region downstream of the cleavage site, termed the histone downstream element (HDE). A protein called stem-loop binding protein (SLBP) or hairpin binding protein (HBP) specifically binds the 3' end of histone mRNA. SLBP is required for histone pre-mRNA processing in vivo and accompanies the mRNA to the cytoplasm, where it promotes the translation of the histone mRNA. The HDE binds U7 snRNP by base-pairing with the 5' end of U7 snRNA. In mammals, SLBP, the U7 snRNP, and a U7 snRNP-associated zinc finger protein called ZFP100 cooperate to recruit an endonuclease complex that cleaves the pre-mRNA. Recent evidence indicates that CPSF73, a component of the complex that mediates AAUAAA-directed cleavage prior to polyadenylation, is the likely endonuclease. This revealed some unexpected overlap in the machinery carrying out histone pre-mRNA processing and canonical polyadenylation (Godfrey, 2006).

The U7 snRNA is a small RNA (55-70 nt) that, like the spliceosomal snRNAs, contains both a trimethyl guanosine cap and an Sm binding site, which is essential for its function. The Sm site in these snRNAs stably binds a complex of seven related proteins of the LSm/Sm family to form the core snRNP particle. Proteins of the LSm/Sm family share a common tertiary structure called the Sm fold that assembles into hexameric or heptameric rings capable of binding single-stranded RNA. The U snRNPs contain a heptameric Sm ring, with each of the seven individual subunits making a specific contact with a residue in the Sm binding site of the snRNA. The heptameric Sm ring of spliceosomal snRNPs contains the proteins SmB/B', SmD1, SmD2, SmD3, SmE, SmF, and SmG. In contrast, the U7 snRNP contains five of these Sm proteins (B/B1, D3, E, F, G) and two novel Sm proteins called LSm10 and LSm11 that replace SmD1 and SmD2 of the spliceosomal snRNPs. The Sm site found in U7 snRNAs is distinct from the Sm site in spliceosomal snRNAs and is responsible for incorporation of LSm10 and LSm11 into the U7 snRNP. In addition to the Sm fold that participates in ring formation, LSm11 contains an NH₂ terminal extension that makes contacts with ZFP100 and possibly other components of the histone pre-mRNA processing machinery (Godfrey, 2006).

The role of U7 snRNP in histone pre-mRNA processing has been examined primarily in nuclear extract systems that support the processing of synthetic histone pre-mRNAs, and by monitoring the processing of histone pre-mRNAs injected into Xenopus ooctyes. Complementary mutations in U7 snRNA and the HDE provided early evidence that base-pairing between the 5' end of U7 and the HDE was an important part of U7 snRNP function. Furthermore, blocking the 5' end of the U7 snRNA with a complementary oligonucleotide specifically inhibits processing of synthetic histone pre-mRNAs in nuclear extracts. However, the contribution of U7 snRNA to endogenous histone mRNA biosynthesis and whether this contribution is important for animal development have not been examined. To explore these issues, U7 snRNA mutations in Drosophila were generated and characterized (Godfrey, 2006).

Drosophila SLBP, U7 snRNA, and U7 snRNP specific proteins Lsm10 and Lsm11, have all been identified, and steps have been taken to characterize them genetically. Mutations in the Drosophila Slbp gene block normal histone pre-mRNA processing during embryonic development and result in production of polyadenylated histone mRNAs as a consequence of read-through past the normal processing site. This occurs because each of the five Drosophila histone genes contains cryptic polyadenylation sites downstream of the HDE that are utilized in the absence of SLBP. Null mutations of Slbp cause lethality during larval and pupal stages, presumably because of the histone processing defects, although the precise cause of lethality is not known. Slbp mutant cells are capable of replicating chromatin, likely because the inappropriate polyadenylated mRNAs are translated. A hypomorphic Slbp mutant allele that produces reduced amounts of SLBP protein results in the production of both normal and poly A⁺ histone mRNAs during embryogenesis, but does not cause lethality. However, these viable mutant females lay eggs that contain reduced amounts of histone mRNA and protein and do not develop. Thus, SLBP is required during both zygotic development and oogenesis (Godfrey, 2006).

This study compared mutations in the U7 snRNA gene, and the resulting phenotypes were compared with those caused by mutation of Slbp. The results indicate that U7 snRNA is required for normal histone mRNA biosynthesis during Drosophila development and that, like Slbp mutations, loss of U7 snRNA results in the production of polyadenylated histone mRNAs. However, unlike Slbp null mutants, U7 null mutants are viable, but both males and females are sterile. This difference in terminal phenotype is most likely because the maternal supply of U7 snRNA delays the onset of the histone processing defect in U7 mutants relative to Slbp mutants, which do not have a significant maternal supply of SLBP protein. Both U7 and SLBP are required for normal histone mRNA biosynthesis in the female germ line, and mutation of either gene disrupts oogenesis. These data indicate that loss of SLBP and U7 cause similar molecular phenotypes in Drosophila and suggest that early expression of this molecular phenotype prevents normal development (Godfrey, 2006).

Differences and similarities between Drosophila and mammalian 3' end processing of histone pre-mRNAs

Nuclear extracts from Drosophila Kc cells were used to characterize 3' end processing of Drosophila histone pre-mRNAs. Drosophila Stem-loop binding protein (SLBP) plays a critical role in recruiting the U7 snRNP (Dominski, 2003) to the pre-mRNA and is essential for processing all five Drosophila histone pre-mRNAs. The Drosophila processing machinery strongly prefers cleavage after a fourth nucleotide following the stem-loop and favors an adenosine over pyrimidines in this position. Increasing the distance between the stem-loop and the histone downstream element (HDE) does not result in a corresponding shift of the cleavage site, suggesting that in Drosophila processing the U7 snRNP does not function as a molecular ruler. Instead, SLBP directs the cleavage site close to the stem-loop. The upstream cleavage product generated in Drosophila nuclear extracts contains a 3' OH, and the downstream cleavage product is degraded by a nuclease dependent on the U7 snRNP, suggesting that the cleavage factor has been conserved between Drosophila and mammalian processing. A 2'O-methyl oligonucleotide complementary to the first 17 nt of the Drosophila U7 snRNA was not able to deplete the U7 snRNP from Drosophila nuclear extracts, suggesting that the 5' end of the Drosophila U7 snRNA is inaccessible. This oligonucleotide selectively inhibited processing of only two Drosophila pre-mRNAs and had no effect on processing of the other three pre-mRNAs. Together, these studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing share common features, there are also significant differences, likely reflecting divergence in the mechanism of 3' end processing between vertebrates and invertebrates (Dominski, 2005).

Metazoan replication-dependent histone pre-mRNAs do not contain introns, and the only processing reaction necessary to generate mature histone mRNAs is a single endonucleolytic cleavage of the mRNA precursors (pre-mRNAs) to form the 3' end. Studies on 3' end processing were initially carried out in Xenopus oocytes using synthetic pre-mRNAs and sea urchin histone genes and later were facilitated by the development of an in vitro system based on nuclear extracts from mammalian cells. Replication-dependent histone pre-mRNAs contain two cis elements required for 3' end processing: a highly conserved stem-loop structure consisting of a 6-bp stem and a 4-nt loop and a less conserved histone downstream element (HDE) located ~15 nt 3' of the stem-loop. Mammalian histone pre-mRNAs are cleaved between the two elements, 5 nucleotides downstream of the stem-loop. The stem-loop is recognized by the stem-loop binding protein (SLBP), also referred to as the hairpin binding protein (HBP). The HDE interacts with the U7 snRNP, which contains an ~60-nt U7 snRNA, and this interaction is primarily mediated by base-pairing between the HDE and the 5' end of U7 snRNA. In vitro studies in mammalian nuclear extracts suggest that SLBP stabilizes binding of the U7 snRNP to the pre-mRNA and is essential in processing of only those pre-mRNAs that do not form sufficiently stable duplexes with the U7 snRNA. This role of SLBP in mammalian processing is most likely mediated by ZFP100, a 100-kDa zinc finger protein associated with the U7 snRNP and interacting with the SLBP/stem-loop complex. In addition to bridging the two factors bound to their respective sequence elements, ZFP100 may also play other roles in 3' end processing, possibly including the recruitment of the cleavage factor (Dominski, 2005).

Purification of the U7 snRNP from mammalian cells resulted in identification of two novel Sm-like proteins: Lsm10 and Lsm11, which replace the D1 and D2 Sm proteins present in the spliceosomal snRNPs. Lsm11 interacts in vitro with ZFP100 and plays a key role in recognizing the unique sequence of the Sm binding site in U7 snRNA. Orthologs of Lsm10 and Lsm11 are also found in the Drosophila U7 snRNP, demonstrating that the unique structure of the U7 snRNP in vertebrates and invertebrates is conserved. A counterpart of ZFP100 has not been yet identified in the Drosophila genome, suggesting that ZFP100 is either weakly conserved between vertebrates and invertebrates or processing of histone pre-mRNAs in Drosophila does not require this protein (Dominski, 2005).

Nuclear extracts from Drosophila S-2 and Kc cultured cells and embryos are capable of 3' end processing of presynthesized Drosophila histone pre-mRNAs. Nuclear extracts from Kc cells are also capable of cotranscriptional processing of histone pre-mRNAs. Unlike the auxiliary role played by SLBP in mammalian in vitro processing, Drosophila SLBP is indispensable for processing of all Drosophila histone pre-mRNAs. This observation suggests that Drosophila SLBP plays a much more important role in recruiting the U7 snRNP to the pre-mRNA than it does in the mammalian processing. This study uses an in vitro system based on Drosophila nuclear extracts to characterize 3' end processing of Drosophila histone pre-mRNAs and to define differences and similarities in processing between this model invertebrate processing system and processing in mammalian nuclear extracts (Dominski, 2005).

These studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing occur with similar chemistry and both require SLBP and the U7 snRNP, the two mechanisms differ significantly in the relative importance of these trans-acting factors and in the specification of the cleavage site (Dominski, 2005).

Drosophila nuclear extracts cleave histone pre-mRNAs after the fourth nucleotide following the stem-loop and prefer an adenosine preceding the cleavage site. Consistent with this, all natural Drosophila histone pre-mRNAs contain an adenosine in this position. If the fourth nucleotide is changed to a pyrimidine, cleavage is also efficient after an adenosine at the third position but not after an adenosine located 5 nt downstream of the stem-loop, i.e., at the site exclusively utilized during mammalian processing. Sea urchin histone mRNAs, the only other invertebrate histone mRNAs with the characterized 3' ends, terminate with an ACCA consensus sequence. Thus, cleavage after the fourth nucleotide following the stem-loop may be a general feature of 3' end processing of invertebrate histone pre-mRNAs. Both Drosophila and mammalian processing machineries are similar in their extreme resistance to EDTA, generation of a 3' hydroxyl group at the end of the upstream cleavage product, and degradation of the downstream cleavage product by a U7 snRNP dependent activity. These results suggest that both processing machineries utilize the same or a highly related cleavage factor in 3' end processing of histone pre-mRNAs (Dominski, 2005).

In mammalian processing, the site of cleavage is determined by the position of the HDE, and moving the HDE, and, hence, the U7 snRNP, away from the stem-loop by as few as 4 nt results in a corresponding shift of the cleavage site. This observation led to the hypothesis that U7 snRNP recruits the cleavage factor to the pre-mRNA and acts as a molecular ruler to specify the cleavage site. SLBP bound to the stem-loop facilitates binding of the U7 snRNP to the HDE but does not play a direct role in recruitment of the cleavage factor. Consistent with this model, removal of SLBP, or using a substrate that cannot bind SLBP, reduces processing activity but does not abolish it (Dominski, 2005).

In contrast to mammalian processing, processing of Drosophila histone pre-mRNA is absolutely dependent on SLBP. In addition, increasing the distance between the stem-loop and the HDE by 4 or 8 nt in Drosophila histone pre-mRNA moved the cleavage site only 1 nt upstream from its normal position and did not abolish processing at the normal site. Larger insertions between the stem-loop and the HDE resulted in low efficiency cleavage further away from the stem-loop, but cleavage at these sites was still dependent on SLBP. This is in direct contrast to mammalian histone processing, where cleavage at the distant sites is independent of SLBP. Thus, in Drosophila processing the U7 snRNP does not function as a molecular ruler, but instead SLBP plays the critical role in specifying the cleavage site (Dominski, 2005).

To explain the observed differences between processing in Drosophila and mammalian nuclear extracts, it is proposed that within the Drosophila processing complex SLBP tightly interacts with the U7 snRNP, and this interaction is essential for bringing the U7 snRNP to the pre-mRNA. The two factors remain associated even if their respective binding sites are separated by a larger distance, likely by looping out the inserted nucleotides. The mutant pre-mRNAs are preferentially cleaved close to the stem-loop, reflecting the critical role of SLBP in forming the processing complex, although the precise position of the cleavage site and efficiency of processing depends on the size of the insert. In mammalian processing, the region between the stem-loop and the HDE is either rigidified, thus precluding looping out the inserted nucleotides, as previously suggested, or the interaction between SLBP and the U7 snRNP is relatively weak and disrupted by larger insertions, so binding of the U7 snRNP to the pre-mRNA depends solely on the base-pairing interaction. It is likely that in Drosophila processing the cleavage factor is recruited to histone pre-mRNA by interaction with both the U7 snRNP and SLBP, and neither factor is competent to carry out this function individually (Dominski, 2005).

In mammalian nuclear extracts, processing of histone pre-mRNAs is efficiently inhibited by relatively short 2'O-methyl oligonucleotides complementary to the 5' end of the mammalian U7 snRNA. These oligonucleotides, including a 10-mer, are also very efficient in depleting the U7 snRNP from nuclear extracts and were successfully used to affinity purify U7 snRNP from mammalian cells, demonstrating that the 5' end of the mammalian U7 snRNA is readily accessible. In contrast, two relatively long oligonucleotides, alphaDa, complementary to the first 17 nt of the Drosophila U7 snRNA, and alphaDb, complementary to nt 4-23, were not effective in depleting the U7 snRNP from Drosophila nuclear extracts. These results suggest that the 5' end of U7 snRNA is not accessible in the Drosophila U7 snRNP (Dominski, 2005).

Surprisingly, the alphaDa 2'O-methyl oligonucleotide abolished processing of the dH3* and dH1* pre-mRNAs (hybrid pre-mRNAs consisting of the stem-loop and cleavage site from the mouse H2a-614 pre-mRNA) but did not significantly affect processing of the other three Drosophila histone pre-mRNAs. Three additional oligonucleotides complementary to the regions of the U7 snRNP located closer to the Sm binding site effectively blocked processing of all five histone pre-mRNAs. It is not understood why processing of only two Drosophila pre-mRNAs is affected by the alphaDa oligonucleotide and which features of the HDEs make processing of the Drosophila pre-mRNAs either sensitive or resistant to this oligonucleotide. Selective inhibition of processing by the alphaDa oligonucleotide depending on the type of pre-mRNA used in the reaction suggests that blocking of the U7 snRNA must occur during processing. One possibility is that the U7 snRNP is initially recruited to the pre-mRNA solely by SLBP bound to the pre-mRNA, and later this interaction is followed by formation of a duplex between the HDE and the U7 snRNA, as a result of unmasking of the 5' end of U7 snRNA. The alphaDa oligonucleotide might block binding of the U7 snRNA to the HDE in the hybrid dH1* and dH3* pre-mRNAs, but not in the other pre-mRNAs, during this later step, while the other oligonucleotides block binding to all the HDEs (Dominski, 2005).

Overall, thes studies indicate that the structure of the 5' end of the Drosophila U7 snRNA and the mechanism of its initial interactions with the HDE differ significantly from the recognition of the HDE in processing of mammalian histone pre-mRNAs (Dominski, 2005).

In vitro processing of all five Drosophila histone pre-mRNAs is absolutely dependent on SLBP. This study has demonstrated that SLBP is essential for recruitment of the U7 snRNP to the pre-mRNA. The necessity of SLBP for recruitment of the U7snRNP to the Drosophila pre-mRNAs suggests that either Drosophila HDEs are unable to form a strong duplex with the U7 snRNA or that the interaction of the U7 snRNP with the SLBP/pre-mRNA complex is necessary to promote base-pairing by making the 5' end of U7 snRNA accessible (Dominski, 2005).

Both the 5' end of the Drosophila U7 snRNA and Drosophila HDEs are AU rich, allowing a number of possible base-pair schemes for making a duplex between the two RNAs. It is hypothesized that the most likely alignment used during processing is the one that allows formation of the largest number of base pairs between the purine core of the HDE and the CUCUUU sequence in the U7 snRNA and not necessarily the alignment that allows formation of the overall most stable duplex. The CUCUUU sequence is highly conserved among all known U7 snRNAs and is involved in recognition of the purine core in sea urchin and mammalian histone pre-mRNAs. A 3-nt mutation within the purine core of the hybrid dH3* pre-mRNA abolishes processing, whereas a 6-nt mutation within the AU-rich region immediately downstream of the purine core only partially inhibits processing. These results support the interpretation that base-pairing between the U7 snRNA and the purine core is critical, whereas formation of additional base in other regions increases the efficiency of Drosophila processing. It is also possible that the base-pairing interaction is limited to the purine core and the CUCUUU sequence in the U7 snRNA, whereas the AU-rich sequences in the U7 snRNA and the HDE are brought together by protein-protein interactions (Dominski, 2005).

This study demonstrated that the HDE of the hybrid dH3* pre-mRNA can abolish processing of the full-length substrate, presumably by sequestering the U7 snRNP, only when present at very high concentrations. Interestingly, this weak interaction of Drosophila HDEs with the U7 snRNP is sufficient to recruit a 5'-3' exonuclease that specifically degrades the downstream cleavage product in a U7 dependent manner. Thus, the endonucleolytic cleavage must require much stronger binding of the U7 snRNP to the pre-mRNA, while degradation of the DCP by an exonuclease may require only loose association of the HDE with the U7 snRNP (Dominski, 2005).

The most notable difference between histone pre-mRNA processing in Drosophila and mammalian nuclear extracts is the absolute dependence of Drosophila processing on SLBP and the role of SLBP in specifying the cleavage site close to the stem-loop. The Drosophila U7 snRNP does not function as a molecular ruler in processing and this feature most likely reflects a critical role of SLBP in recruiting the cleavage factor as well as the U7 snRNP, to histone pre-mRNA. These data suggest that SLBP and the U7 snRNP may form a tight complex on the histone pre-mRNA, and this complex remains stable even in the presence of large insertions between the stem-loop and the HDE (Dominski, 2005).

The similarities in the chemistry of the cleavage reaction, including preference for an adenosine preceding the cleavage site and generation of the 3'OH group in the presence of EDTA, as well as degradation of the downstream cleavage product by a U7-dependent 5'-3' exonuclease suggest that the cleavage factor has been conserved between Drosophila and mammalian processing. It will be of interest to determine whether there are factors unique to only one of these two types of organisms emphasizing long evolutionary distance and the divergence between vertebrates and invertebrates (Dominski, 2005).

3' end processing of Drosophila melanogaster histone pre-mRNAs: requirement for phosphorylated Drosophila stem-loop binding protein and coevolution of the histone pre-mRNA processing system

Synthetic pre-mRNAs containing the processing signals encoded by Drosophila histone genes undergo efficient and faithful endonucleolytic cleavage in nuclear extracts prepared from Drosophila cultured cells and 0- to 13-h-old embryos. Biochemical requirements for the in vitro cleavage are similar to those previously described for the 3' end processing of mammalian histone pre-mRNAs. Drosophila 3' end processing does not require ATP and occurs in the presence of EDTA. However, in contrast to mammalian processing, Drosophila processing generates the final product ending four nucleotides after the stem-loop. Cleavage of the Drosophila substrates is abolished by depleting the extract of the Drosophila stem-loop binding protein (dSLBP), indicating that both dSLBP and the stem-loop structure in histone pre-mRNA are essential components of the processing machinery. Recombinant dSLBP expressed in insect cells by using the baculovirus system efficiently complements the depleted extract. Only the RNA-binding domain plus the 17 amino acids at the C terminus of dSLBP are required for processing. The full-length dSLBP expressed in insect cells is quantitatively phosphorylated on four residues in the C-terminal region. Dephosphorylation of the recombinant dSLBP reduces processing activity. Human and Drosophila SLBPs are not interchangeable and strongly inhibit processing in the heterologous extracts. The RNA-binding domain of the dSLBP does not substitute for the RNA-binding domain of the human SLBP in histone pre-mRNA processing in mammalian extracts. In addition to the stem-loop structure and dSLBP, 3' processing in Drosophila nuclear extracts depends on the presence of a short stretch of purines located ca. 20 nucleotides downstream from the stem, and an Sm-reactive factor, most likely the Drosophila counterpart of vertebrate U7 snRNP (Dominski, 2002).

Development of an in vitro system based on nuclear extracts from human and mouse cells was a major advance that allowed a molecular analysis of 3' end processing of mammalian histone pre-mRNA. A Drosophila in vitro system based on the mammalian system has been developed using nuclear extract from Drosophila S-2 cells and Drosophila histone pre-mRNAs. This system was utilized for mapping the structural features in Drosophila histone pre-mRNA and dSLBP essential for 3' end processing (Dominski, 2002).

Drosophila cultured cells are a convenient and relatively inexpensive source of nuclear extracts proficient in transcription or splicing and have been used for large-scale purification of spliceosomal snRNPs. The nuclear extract from Drosophila S-2 cells and 0- to 20-h-old embryos are also very efficient in 3' end processing of all five Drosophila histone pre-mRNAs. Since Drosophila contains only one gene (present in multiple copies) for each of the five different histone proteins, it is essential that all five Drosophila histone pre-mRNAs be efficiently processed. In mammalian cells there are multiple nonallelic copies of each histone gene, and the processing efficiency of different pre-mRNAs encoded by these copies significantly varies in vivo and in vitro (Dominski, 2002).

Drosophila histone pre-mRNA processing in vitro has biochemical properties similar to processing in mammalian cells. The reaction does not require divalent ions or ATP and generates the final product without a significant lag time, suggesting that a relatively small number of factors assemble to form a functional processing complex. The presence of the cleaved 3' fragment indicates that generation of the mature 3' end in Drosophila histone mRNAs occurs through endonucleolytic cleavage and not by the activity of a 3' exonuclease. In dNE, histone pre-mRNAs are processed four nucleotides after the stem-loop, whereas in mammalian nuclear extracts cleavage occurs one nucleotide farther downstream. There is an adenosine residue four nucleotides after the stem-loop in all five Drosophila histone pre-mRNAs, whereas most mammalian histone mRNAs end in ACCCA, suggesting that cleavage after an A has been conserved in evolution (Dominski, 2002).

Nuclear extract from Drosophila S-2 cells was very active in cleaving histone pre-mRNAs containing the downstream element encoded by all five Drosophila histone genes. In sea urchins an invariant sequence (CAAGAAAGA) has been identified in the downstream element of all histone pre-mRNAs, and this sequence base pairs with the 5' end of sea urchin U7 snRNA. The Drosophila histone genes do not share any highly conserved sequences downstream of the stem-loop, although they are all generally purine-rich. Mutagenesis studies of the downstream sequence from Drosophila H3 pre-mRNA have revealed that a GAGAUA element plays a critical role in processing; substitution of this sequence with the complementary nucleotides (mutant M1) abolishes in vitro processing, whereas mutation of adjacent nucleotides has no effect. In addition to identifying the downstream processing element in Drosophila H3 pre-mRNA, the M1 mutant provides further evidence that the in vitro system reproduces a genuine processing event and is not a result of 3' to 5' exonucleases activity stalled by the stem-loop associated with dSLBP. The sequence requirements of the downstream element must be more complex than simply the presence of the purine-rich element at a proper distance from the stem-loop since the mouse H2a pre-mRNA contains a similar purine-rich sequence in the same location and is processed in the Drosophila extract very inefficiently, in contrast to all five Drosophila histone pre-mRNAs. Perhaps there are other sequence elements in Drosophila histone pre-mRNAs that are not conserved in pre-mRNAs of higher organisms and which contribute to high efficiency of processing in dNE (Dominski, 2002).

While the low efficiency of processing of mouse H2a pre-mRNA in Drosophila extract is puzzling, it is easier to explain the inability of the mNE to process Drosophila H3 pre-mRNA. The downstream element from Drosophila H3 pre-mRNA has a very limited complementarity to the 5' end of mouse U7 snRNA and in the optimal configuration the two RNAs can form only 10 bp over a 19-nucleotide region, with the longest uninterrupted stretch of duplex RNA consisting of only 4 bp. For comparison, the mouse H1t pre-mRNA, previously shown to be a poor and completely SLBP-dependent substrate, can form either 11 or 13 bp in two alternative alignments with the U7 snRNA, and the longest uninterrupted duplex consists of 7 and 6 bp, respectively. In contrast, the mouse H2a-614 pre-mRNA, a good mammalian processing substrate, forms 14 base pairs with mouse U7 snRNA interrupted by only one mismatch. Thus, given the requirement for an extensive duplex between U7 snRNA and the downstream element for mammalian 3' end processing, the inability of the mNE to process Drosophila H3 pre-mRNA is not surprising (Dominski, 2002).

The downstream purine-rich sequence identified in these studies as essential for processing in the dNE is most likely recognized by the Drosophila equivalent of U7 snRNP. This interpretation is supported by the finding that Sm antibodies, but not control monoclonal antibodies, reproducibly reduce the efficiency of histone pre-mRNA processing. However, despite sequencing the entire Drosophila genome, U7 snRNA has not yet been identified in this organism. Both the small size and the limited evolutionary conservation precludes a search for this RNA based on sequence similarity to known vertebrate and sea urchin U7 snRNAs (Dominski, 2002).

In mammalian extracts the importance of SLBP in 3' end processing in vitro varies considerably between multiple histone pre-mRNAs and depends on the strength with which U7 snRNA base pairs with the downstream element. When there is limited complementarity between the downstream element and 5' end of U7 snRNA, as in the case of mouse H1t pre-mRNA, there is a complete dependence of processing on SLBP. Processing of some mammalian histone mRNAs that are capable of extensive base pairing with the U7 snRNA can occur in the absence of SLBP. In contrast, in vitro processing of all five Drosophila histone pre-mRNAs is virtually completely dependent on the presence of SLBP. This dependence most likely results from the relatively short downstream element in Drosophila histone pre-mRNA, which in dH3 pre-mRNA appears to be ca. six nucleotides long, and the 5' end of a putative Drosophila U7 snRNA. In sea urchins the region of complementarity between pre-mRNA and U7 snRNA is limited to only 6 bp and the formation of rather short duplexes between the two RNAs and thus a requirement for additional interaction involving U7 snRNP and SLBP may be a general feature of 3' end processing in lower metazoans (Dominski, 2002).

A series of mutations in the dSLBP gene results in a large reduction in dSLBP concentration in vivo. In the mildest of the mutants, which are viable and female sterile and still express some dSLBP, there is a great reduction in the amount of histone mRNA synthesized during oogenesis, resulting in embryonic lethality due to a lack of histone proteins and the inability to complete the syncytial cell cycles. More severe mutants are zygotically lethal, with death occurring in the larval stages. Interestingly, zygotic mutants express a significant amount of polyadenylated histone mRNA during embryogenesis as a result of transcription past the stem-loop and usage of cryptic polyadenylation sites that are present 3' of each of the Drosophila histone genes. However, even the most severe dSLBP mutants generate, during embryogenesis, a substantial amount of histone mRNA that ends at or near the stem-loop. Whether these histone mRNAs are formed by a small amount of dSLBP remaining in these embryos (which in at least one mutant cannot be detected by biochemical assays) or whether there is an alternative mechanism for forming and stabilizing mRNAs ending at the stem-loop is not known. If the latter situation is true, then this mechanism does not function in nuclear extracts from Drosophila cultured cells (Dominski, 2002).

A striking feature of dSLBP not shared by vertebrate SLBPs is its hyperphosphorylation. dSLBP overexpressed in insect cells is quantitatively phosphorylated on four sites within the C-terminal region. Based on electrophoretic mobility, a similar level of phosphorylation is present both in embryonic dSLBP and dSLBP expressed in Drosophila cultured cells. Phosphorylation of dSLBP is essential for complete processing activity of the protein in vitro. Since the dephosphorylated and the phosphorylated dSLBP bind to the stem-loop with similar affinity, the phosphorylation must be required for interaction of dSLBP with other factors involved in histone pre-mRNA processing. In the last 13 amino acids of dSLBP there are four serines, which alternate with aspartic acid residues, and it is likely that dSLBP is phosphorylated on these four serines, creating a highly acidic C terminus. Full-length dSLBP also contains at least two partially phosphorylated sites. Detailed mutational analysis will be required to determine which of these sites are critical for histone pre-mRNA processing (Dominski, 2002).

Reversible changes in phosphorylation status of dSLBP would provide an attractive mechanism for regulating function of dSLBP during embryogenesis and/or the cell cycle. Dephosphorylation would convert the active o an inactive form that would effectively inhibit processing. In mammals SLBP accumulates to the highest level in S phase and is degraded by the proteasome pathway immediately after completion of DNA replication. It is not known whether dSLBP displays the same pattern of accumulation and disappearance during the cell cycle in Drosophila cells, but reversible changes in phosphorylation status could provide an equally efficient mechanism of adjusting dSLBP activity. While regulation of dSLBP phosphorylation is a possible attractive mechanism that could contribute to regulation of histone pre-mRNA processing, only hyperphosphorylated SLBP has been observed in Drosophila cultured cells and embryos. However, changes in dSLBP phosphorylation that affected only one or two sites would not necessarily have been detected, particularly if the modification did not alter the electrophoretic mobility (Dominski, 2002).

The initial characterization of histone pre-mRNA processing in sea urchins was made possible because one of the sea urchin histone pre-mRNAs was not processed in frog oocytes as a result of differences in the HDE. The Drosophila histone pre-mRNAs are not processed in mammalian extracts, and the mammalian mRNAs are processed very inefficiently in Drosophila extracts. Moreover, Drosophila and human SLBPs are not interchangeable: dSLBP does not function in 3' end processing in mammalian nuclear extracts, and human SLBP fails to complement dSLBP-depleted nuclear extract from Drosophila S-2 cells. Instead, each protein has a strong inhibitory effect on processing in the heterologous nuclear extract by competing with the endogenous SLBP for binding to the stem-loop in histone pre-mRNA. The processing activity of both dSLBP and human SLBP requires the RBD and the adjacent amino acids of the C-terminal region. Amino acid conservation between Drosophila and mammalian SLBPs is limited only to the RBD and does not extend into the C-terminal region, thus explaining the inability of each protein to substitute for each other. Interestingly, the RBDs are also not interchangeable. The hybrid H-D-H SLBP containing both flanking domains from human SLBP and the RBD from dSLBP does not support processing in mammalian nuclear extracts. The human RBD contains a nine-amino-acid region dispensable for RNA binding but necessary for processing. This region, together with the C-terminal residues, is involved in interaction of the SLBP-pre-mRNA complex with a novel 100-kDa zinc finger protein (hZFP100) associated with the U7 snRNP. The nine-amino-acid region includes the critical DR dipeptide, which is changed in dSLBP to ER. It is possible that this single amino acid substitution, while allowing dSLBP to function efficiently in dNE, is largely responsible for inability of the hybrid H-D-H protein to function in mammalian nuclear extract. These data are consistent with coevolution of the machinery independently in the vertebrate and invertebrate lineages (Dominski, 2002).

The data presented in this study suggest that there are many similarities between the processing machineries in Drosophila and higher organisms, including the requirement for SLBP and the purine-rich sequence downstream from the cleavage site. However, the components have diverged significantly during evolution and are no longer recognized in the heterologous systems. Formally, it is still possible that there is no U7 snRNA in Drosophila, since there is no U12 snRNA, although there are ATAC introns and a U11 snRNA. This would imply that the mechanism of histone pre-mRNA processing in Drosophila is substantially different from that in sea urchins and vertebrates. It might be significant that three other proteins required for mammalian histone pre-mRNA processing hZFP100 and two U7 specific proteins, Lsm10 and Lsm11, have no obvious homologues in Drosophila genomes. Undoubtedly, future studies with dNE will be very helpful in providing more information about the mechanism of histone pre-mRNA processing in this organism (Dominski, 2002).

Developmental control of histone mRNA and dSLBP synthesis during Drosophila embryogenesis and the role of dSLBP in histone mRNA 3' end processing in vivo

In metazoans, the 3' end of histone mRNA is not polyadenylated but instead ends with a stem-loop structure that is required for cell cycle-regulated expression. The sequence of the stem-loop in the Drosophila melanogaster histone H2b, H3, and H4 genes is identical to the consensus sequence of other metazoan histone mRNAs, but the sequence of the stem-loop in the D. melanogaster histone H2a and H1 genes is novel. dSLBP binds to these novel stem-loop sequences as well as the canonical stem-loop with similar affinity. Eggs derived from females containing a viable, hypomorphic mutation in dSLBP store greatly reduced amounts of all five histone mRNAs in the egg, indicating that dSLBP is required in the maternal germ line for production of each histone mRNA. Embryos deficient in zygotic dSLBP function accumulate poly(A)(+) versions of all five histone mRNAs as a result of usage of polyadenylation signals located 3' of the stem-loop in each histone gene. Since the 3' ends of adjacent histone genes are close together, these polyadenylation signals may ensure the termination of transcription in order to prevent read-through into the next gene, which could possibly disrupt transcription or produce antisense histone mRNA that might trigger RNA interference. During early wild-type embryogenesis, ubiquitous zygotic histone gene transcription is activated at the end of the syncytial nuclear cycles during S phase of cycle 14, silenced during the subsequent G(2) phase, and then reactivated near the end of that G(2) phase in the well-described mitotic domain pattern. There is little or no dSLBP protein provided maternally in wild-type embryos, and zygotic expression of dSLBP is immediately required to process newly made histone pre-mRNA (Lanzotti, 2002).

Search PubMed for articles about Drosophila Slbp

Amodeo, A. A., Jukam, D., Straight, A. F. and Skotheim, J. M. (2015). Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition. Proc Natl Acad Sci U S A 112(10): E1086-1095. PubMed ID: 25713373

Berloco, M., Fanti, L., Breiling, A., Orlando, V. and Pimpinelli, S. (2001). The maternal effect gene, abnormal oocyte (abo), of Drosophila melanogaster encodes a specific negative regulator of histones. Proc Natl Acad Sci U S A 98(21): 12126-12131. PubMed ID: 11593026

Dominski, Z., Zheng, L. X., Sanchez, R. and Marzluff, W. F. (1999). Stem-loop binding protein facilitates 3'-end formation by stabilizing U7 snRNP binding to histone pre-mRNA. Mol Cell Biol 19(5): 3561-3570. PubMed ID: 10207079

Dominski, Z., Yang, X. C., Raska, C. S., Santiago, C., Borchers, C. H., Duronio, R. J. and Marzluff, W. F. (2002). 3' end processing of Drosophila melanogaster histone pre-mRNAs: requirement for phosphorylated Drosophila stem-loop binding protein and coevolution of the histone pre-mRNA processing system. Mol Cell Biol 22(18): 6648-6660. PubMed ID: 12192062

Dominski, Z., Yang, X. C., Purdy, M. and Marzluff, W. F. (2005). Differences and similarities between Drosophila and mammalian 3' end processing of histone pre-mRNAs. RNA 11(12): 1835-1847. PubMed ID: 16251385

Fan, J., Wang, K., Du, X., Wang, J., Chen, S., Wang, Y., Shi, M., ′, L., Wu, X., Zheng, D., Wang, C., Wang, L., Tian, B., Li, G., Zhou, Y. and Cheng, H. (2019). ALYREF links 3'-end processing to nuclear export of non-polyadenylated mRNAs. EMBO J 38(9). PubMed ID: 30858280

Godfrey, A. C., Kupsco, J. M., Burch, B. D., Zimmerman, R. M., Dominski, Z., Marzluff, W. F. and Duronio, R. J. (2006). U7 snRNA mutations in Drosophila block histone pre-mRNA processing and disrupt oogenesis. RNA 12(3): 396-409. PubMed ID: 16495235

He, W. X., Wu, M., Liu, Z., Li, Z., Wang, Y., Zhou, J., Yu, P., Zhang, X. J., Zhou, L. and Gui, J. F. (2018). Oocyte-specific maternal Slbp2 is required for replication-dependent histone storage and early nuclear cleavage in zebrafish oogenesis and embryogenesis. RNA 24(12): 1738-1748. PubMed ID: 30185624

Iampietro, C., Bergalet, J., Wang, X., Cody, N. A., Chin, A., Lefebvre, F. A., Douziech, M., Krause, H. M. and Lecuyer, E. (2014). Developmentally regulated elimination of damaged nuclei involves a Chk2-dependent mechanism of mRNA nuclear retention. Dev Cell [Epub ahead of print]. PubMed ID: 24835465

Kolev, N. G. and Steitz, J. A. (2005). Symplekin and multiple other polyadenylation factors participate in 3'-end maturation of histone mRNAs. Genes Dev 19(21): 2583-2592. PubMed ID: 16230528

Lanzotti, D. J., Kaygun, H., Yang, X., Duronio, R. J. and Marzluff, W. F. (2002). Developmental control of histone mRNA and dSLBP synthesis during Drosophila embryogenesis and the role of dSLBP in histone mRNA 3' end processing in vivo. Mol Cell Biol 22(7): 2267-2282. PubMed ID: 11884612

Lefebvre, F. A., Benoit Bouvrette, L. P., Bergalet, J. and Lecuyer, E. (2017). Biochemical fractionation of time-resolved Drosophila embryos reveals similar transcriptomic alterations in replication checkpoint and histone mRNA processing mutants. J Mol Biol [Epub ahead of print]. PubMed ID: 28167048

Lu, X., Li, J. M., Elemento, O., Tavazoie, S. and Wieschaus, E. F. (2009). Coupling of zygotic transcription to mitotic control at the Drosophila mid-blastula transition. Development 136(12): 2101-2110. PubMed ID: 19465600

Martin, F., Schaller, A., Eglite, S., Schumperli, D. and Muller, B. (1997). The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein. EMBO J 16(4): 769-778. PubMed ID: 9049306

Michalski, D. and Steiniger, M. (2015). In vivo characterization of the Drosophila mRNA 3' end processing core cleavage complex. RNA [Epub ahead of print]. PubMed ID: 26081560

Sabath, I., Skrajna, A., Yang, X. C., Dadlez, M., Marzluff, W. F. and Dominski, Z. (2013). 3'-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors. RNA 19(12): 1726-1744. PubMed ID: 24145821

Skrajna, A., Yang, X. C., Bucholc, K., Zhang, J., Hall, T. M. T., Dadlez, M., Marzluff, W. F. and Dominski, Z. (2017). U7 snRNP is recruited to histone pre-mRNA in a FLASH-dependent manner by two separate regions of the stem-loop binding protein. RNA 23(6): 938-951. PubMed ID: 28289156

Skrajna, A., Yang, X. C., Dadlez, M., Marzluff, W. F. and Dominski, Z. (2018). Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker. Nucleic Acids Res. PubMed ID: 29529248

Sullivan, K. D., Steiniger, M. and Marzluff, W. F. (2009). A core complex of CPSF73, CPSF100, and Symplekin may form two different cleavage factors for processing of poly(A) and histone mRNAs. Mol Cell 34(3): 322-332. PubMed ID: 19450530

Tan, D., Marzluff, W. F., Dominski, Z. and Tong, L. (2013). Structure of histone mRNA stem-loop, human stem-loop binding protein, and 3'hExo ternary complex. Science 339(6117): 318-321. PubMed ID: 23329046

Turner, K. J., Hoyle, J., Valdivia, L. E., Cerveny, K. L., Hart, W., Mangoli, M., Geisler, R., Rees, M., Houart, C., Poole, R. J., Wilson, S. W. and Gestri, G. (2019). Abrogation of Stem Loop Binding Protein (Slbp) function leads to a failure of cells to transition from proliferation to differentiation, retinal coloboma and midline axon guidance deficits. PLoS One 14(1): e0211073. PubMed ID: 30695021

Wang, Z. F., Whitfield, M. L., Ingledue, T. C., 3rd, Dominski, Z. and Marzluff, W. F. (1996). The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing. Genes Dev 10(23): 3028-3040. PubMed ID: 8957003

Wilky, H., Chari, S., Govindan, J. and Amodeo, A. A. (2019). Histone concentration regulates the cell cycle and transcription in early development. Development 146(19). pii: dev177402. PubMed ID: 31511251

Yang, X. C., Sabath, I., Debski, J., Kaus-Drobek, M., Dadlez, M., Marzluff, W. F. and Dominski, Z. (2013). A complex containing the CPSF73 endonuclease and other polyadenylation factors associates with U7 snRNP and is recruited to histone pre-mRNA for 3'-end processing. Mol Cell Biol 33(1): 28-37. PubMed ID: 23071092

Zhang, J., Tan, D., DeRose, E. F., Perera, L., Dominski, Z., Marzluff, W. F., Tong, L. and Hall, T. M. (2014). Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation. Proc Natl Acad Sci U S A 111(29): E2937-2946. PubMed ID: 25002523

date revised: 15 November 2020

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