InteractiveFly: GeneBrief

Helicase at 25E: Biological Overview | References

BIOLOGICAL OVERVIEW

mRNA export from the nucleus requires the RNA helicase UAP56 (Helicase at 25E) and involves remodeling of ribonucleo-protein complexes in the nucleus. This study shows that UAP56 is required for bulk mRNA export from the nurse cell nuclei that supply most of the material to the growing Drosophila oocyte and for the organization of chromatin in the oocyte nucleus. Loss of UAP56 function leads to patterning defects that identify uap56 as a spindle-class gene similar to the RNA helicase Vasa. UAP56 is required for the localization of gurken, bicoid and oskar mRNA as well as post-translational modification of Osk protein. By injecting grk RNA into the oocyte cytoplasm, this study shows that UAP56 plays a role in cytoplasmic mRNA localization. It is proposed that UAP56 has two independent functions in the remodeling of ribonucleo-protein complexes. The first is in the nucleus for mRNA export of most transcripts from the nucleus. The second is in the cytoplasm for remodeling the transacting factors that decorate mRNA and dictate its cytoplasmic destination (Meignin, 2008).

UAP56 is a conserved member of the DExH/D RNA helicase superfamily implicated in many aspects of RNA metabolism (Fairman, 2004; Fleckner, 1997) including general mRNA export from the nucleus (Gatfield, 2001; Herold, 2003; Libri, 2001). In human cells, UAP56 is preferentially associated with spliced mRNA and has a major role in bulk mRNA export (Gatfield, 2001). Furthermore, Xenopus UAP56 and its yeast homologue Sub2p are thought to be required co-transcriptionally for the recruitment of the mRNA export factor Aly/REF to mRNA (Luo, 2001). In yeast, the THO complex, which functions in transcription elongation, interacts with mRNA export factors to form the TREX (TRanscription/EXport) complex, linking transcription and export (Strasser, 2002). The human THO complex becomes associated with spliced mRNA during the course of splicing (Masuda, 2005). In Drosophila cells, UAP56 has been shown to be essential for the export of both spliced and intronless poly(A)+ mRNAs (Gatfield, 2001). Drosophila uap56 is an essential gene that was first identified as an enhancer of position effect variegation and encodes a nuclear protein named Hel25E/UAP56 (Eberl, 1997). UAP56 is proposed to promote an open chromatin structure by unwinding or releasing the mRNA from the site of transcription. It is also thought to be involved in regulating the spread of heterochromatin (Meignin, 2008).

During Drosophila oogenesis, the antero-posterior and dorso-ventral axes of the future embryo are specified through the cytoplasmic localization and translational regulation of a large number of specific transcripts. The most extensively studied mRNAs are gurken (grk) that encodes a TGFβ signal, bicoid (bcd) that encodes the anterior morphogen, and oskar (osk), which specifies posterior structures and the future germ line. All transcripts in the oocyte are thought to be transcribed in the nurse cell nuclei and transported through actin-rich ring canals into the oocyte, where they are selectively localized by transport on microtubules (MTs) by molecular motors. Some of these mRNAs are then localized within the oocyte cytoplasm. During their complex path of localization, these transcripts are thought to be present in large RNP complexes that contain a variety of RNA binding proteins. The composition of these complexes is thought to vary during the different steps of the biosynthesis, export and localization of the transcripts, and RNA helicases play essential roles in remodeling the RNPs during RNA processing, transport, localization, anchoring and translation (Meignin, 2008).

RNA helicases are also likely to be involved in remodeling a diverse range of trans-acting factors required for mRNA localization. These include cytoplasmic determinants and motor cofactors such as BicaudalD (BicD) and Egalitarian (Egl) as well as nuclear components such as hnRNPs or splicing factors. Such trans-acting factors are thought to dictate which molecular motor the transcripts associate with and therefore their cytoplasmic destination. For example, in the Drosophila oocyte, oskar (osk) mRNA requires Kinesin 1 to transport it to the plus ends of MTs while grk mRNA requires cytoplasmic Dynein (Dynein) to transport it to the minus ends of MTs (Meignin, 2008).

In some cases, factors recruited to the RNA in the nucleus remain with the RNA and function in the cytoplasm. For example, the nuclear exon-exon junction components (EJC) Mago nashi, Y14 and eIF4AIII are recruited by osk transcripts in the nucleus and then play a role in its cytoplasmic localization. Once at its final destination at the posterior pole, osk mRNA is translated using two distinct initiation codons, resulting in two different proteins. The short form of Osk promotes the assembly of polar granules by recruiting Vasa, a member of DExH/D-box family of putative RNA helicases at the posterior pole of the oocyte. Vasa is also required for promoting translation of grk through an interaction with the translation factor eIF5B/dIF2. In contrast, grk transcripts are able to recruit in the cytoplasm all the factors required for their localization (Meignin, 2008).

In Drosophila, at least 12 genes are members of the DExH/D RNA helicase superfamily. One of the best described, vasa, was originally identified as a member of the posterior class of maternal effect genes. Vasa is required for the translation of osk and nos mRNAs during the assembly of the pole plasm and for the localization and translation of grk mRNA during oogenesis. Other RNA helicases play a role during Drosophila oogenesis; belle (bel), hel25E or uap56, spindle E (spn-E) and eIF-4AIII. In addition, the small repeat-associated siRNAs (rasiRNAs) pathway, containing spn-E, aubergine and armitage, is required for axis specification. In many of these cases, the intracellular localization and translational regulation of bcd, osk and grk mRNA are affected to varying degrees (Meignin, 2008).

This study tested whether the RNA helicase UAP56 is required in the cytoplasm for mRNA localization and post-translational modification in addition to its well-studied roles in the nucleus in splicing and mRNA export. By creating new alleles of the gene, it was shown that uap56 is a spindle-class gene. uap56 mutants have strong Dorso-ventral egg shell defects caused by a mislocalization of grk mRNA and its incorrect translational regulation. grk mRNA injected into the oocyte cytoplasm of uap56 mutants fails to localize correctly, suggesting that the uap56 phenotype is due to a lack of a factor required in the cytoplasm for mRNA localization. It was also shown that UAP56 plays a role in osk and bcd mRNA localization and Osk post-translational modification. Thus UAP56 plays multiple roles in mRNA localization and post-translational modification in the oocyte cytoplasm. It is proposed that UAP56 is required for remodeling of cytoplasmic RNP complexes required for mRNA localization and post-translational modification (Meignin, 2008).

UAP56 is an RNA helicase that has been shown to play important roles in mRNA metabolism in the nucleus. In Drosophila, UAP56 is a component of the Exon Junction Complex (EJC) (Gatfield, 2001) and is required in tissue culture cells for bulk mRNA export from the nucleus. Using new mutations in the gene, this study has shown that UAP56 is also required for bulk mRNA export during Drosophila oogenesis. UAP56 also has a novel and unexpected role in mRNA localization and post-translational modification in the cytoplasm since uap56 mutants have defects in grk, osk and bcd mRNA localization and Osk post-translational modification. uap56 mutants show strong dorso-ventral egg shell defects due to disruption in cytoplasmic transport of grk mRNA, in addition to other phenotypes that display phenotypes that define uap56 as a spindle-class gene, like the RNA helicase and posterior group gene vasa. Therefore, these data have uncovered a new cytoplasmic role for UAP56 in mRNA transport and post-translational modification. It is proposed that UAP56 is required in the oocyte cytoplasm to remodel RNP complexes involved in mRNA localization and post-translational modification (Meignin, 2008).

The analysis of new alleles of the uap56 gene has revealed that UAP56 is required for general nuclear mRNA export from the nurse cell nuclei. This explains why it was impossible to study the uap56 null mutants during oogenesis since a total block in mRNA export causes cell lethality. The current observations are in good agreement with the previous work showing that UAP56 is essential for bulk mRNA export in Drosophila tissue culture cells (Gatfield, 2001) and other model systems (Jensen, 2001; MacMorris, 2003; Zhao, 2004). In wild-type, the intracellular distribution of bulk polyadenylated RNAs reveals a cytoplasmic localization with an accumulation in the Nuage. In contrast, this distribution is disrupted in uap56 mutants. During Drosophila oogenesis, the Nuage is characterized by electron dense germ line specific structures that form around the nurse cell nuclei. It is suggested that the Nuage someway facilitates the assembly of mRNP particles that mediate the transport, translational regulation and storage of specific transcripts. The Nuage contains Vasa, Maelstrom, Aubergine and Belle, all thought to be required for axis specification in the oocyte. Most components of the Nuage are also localized at the posterior pole of the Drosophila oocyte. This study found that poly(A)+ RNA is present at high levels in particles in the Nuage and that UAP56 is involved in the formation of the Nuage since Vasa protein is absent from the Nuage in uap56 mutants. However, UAP56 is not specifically localized in the Nuage nor at the posterior pole, so UAP56 is neither a posterior group gene nor a component of the Nuage. These observations are interpreted as indicating that UAP56 is required upstream of Nuage and pole plasm formation. It is proposed that UAP56 is required to facilitate the correct assembly of different mRNAs, including grk and osk with the appropriate RNA binding proteins in RNPs, before, during and possibly after export from the nurse cell nuclei. This assembly is crucial for the downstream events responsible for the transport and assembly of the RNPs into the Nuage (Meignin, 2008).

Interestingly, a small fraction of poly(A)+ RNA was found in the oocyte nucleus, an observation that could be explained in two possible ways. Either a sub-population of RNA is transcribed in the oocyte nucleus or a sub-population of poly(A)+ RNA is transported from nurse cells to oocyte and then imported into the oocyte nucleus. The second explanation is favored for the following reasons. First, it is thought that the oocyte nucleus is transcriptionally inactive. Second, the I factor mRNA is known to be synthesized in the nurse cells, transported into the oocyte and then imported into the oocyte nucleus (Van De Bor, 2005). While the genetic background that was studied is not very active for I factor transcription, it is possible that the poly(A)+ RNA detected in the oocyte nucleus represents transcripts of other transposable elements or some endogenous transcripts that follow the same pattern of biogenesis and import into the oocyte nucleus (Meignin, 2008).

During oogenesis and early embryogenesis, the majority of UAP56 protein is present in the nucleus and is probably associated with DNA. These results are consistent with previous work showing that UAP56 is closely associated with salivary gland chromosomes and localized to the nuclei of Drosophila embryos and ovaries (Eberl, 1997) as well as acting as an enhancer of position affect variegation in Drosophila (Eberl, 1997) and affecting heterochromatic gene expression in yeast (Lahue, 2005). These data together with the conclusion that uap56 is a spindle-group gene (Eberl, 1997; Lahue, 2005; Strasser, 2002) strongly suggest that the gene is involved in chromatin organization (Meignin, 2008).

In yeast, Sub2p associates with the TREX (TRansport-EXport) complex (Fischer, 2002; Strasser, 2002), which is required directly for transcription elongation, splicing and export and is recruited during transcription (Abruzzi, 2004). In contrast, the mammalian TREX complex is recruited during splicing (Reed, 2005). It is proposed that, in Drosophila, UAP56 has an intermediate role between yeast and human cells. It is likely to bind mRNA during transcription and be involved in splicing in a similar manner to nonsense-mediated mRNA decay (NMD) (Meignin, 2008).

The new alleles of uap56 reveal a role of UAP56 in mRNA localization and translational modification in the cytoplasm, key processes in axis specification. A reduction in the intensity of tau GFP staining in the oocyte was found, although the antero-posterior gradient, which is essential for mRNA localization, was unaffected. While the possibility cannot be excluded that the reduction in efficiency of localization of injected grk mRNA is due to a lower density of MTs in the oocyte, the alternative interpretation is favored, in which UAP56 plays a more direct cytoplasmic role in promoting remodeling of factors required for grk RNA localization (Meignin, 2008).

The observations in the context of the previous work on UAP56 suggest that, as well as being a general RNA export factor in Drosophila, the protein has a specific role in the localization and post-translational modification of a sub-population of key transcripts. In this respect, UAP56 is similar to some other components of the EJC, such as Mago nashi, Y14 and eIF4AIII, which are involved in mRNA localization and post-translation control of a subset of Drosophila transcripts. Interestingly, mutants in the small repeat-associated siRNAs (rasiRNAs) pathway also show similar defects in axis specification to uap56 mutants, raising the possibility that UAP56 is a component of the rasiRNA pathway. Nevertheless, the results are surprising, given that UAP56 is thought of as a ubiquitous house keeping gene with an essential function in the export of all mRNAs. A partial loss-of-function of an equivalent essential general mRNA export factor, NXF1, does not give rise to specific developmental defects, such as the ones observed with uap56 alleles. rasiRNA mutants show an accumulation of double strand breaks in egg chambers. However, no differences were found in the accumulation of H2A staining of double strand breaks between wild type and mutant germaria, in contrast to armi, aub and spn-D mutants. It is concluded that the defects observe in axis specification in uap56 mutants are not due to double strand breaks perturbing signaling in the germ line. Therefore, it is proposed that, unlike NXF1, UAP56 is likely to remain on the RNA after export and has a role in the remodeling of RNP complexes in the cytoplasm. This idea is supported by the fact that UAP56 is detected in the cytoplasm as well as the nucleus. However, no UAP56 colocalized is observed with mRNA in the cytoplasm or recruited by injected RNA. These observation are interpreted as indicating that UAP56 may only associate with RNA transiently in the cytoplasm, while acting as a cytoplasmic RNA remodeling factor. Such a role is novel for UAP56, and it remains to be discovered whether it is a general feature of this RNA dependent helicase in a variety of model systems (Meignin, 2008).

A heterochromatin-specific RNA export pathway facilitates piRNA production

PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30 nt piRNAs are processed in the cytoplasm from long non-coding RNAs that often lack RNA processing hallmarks of export-competent transcripts. By studying how these transcripts achieve nuclear export, this study uncovered an RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1 and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. These findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to export unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses (ElMaghraby, 2019).

Integrity of the germline genome is essential for the survival of multicellular species. In animal gonads, a class of small regulatory RNAs, called PIWI-interacting RNAs (piRNAs), guide Argonaute effector proteins to silence transposable elements, thereby counteracting their mutagenic impact. piRNAs are processed from single-stranded precursors, which are transcribed by RNA polymerase II from transposon-rich loci termed piRNA clusters. As piRNA biogenesis occurs in peri-nuclear processing centers, the long non-coding precursor transcripts must be exported from the nucleus to the cytoplasm (ElMaghraby, 2019).

mRNAs, the most prominent RNA polymerase II transcripts, are exported through nuclear pore complexes primarily by the nuclear RNA export factor 1 (Nxf1/Tap) and its binding partner Nxt1/p15. The Nxf1-Nxt1 heterodimer is recruited to export-competent mRNAs after successful completion of RNA-processing events such as 5' capping, splicing, and 3' end formation (Heath, 2016). Nuclear mRNA surveillance systems ensure that unprocessed transcripts are not exported but instead are degraded within the nucleus. mRNA processing is therefore a critical step in licensing export of RNA polymerase II transcripts to the cytoplasm (ElMaghraby, 2019).

In Drosophila, only few piRNA precursors are spliced and poly-adenylated, and these require the mRNA export receptor Nxf1-Nxt1 for nuclear exit. Most piRNA precursors instead lack RNA processing marks characteristic for mRNAs. These precursors originate from heterochromatic loci, whose expression depends on Rhino, a variant of the conserved heterochromatin protein 1 (HP1). Rhino, via its adaptor protein Deadlock, recruits effector proteins that stimulate transcription initiation within heterochromatin (through the TFIIA-L homolog Moonshiner) (Andersen, 2017), and that suppress co-transcriptional RNA processing events such as splicing or 3' cleavage and polyadenylation (through the Rai1 homolog Cutoff). Rather than imposing transcriptional silencing similar to HP1, Rhino thereby facilitates non-canonical transcription of heterochromatic piRNA source loci to allow the production of non-coding precursors for transposon-targeting piRNAs. How the resulting unprocessed piRNA precursors escape nuclear RNA quality control and how they are transported to cytoplasmic piRNA processing centers are central open questions (ElMaghraby, 2019).

This study has identified a nuclear export pathway dedicated to piRNA precursors. It involves Nuclear export factor 3 (Nxf3), which is required for piRNA production, transposon silencing, and fertility and which evolved from the principal mRNA-export receptor Nxf1. Instead of relying on RNA processing events for recruitment, Nxf3 is targeted to nascent piRNA precursors via Rhino, Deadlock, and CG13741/Bootlegger, a previously unknown effector protein of Rhino-dependent heterochromatin expression. After cargo binding, Nxf3 mediates piRNA precursor export in a Crm1/Xpo1-dependent manner, allowing Nxf3 and Bootlegger to deliver piRNA precursors to peri-nuclear nuage where piRNA biogenesis factors accumulate. This dedicated RNA export pathway (formed by Nxf1 duplication and neo-functionalization) therefore bypasses nuclear RNA surveillance systems and transports piRNA precursors from their heterochromatic origins to their cytoplasmic processing sites (ElMaghraby, 2019).

This work uncovered the Nxf3-Bootlegger pathway, which transports RNA Polymerase II-generated transcripts emanating from heterochromatin to peri-nuclear nuage where they are processed into piRNAs. This pathway (1) bypasses nuclear RNA surveillance mechanisms that would otherwise degrade the unprocessed precursor RNAs and (2) enables nuclear export and delivery of the piRNA precursors to nuage. Retroviruses also must bypass nuclear RNA quality-control mechanisms to export their unprocessed genomic RNA. Specific hairpins in the retroviral RNA recruit the mRNA export receptor Nxf1/Tap (e.g., Mason-Pfizer monkey virus) or exploit the exportin Crm1 through a virally encoded adaptor protein (e.g., HIV). Due to their high sequence diversity, piRNA precursors are unlikely to recruit export machinery via sequence or structural features. Instead, this work suggests that specificity in piRNA precursor export is achieved through Rhino, which recruits the specialized NXF variant, Nxf3, to nascent transcripts at heterochromatic piRNA clusters. The Nxf3-Bootlegger pathway highlights Rhino's central role in facilitating productive expression of transposon sequence information within heterochromatin. Via its adaptor protein Deadlock, Rhino connects H3K9me3 marks at piRNA clusters to a set of effector proteins that allow transcription initiation (through the TFIIA-L paralog Moonshiner), prevents transcription termination (through the Rai1/DXO paralog Cutoff), and directs nuclear export of the emerging unprocessed RNAs (through the Nxf1 paralog Nxf3). In this, Rhino provides an epigenetic specificity anchor at heterochromatic piRNA clusters where the canonical layers of specificity are either inaccessible (transcription factor binding sites for transcription initiation) or absent (RNA maturation signatures for nuclear export). Thus, central dogmas of gene expression control are bypassed at piRNA clusters through the action of physically connected paralogs of core gene expression factors (ElMaghraby, 2019).

Notably, another NXF paralog, Drosophila Nxf2, is also an essential piRNA pathway factor as it licenses heterochromatin formation downstream of nuclear Piwi. This points to the NXF protein family as a hotspot for genetic innovation through gene duplication and neo-functionalization. In vertebrates, Nxf1/Tap has also diversified into multiple NXF variants. Mammalian NXF2 and NXF3 (both not directly related to Drosophila Nxf2 and Nxf3) are preferentially expressed in gonads, and Nxf2 mutant mice are male sterile. It is proposed that neo-functionalized NXF variants have evolved throughout the animal kingdom at least in part to serve key functions in genome defense (ElMaghraby, 2019).

In order to modulate gene expression control, neo-functionalized protein paralogs must connect to the central gene expression machinery. Indeed, Nxf3 and Bootlegger co-purify with UAP56, a nuclear DEAD-box ATPase that facilitates loading of Nxf1-Nxt1 onto export-competent mRNA. In the context of mRNA export, UAP56 functions together with the THO complex. Recent work indicates a role for UAP56 and THO in piRNA cluster transcription and in defining Rhino's chromatin occupancy (Zhang, 2018). Based on the current findings, it is speculated that UAP56 and THO, analogous to their function in mRNA export, also license loading of Nxf3-Nxt1 onto piRNA precursors. In this process, Bootlegger might function analogous to SR proteins, canonical export adaptors that recruit Nxf1-Nxt1 to export-competent mRNA, or analogous to the adaptor Aly/Ref that interacts with UAP56 and Nxf1-Nxt1. It is proposed that the mechanistic investigation of specialized paralog-based gene expression pathways, such as the Nxf3-Bootlegger pathway, will provide important insight also into the molecular principles of canonical gene expression (ElMaghraby, 2019).

While the Nxf3-Bootlegger pathway evolved from the general mRNA export machinery, it also shares similarities to pre-microRNA export, which is dependent on Exportin-5. In both cases, the export machinery not only facilitates passage through the NPC, but binding of the export factors to cargo RNA also protects against nuclear RNA degradation. Similarly, and in contrast to mRNA export, both pathways utilize the Ran-GTP gradient, either directly (Exportin-5) or indirectly via Crm1 (Nxf3). As the Ran-GTP gradient confers directionality to nuclear transport pathways (here via dissociation of Crm1 from Nxf3 in the cytoplasm), it is suggested that Crm1-mediated export allows Nxf3 to remain bound to its own RNA cargo after nuclear export. Staying RNA bound enables Nxf3 and Bootlegger to escort piRNA precursors to nuage, a peri-nuclear biomolecular condensate (Nott, 2015) where the piRNA biogenesis machinery is concentrated. The identification of the Nxf3-Bootlegger pathway thus provides key insight into the long sought-after mechanism that contributes specificity to piRNA biogenesis. Remarkably, human NXF3 has independently evolved an export dependency on Crm1, suggesting that it also holds on to its RNA cargo after NPC translocation. NXF variants might therefore more broadly enable delivery of RNA cargo to specific subcellular compartments. The Nxf3-Bootlegger pathway provides a glimpse into how biological information is sorted and spatially distributed in cells. It is noted that similar findings on the Nxf3-Bootlegger pathway have been published by Kneuss and colleagues (Kneuss et al., 2019) (ElMaghraby, 2019).

Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export

The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. This study reports that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. These data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production (Kneuss, 2019).

The transcription and export of piRNA precursor transcripts require a highly specialized machinery that must assemble correctly at dual-strand cluster loci, initiate noncanonical transcription, and license and transport these transcripts (which lack features of processed and export-competent mRNAs) to the cytoplasm, where they are processed into mature piRNAs. How each step is achieved and how the elements involved interact are yet to be fully understood (Kneuss, 2019).

This study shows that export of piRNA precursors from dual-strand clusters in nurse cells depends on a specific mechanism that requires Bootlegger, a protein without known domains, and the nuclear export factor Nxf3. This study found that Bootlegger is important for either the synthesis or stability of transcripts from dual-strand piRNA clusters and is required for Nxf3 recruitment to dual-strand piRNA cluster loci. Analysis of RNA-seq, small RNA-seq, and RIP-seq for Nxf3, combined with immunofluorescence and RNA-FISH analyses, provides evidence that Nxf3 binds to and transports piRNA precursor transcripts from their sites of transcription (piRNA clus-ters) to the sites where piRNA processing takes place (nuage). It was further found that Nxf3-mediated export depends on Crm1. These findings are all in agreement with those of ElMaghraby (2019), who also identified Nxf3 and Bootlegger as critical facilitators of piRNA precursor export (Kneuss, 2019).

How Bootlegger is recruited to piRNA cluster loci remains to be determined. One plausible mechanism is via a direct protein-protein interaction with one or more components of the RDC complex, which specifies piRNA clusters upon recognition of H3K9me3 marks by Rhi. In support of this hypothesis, an interaction between Boot- legger and Del was observed by a yeast two-hybrid screen (ElMaghraby. 2019); however, whether this complex forms in ovaries requires further examination. Recruitment of Nxf3 in turn requires Bootlegger, likely also through direct protein-protein interaction. It will be important to uncover the precise contacts that drive this recruitment and probe whether RNA binding by Nxf3 (and/or Bootlegger) might contribute to complex formation. Nxf3 likely binds cluster RNAs through its cargo-binding domain, which is composed of an RNA-binding domain and LRRs. However, how specific binding of piRNA precursors is achieved remains a mystery. In fact, how exclusivity for the three nuclear export factor proteins with annotated functions (Nxf1-3) is achieved is a critical question for the future (Kneuss, 2019).

Eukaryotic cells use various different RNA export machineries, depending on the class of cargo RNA. Canonical mRNAs, which carry 5' caps, have undergone splicing, and carry 3' poly(A) tails, are exported via the Nxf1-Nxt1 pathway. Another extreme example are pre- microRNAs (pre-miRNAs), which are specifically recognized via their secondary structure and exported by Exportin-5. A subset of RNAs is exported via a Crm1-dependant mechanism. Although Crm1 is typically involved in protein export, it can function in RNA export via adapter proteins. For example, Crm1 binds to the Cap-binding complex and the adapter protein PHAX to export ~200-nt sized small nuclear RNAs. This study shows that Nxf3-mediated export of piRNA precursor transcripts also requires Crm1. The role of Nxt1 in piRNA precursor export is not yet clear. It is plausible that Nxt1 is required for export by Nxf3 or might function in cotranscriptional recruitment to cargo RNAs. In this regard, it is striking that Nxt1 and also UAP56 and THO complex components are concentrated at piRNA clusters even though these proteins are reportedly essential for general mRNA export (Kneuss, 2019).

The work presented in this and other studies begin to paint a picture in which the evolutionary pressures exerted by the need to control mobile genetic elements have resulted in exaptation and dedication of nuclear export factor family members to the piRNA pathway. This specialization has taken different forms. The most easily understood is the adaptation of Nxf3 to export a specific class of noncanonically transcribed RNAs (ElMaghraby, 2019), whereas the precise mechanism by which Nxf2 acts as a cotranscriptional silencing factor remains more mysterious. Ultimately, an understanding of how these proteins have assumed new roles through evolution will require a detailed understanding of how they are recruited to particular transcripts and how they mediate their downstream effects (Kneuss, 2019).

REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export

The metazoan proteins UAP56, REF1, and NXF1 are thought to bind sequentially to mRNA to promote its export to the cytoplasm: UAP56 is thought to recruit REF1 to nascent mRNA; REF1 acts as an adaptor protein mediating the association of NXF1 with mRNA, whereas NXF1 translocates the mRNA across the nuclear pore complex. REF1 is a component of the exon-exon junction complex (EJC); thus, the EJC is thought to play a role in the export of spliced mRNA. NXF1 and UAP56 are essential for mRNA export. An essential role for metazoan REF1 or the additional EJC proteins in this process has not been established. Contrary to expectation, this study shows that REF1 and the additional components of the EJC are dispensable for export of bulk mRNA in Drosophila cells. Only when REF1 and RNPS1 are codepleted, or when all EJC proteins are simultaneously depleted is a partial nuclear accumulation of polyadenylated RNAs observed. Because a significant fraction of bulk mRNA is detected in the cytoplasm of cells depleted of all EJC proteins, it is concluded that additional adaptor protein(s) mediate the interaction between NXF1 and cellular mRNAs in metazoa. The results imply that the essential role of UAP56 in mRNA export is not restricted to the recruitment of REF1 (Gatfield, 2002).

Before this study, REF1 and the additional components of the EJC were largely believed to provide a molecular link between splicing and mRNA export. However, this study shows that neither REF1 nor the individual EJC proteins are essential for export of bulk mRNA. Furthermore, simultaneous depletion of REF1, RNPS1, SRm160, and Y14 results in only a partial accumulation of polyadenylated RNAs within the nucleus, indicating that besides REF1 or the EJC proteins, additional adaptor protein(s) bridge the interaction between NXF1:p15 and cellular mRNAs in higher eukaryotes (Gatfield, 2002).

Although REF1 is not essential for mRNA export in vivo, several lines of evidence indicate that REF1 can nevertheless contribute to the recruitment of NXF1 to cellular mRNAs. First, antibodies specific to REFs that prevent their interaction with RNA in vitro reduced the export rate of mRNA after microinjection into Xenopus oocytes. Second, microinjection of recombinant REF1 into Xenopus oocytes stimulates the export of mRNAs that are otherwise exported inefficiently. Finally, it has recently been shown that the Herpes virus ICP27 protein promotes export of viral RNAs by recruiting REF1 directly and NXF1 indirectly. Thus, REF1 is able to promote NXF1 binding to RNA cargoes. Together with these observations, the results suggest that in higher eukaryotes REF1 is not essential for the export of bulk mRNA (i.e., mRNA export can still take place in its absence), but may contribute to the overall efficiency of this process. It cannot be ruled out that REF1 plays in addition a crucial role in the export of specific mRNAs. Moreover, REF1 may play a role in other posttranscriptional processes (Gatfield, 2002).

Depletion of the individual EJC proteins leads to different effects on cell proliferation, mRNA export, and protein synthesis. In particular, individual depletion of RNPS1 or Y14 inhibits cell proliferation without significantly affecting bulk mRNA export (as judged by oligo[dT] in situ hybridizations). This suggests that these proteins may play a crucial role in the posttranscriptional metabolism of specific transcript(s), which in turn are required for cell proliferation or survival (Gatfield, 2002).

Simultaneous depletion of REF1 and RNPS1 or of these two proteins together with SRm160 and Y14 significantly inhibits both cell proliferation and the incorporation of [35S]Met into newly synthesized proteins. The inhibition of protein synthesis may be in part due to a partial inhibition of mRNA export, but a reduction in steady-state mRNA levels also contributes to the observed phenotype. Alternatively, the overall reduction of protein synthesis could be due to a role of these proteins in the translation activity of exported mRNAs rather than in the export process itself. Consistently, a role for Y14 in modulating the translation activity of cytoplasmic mRNAs has been proposed. Finally, the accumulation of poly(A)+ RNA within the nucleus may not only reflect an export block but the nuclear retention of pre-mRNAs whose processing is inhibited. Although splicing is not generally inhibited in the absence of REF1, RNPS1, SRm160, and Y14, removal of specific introns and/or 3'-end processing of specific transcripts may require the activity of some of these proteins. In particular, both RNPS1 and SRm160 were originally identified as activators of pre-mRNA splicing and SRm160 has been reported to influence polyadenylation in mammalian cells (Gatfield, 2002).

Although the Drosophila genome encodes homologues of all known vertebrate EJC proteins, it has not been established whether the EJC is assembled in this organism, so the conclusions are applicable to the individual proteins and not to the EJC. The possibility that in vertebrates these proteins would play an essential role in bulk mRNA export through their assembly into the EJC is nonetheless unlikely. Indeed, the effect of splicing (and thereby of EJC assembly) on multiple steps of gene expression including transcription, polyadenylation, mRNA export, translation efficiency, and mRNA decay has recently been systematically analyzed in mammalian cells. These studies show that mRNAs generated through the splicing pathway are more efficiently incorporated into polyribosomes. In contrast, splicing and EJC formation has no significant effect on nuclear mRNA export. In agreement with these observations in mammalian cells, in this study it was show that REF1/Aly, RNPS1, SRm160, and Y14 are not essential for the formation of a export-competent mRNP in Drosophila cells (Gatfield, 2002).

Is UAP56 a specific recruitment factor or a general mRNP remodeling protein? Although in S. cerevisiae the role of Sub2p in mRNA export has been shown to be related to its ability to recruit Yra1p (Strässer, 2001), further work is needed to clarify the role of this protein in higher eukaryotes. Analysis of the association of UAP56 and REF1 with the Balbiani ring RNPs of Chironomus tetans has shown that UAP56 is recruited cotranscriptionally along the mRNA independently of the presence of introns (Kiesler, 2002). In contrast, the recruitment of REF1 is restricted to the regions of the transcript in which introns have been removed. This indicates that binding of UAP56 to nascent transcripts does not necessarily lead to the recruitment of REF1 (Gatfield, 2002).

In this and previous studies (Gatfield, 2001) it was found that although UAP56 is essential for the export of bulk mRNA, REF1 is dispensable. There are several possible explanations for this observation. In higher eukaryotes, UAP56 may recruit an unidentified essential protein (besides REF1), which in turn promotes binding of NXF1:p15 heterodimers. Alternatively, UAP56 may not act as a recruiting factor but its role in mRNA export could be more general. Indeed, as expected for a helicase, UAP56 could trigger ATP-dependent rearrangements of the mRNP at specific stages of the export process. This may then facilitate binding of proteins that act as adaptors for NXF1. Although this is an attractive hypothesis, it has not yet been shown whether UAP56 has helicase activity or whether its putative ATPase activity is required for its essential export function (Gatfield, 2002).

The essential role of NXF1 or Mex67p in the export of poly(A)+ RNA is well established in S. cerevisiae, C. elegans, and D. melanogaster. In these organisms, inactivation or depletion of these proteins results in a strong accumulation of bulk mRNA within the nucleoplasm. In yeast and Drosophila cells, this nuclear accumulation of poly(A)+ RNA correlates with a strong inhibition of protein synthesis, indicating that export of most mRNAs is affected. Similarly, as mentioned above, both Dm UAP56 and its yeast homologue Sub2p are essential for export of bulk mRNA (Gatfield, 2002).

The link between Sub2p and Mex67p is provided by Yra1p, which is also essential for the export of poly(A)+ RNA in S. cerevisiae. This paper shows that, despite strong conservation, REF1, the metazoan homologue of Yra1p, is dispensable for bulk mRNA export. This raises the possibility that another essential adaptor protein or (alternatively) multiple and partially redundant adaptor proteins link UAP56 and NXF1 in higher eukaryotes. The identification of this additional adaptor protein(s) will provide important insights into the molecular mechanisms by which mRNAs are exported in higher eukaryotic cells (Gatfield, 2002).

The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila

Dbp5 is the only member of the DExH/D box family of RNA helicases that is directly implicated in the export of messenger RNAs from the nucleus of yeast and vertebrate cells. Dbp5 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore complex (NPC). In an attempt to identify proteins present in a highly enriched NPC fraction, two other helicases were detected: RNA helicase A (RHA) and UAP56. This suggested a role for these proteins in nuclear transport. Contrary to expectation, the Drosophila homolog of Dbp5 is not essential for mRNA export in cultured Schneider cells. In contrast, depletion of HEL, the Drosophila homolog of UAP56, inhibits growth and results in a robust accumulation of polyadenylated RNAs within the nucleus. Consequently, incorporation of [³⁵S]methionine into newly synthesized proteins is inhibited. This inhibition affects the expression of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs. In HeLa nuclear extracts, UAP56 preferentially, but not exclusively, associates with spliced mRNAs carrying the exon junction complex (EJC). It is concluded that HEL is essential for the export of bulk mRNA in Drosophila. The association of human UAP56 with spliced mRNAs suggests that this protein might provide a functional link between splicing and export (Gatfield, 2001: Full text of article).

While the knock-downs of DBP80 and MLE have no apparent phenotype in SL2 cells, depletion of HEL inhibits growth and results in a robust nuclear accumulation of bulk poly(A)+ mRNA. Although the human and yeast homologs of HEL have been implicated in pre-mRNA splicing, this study found that the mRNA export block observed after its depletion is not a consequence of a general inhibition of splicing. Moreover, despite its preferential association with spliced mRNAs carrying the EJC in vitro, in vivo HEL/UAP56 is involved in the export of both intron-containing and intronless mRNAs. Similar results were obtained in yeast upon the depletion of Sub2p (Jensen (2001). The mechanism by which HEL/UAP56/Su2p is implicated in export needs to be elucidated, but like other helicases, these proteins can be considered to be ATP-driven switches that can produce conformational changes of associated proteins and trigger ATP-dependent rearrangements of the RNP fiber at specific stages of the export process. More generally, the results indicate that both in yeast and metazoans export of intron-containing and intronless mRNAs is mediated by a common set of proteins including HEL/UAP56/Sub2p, the heterodimeric export receptor TAP/p15 (also known as NXF1/p15 or Mex67p/Mtr2p in yeast), and the hnRNP-like proteins REFs/Yra1 (Gatfield, 2001).

A new enhancer of position-effect variegation in Drosophila melanogaster encodes a putative RNA helicase that binds chromosomes and is regulated by the cell cycle

In Drosophila, position-effect variegation of the white gene has been a useful phenomenon by which to study chromosome structure and the genes that modify it. A new enhancer of variegation locus, Dmrnahel (hel), has been identified. Deletion or mutation of hel enhances white variegation, and this can be reversed by a transformed copy of hel⁺. In the presence of two endogenous copies, the transformed hel⁺ behaves as a suppressor of variegation. he1 is an essential gene and functions both maternally and zygotically. The HEL protein is similar to known RNA helicases, but contains an unusual variant (DECD) of the DEAD motif common to these proteins. Potent HEL homologues have been found in mammals, yeast and worms. HEL protein associates with salivary gland chromosomes and locates to nuclei of embryos and ovaries, but disappears in mitotic domains of embryos as chromosomes condense. It is proposed that the HEL protein promotes an open chromatin structure that favors transcription during development by regulating the spread of heterochromatin, and that HEL is regulated by, and may have a role in, the mitotic cell cycle during embryogenesis (Eberl, 1997; Full text of article).

Search PubMed for articles about Drosophila UAP56

Abruzzi, K. C., Lacadie, S. and Rosbash, M. (2004). Biochemical analysis of TREX complex recruitment to intronless and intron-containing yeast genes. EMBO J. 23: 2620-2631. PubMed ID: 15192704

Andersen, P. R., Tirian, L., Vunjak, M. and Brennecke, J. (2017). A heterochromatin-dependent transcription machinery drives piRNA expression. Nature 549(7670): 54-59. PubMed ID: 28847004

Eberl, D. F. et al. (1997). A new enhancer of position-effect variegation in Drosophila melanogaster encodes a putative RNA helicase that binds chromosomes and is regulated by the cell cycle. Genetics 146: 951-963. PubMed ID: 9215899

ElMaghraby, M. F., Andersen, P. R., Puhringer, F., Hohmann, U., Meixner, K., Lendl, T., Tirian, L. and Brennecke, J. (2019). A heterochromatin-specific RNA export pathway facilitates piRNA production. Cell 178(4): 964-979. PubMed ID: 31398345

Fairman, M. E., et al. (2004). Protein displacement by DExH/D “RNA helicases” without duplex unwinding. Science 304: 730-734. PubMed ID: 15118161

Fischer, K., et al. (2002). The mRNA export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores. EMBO J. 21: 5843-5852. PubMed ID: 12411502

Fleckner, J., et al. (1997). U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction. Genes Dev. 11: 1864-1872. PubMed ID: 9242493

Gatfield, D., Le Hir, H., Schmitt, C., Braun, I. C., Köcher, T., Wilm, M. and Izaurralde, E. (2001). The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila. Curr. Biol. 11(21): 1716-21. PubMed ID: 11696332

Gatfield, D. and Izaurralde, E. (2002). REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export. J. Cell Biol. 159(4): 579-88. PubMed ID: 12438415

Heath, C. G., Viphakone, N. and Wilson, S. A. (2016). The role of TREX in gene expression and disease. Biochem J 473(19): 2911-2935. PubMed ID: 27679854

Herold, A., Teixeira, L. and Izaurralde, E. (2003). Genome-wide analysis of nuclear mRNA export pathways in Drosophila. EMBO J. 22(10): 2472-83. PubMed ID: 12743041

Jensen, T. H., Boulay, J., Rosbash, M. and Libri, M. (2001). The DECD box putative ATPase Sub2p is an early mRNA export factor. Curr. Biol. 11: 1711-1715. PubMed ID: 11696331

Kiesler, E., Miralles, F. and Visa, N. (2002). HEL/UAP56 binds cotranscriptionally to the Balbiani Ring Pre-mRNA in an intron-independent manner and accompanies the BR mRNP to the nuclear pore. Curr. Biol. 12: 859-862. PubMed ID: 12015125

Kneuss, E., Munafo, M., Eastwood, E. L., Deumer, U. S., Preall, J. B., Hannon, G. J. and Czech, B. (2019). Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export. Genes Dev 33(17-18): 1208-1220. PubMed ID: 31416967

Lahue, E., et al. (2005). The Saccharomyces cerevisiae Sub2 protein suppresses heterochromatic silencing at telomeres and subtelomeric genes. Yeast 22: 537-551. PubMed ID: 15942929

Libri, D., Graziani, N., Saguez, C. and Boulay, J. (2001). Multiple roles for the yeast SUB2/yUAP56 gene in splicing, Genes Dev. 15: 36-41. PubMed ID: 11156603

Luo, M. L. et al. (2001). Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly. Nature 413: 644-647. PubMed ID: 11675789

Masuda, S. et al. (2005). Recruitment of the human TREX complex to mRNA during splicing. Genes Dev. 19: 1512-1517. PubMed ID: 15998806

MacMorris, M., Brocker, C. and Blumenthal, T. (2003). UAP56 levels affect viability and mRNA export in Caenorhabditis elegans. RNA 9: 847-857. PubMed ID: 12810918

Meignin, C. and Davis, I. (2008). UAP56 RNA helicase is required for axis specification and cytoplasmic mRNA localization in Drosophila. Dev. Biol. 315(1): 89-98. PubMed ID: 18237727

Nott, T. J., Petsalaki, E., Farber, P., Jervis, D., Fussner, E., Plochowietz, A., Craggs, T. D., Bazett-Jones, D. P., Pawson, T., Forman-Kay, J. D. and Baldwin, A. J. (2015). Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol Cell 57(5): 936-947. PubMed ID: 25747659

Reed, R. and Cheng, H. (2005). TREX, SR proteins and export of mRNA. Curr. Opin. Cell Biol. 17: 269-273. PubMed ID: 15901496

Strässer, K. and Hurt, E. (2001). Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yra1p. Nature 413: 648-652. PubMed ID: 11675790

Strässer, K., et al. (2002). TREX is a conserved complex coupling transcription with messenger RNA export, Nature 417: 304-308. PubMed ID: 11979277

Van De Bor, V., et al. (2005). Gurken and the I factor retrotransposon RNAs share common localization signals and machinery. Dev. Cell 9: 51-62. PubMed ID: 15992540

Zhang, G., Tu, S., Yu, T., Zhang, X. O., Parhad, S. S., Weng, Z. and Theurkauf, W. E. (2018). Co-dependent assembly of Drosophila piRNA precursor complexes and piRNA cluster heterochromatin. Cell Rep 24(13): 3413-3422 e3414. PubMed ID: 30257203

Zhao, R., et al. (2004). Crystal structure of UAP56, a DExD/H-box protein involved in pre-mRNA splicing and mRNA export. Structure 12: 1373-1381. PubMed ID: 15296731

date revised: 3 January 2020

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