The observed expression pattern of aly mRNA is consistent with the earliest known phenotype of aly mutant testes: failure to initiate transcription of a set of genes in early primary spermatocytes. In wild-type testes, the aly transcript is detected at high levels in early primary spermatocytes by RNA in situ hybridization using an antisense RNA probe generated from a cDNA clone. The level of expression decreases as the spermatocytes mature, and is undetectable in meiotic cysts. No staining was observed in the most apical region of the testis, which contains mitotically proliferating cells (White-Cooper, 2000).
The lin-9 gene of C. elegans has been identified as part of a genetic pathway that antagonizes the EGFR-Ras-MAPK-based induction of vulval cell fate during larval development. The lin-9 (SynMuvB) pathway is postulated to involve signalling between the hypodermis and the vulval precursor cells, based on mosaic analysis with different genes in the pathway. Many components of the pathway have now been shown to be nuclear; some ubiquitously expressed, e.g., TAM-1 and LIN-53, others restricted to the vulval precursor cells and not hypodermis, e.g. LIN-36, and still others restricted to the hypodermis and not vulval precursor cells, e.g. LIN-13. The location in the cell at which LIN-9 acts, or even in which cell the protein is required is still unknown. How the lin-9 pathway interacts with the EGFR-Ras-MAPK pathway also remains unknown. The lin-9 pathway may act directly by inhibiting one of the components of the EGFR-Ras-MAPK cascade, or in parallel, perhaps controlling transcription of downstream targets of MAPK activation. To explore how aly, and by analogy lin-9 and other aly homologs, may act, the subcellular distribution of Aly protein was examined (White-Cooper, 2000).
Indirect immunofluorescence using an antibody raised against a bacterially expressed Aly protein has revealed that Aly is nuclear, and mostly localized on the chromatin of primary spermatocytes, consistent with the predicted nuclear localization signals in the protein sequence. The protein is not detected in the nuclei of the somatic cyst cells that surround each cyst of 16 germ cells. This result was expected from Northern blot analysis, that indicated the aly transcript is germline dependent. The subcellular localization of Aly protein depends on the stage of spermatocyte growth. Staining of wild-type testes by whole-mount immunohistochemistry revealed that in very early primary spermatocytes Aly protein is both nuclear and cytoplasmic. When the cysts start to mature, Aly protein becomes predominantly nuclear and is concentrated on chromatin as seen by immunofluorescence staining of cells at this developmental stage in squashed preparations. Aly protein staining becomes weaker as the primary spermatocytes mature and is undetectable in cells undergoing the meiotic divisions and all stages thereafter. Aly protein is not detected at the very apical tip of the testis where spermatogonia are undergoing mitotic amplification divisions, as expected from the lack of aly mRNA in those cells (White-Cooper, 2000).
The shift of Aly protein from cytoplasm to nucleus in early primary spermatocytes in wild-type testes suggests that the nuclear localization of the protein may be regulated, perhaps in response to a signalling event. In testes from a loss-of-function allele, the defective Aly protein is exclusively cytoplasmic, both in early and later primary spermatocytes, supporting the possibility that the cytoplasmic form of Aly is inactive (White-Cooper, 2000).
The Aly protein migrates as a 68 kDa doublet in polyacrylamide gels. Western blot analysis shows that the 68 kDa doublet is present in wild-type, and in can, mia and sa mutant testes, but not in null alleles. The Aly doublet is detected in testes from alyz3-1393 homozygotes. The upper band of the doublet is consistently weaker than the faster migrating band, but the relative abundance of the two isoforms is somewhat variable. The slower migrating isoform is relatively more abundant in testes that contain arrested cells, suggesting that there may be a stage dependent modification of the protein. A second band is sometimes detected at about 90 kDa; this band does not represent the Aly protein since it is not consistently present in wild-type samples, and is also occasionally detected in transcript null allele aly5. Additionally this band was not detected by serum from the second rabbit immunized with the same fusion protein. The aly1 allele is somewhat temperature sensitive: whereas most cysts arrest as primary spermatocytes, a few cysts complete the meiotic divisions and proceed through some differentiation in homozygous males raised at 18°C. In contrast, homozygous males raised at 25°C show a spermatocyte arrest phenotype indistinguishable from that of the null alleles (Lin, 1996). The 68 kDa Aly doublet is present in testes from aly1 homozygotes raised at 18°C, but not in testes extracts from aly1 males raised at 25°C. Consistent with this Western blotting result, no staining above background levels was detected by whole-mount immunohistochemistry in testes of aly1 males reared at 25°C, whereas strong staining was observed in testes of aly1 males reared at 18°C. In testes from aly1 males reared at 18°C testes, as in wild type, Aly protein is first detected throughout the nucleus and cytoplasm in early primary spermatocytes. In slightly older spermatocytes, the aly1 protein shows strong localization on the chromatin, where it persists in the arrested cells that accumulate at 18°C. Aly protein is not detected in the occasional cysts where meiotic divisions have occurred. The localization of Aly protein in can1 mutant testes and in mia and sa1 is essentially the same as in aly1 reared at 18°C, except that no meiotic or post-meiotic cysts are present (White-Cooper, 2000).
Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? This study investigated how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. The silenced state, set up in precursor cells, was found to be relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components [specifically E(z) and Su(z)12], recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding protein-associated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC), a tissue-specific version of the MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription (Chen, 2011).
The results suggest a stepwise series of developmentally programmed events as terminal differentiation genes convert from a transcriptionally silent state in precursor cells to full expression in differentiating spermatocytes (see Model for the developmentally programmed steps that oppose PcG repression and turn on terminal differentiation gene expression). In precursor cells, differentiation genes are repressed and associated with background levels of hypophosphorylated Pol II and H3K4me3. These genes also display elevated levels of H3K27me3 and Polycomb at the promoter region, suggesting that they are acted upon by the PcG transcriptional silencing machinery. Notably, the differentiation genes studied in precursor cells here did not show the hallmark bivalent chromatin domains enriched for both the repressive H3K27me3 mark and the active H3K4me3 mark that have been characterized for a cohort of differentiation genes in mammalian ESCs (Chen, 2011).
The cell fate switch from proliferating spermatogonia to the spermatocyte differentiation program initiates both global and local changes in the transcriptional regulatory landscape, starting a cell type-specific gene expression cascade that eventually leads to robust transcription of the terminal differentiation genes. Globally, soon after the switch from spermatogonia to spermatocytes, core subunits of the PRC2 complex are downregulated, including E(z), the enzyme that generates the H3K27me3 mark. Locally, after male germ cells become spermatocytes, Pol II accumulates at the terminal differentiation gene promoters, although these genes still remain transcriptionally silent, with low H3K4me3 and high Polycomb protein levels near their promoters (Chen, 2011).
The next step awaits the expression of spermatocyte-specific forms of core transcription machinery and chromatin-associated regulators, including homologs of subunits of both the general transcription factor TFIID (tTAFs) and the MIP/dREAM complex (Aly and other testis-specific components of tMAC). The tMAC complex acts either locally or globally, perhaps at the level of chromatin or directly through interaction with tTAFs, to allow recruitment of tTAFs to promoters of target terminal differentiation genes. The action of tTAFs then allows full and robust transcription of the terminal differentiation genes, partly by displacing Polycomb from their promoters (Chen, 2011).
Strikingly, the two major PcG protein complexes appear to be regulated differently by the germ cell developmental program: whereas the PRC2 components E(z) and Su(z)12 are downregulated, the PRC1 components Polycomb, Polyhomeotic and dRing continue to be expressed in spermatocytes. The global downregulation of the epigenetic 'writer' E(z) in spermatocytes might facilitate displacement of the epigenetic 'reader', the PRC1 complex, from the differentiation genes, with the local action of tTAFs at promoters serving to select which genes are relieved of PRC1. In addition, the tTAFs act at a second level to regulate Polycomb by recruiting and accompanying Polycomb and several other PRC1 components to a particular subnucleolar domain in spermatocytes. It is not yet known whether sequestering of PRC1 to the nucleolus by tTAFs plays a role in the activation of terminal differentiation genes, perhaps by lowering the level of PRC1 that is available to exchange back on to differentiation gene promoters. Conversely, recruitment of PRC1 to the nucleolar region might have a separate function, such as in chromatin silencing in the XY body as observed in mammalian spermatocytes (Chen, 2011).
The findings indicate that, upon the switch from spermatogonia to spermatocytes, the terminal differentiation genes go through a poised state, marked by presence of both active Pol II and repressive Polycomb, before the genes are actively transcribed. Stalled Pol II and abortive transcript initiation are emerging as a common feature in stem/progenitor cells. This mechanism may prime genes to rapidly respond to developmental cues or environmental stimuli. Stalled Pol II could represent transcription events that have initiated elongation but then pause and await further signals, as in the regulation of gene expression by the androgen receptor. Alternatively, Pol II might be trapped at a nascent preinitiation complex, without melting open the DNA, as found in some instances of transcriptional repression by Polycomb. Although ChIP analyses did not have the resolution to distinguish whether Pol II was stalled at the promoter or had already initiated a short transcript, the results with antibodies specific for unphosphorylated Pol II suggest that Pol II is trapped in a nascent preinitiation complex. The PRC1 component dRing has been shown to monoubiquitylate histone H2A on Lys119 near or just downstream of the transcription start site. It is proposed that in early spermatocytes, before expression of the tTAFs and tMAC, the local action of PRC1 in causing H2AK119ub at the terminal differentiation gene promoters might block efficient clearing of Pol II from the preinitiation complex and prevent transcription elongation (Chen, 2011).
Removal of PRC1 from the promoter and full expression of the terminal differentiation genes in spermatocytes require the expression and action of tMAC and tTAFs. Cell type-specific homologs of TFIID subunits have been shown to act gene-selectively to control developmentally programmed gene expression. For example, incorporation of one subunit of the mammalian TAF4b variant into TFIID strongly influences transcriptional activation at selected promoters, directing a generally expressed transcriptional activator to turn on tissue-specific gene expression (Chen, 2011).
The local action of the tTAFs to relieve repression by Polycomb at target gene promoters provides a mechanism that is both cell type specific and gene selective, allowing expression of some Polycomb-repressed genes while keeping others silent. Similar developmentally programmed mechanisms may also reverse PcG-mediated epigenetic silencing in other stem cell systems. Indeed, striking parallels between the current findings and recent results from mammalian epidermis suggest that molecular strategies are conserved from flies to mammals. In mouse epidermis, the mammalian E(z) homolog Ezh2 is expressed in stem/precursor cells at the basal layer of the skin. Strikingly, as was observed for E(z) and Su(z)12 in the Drosophila male GSC lineage, the Ezh2 level declines sharply as cells cease DNA replication and the epidermal differentiation program is turned on. Overexpression of Ezh2 in epidermal precursor cells delays the onset of terminal differentiation gene expression (Ezhkova, 2009), and removal of the Ezh2-generated H3K27me3 mark by the Jmjd3 (Kdm6b) demethylase is required for epidermal differentiation (Chen, 2011).
In particular, the results suggest a possible explanation for the conundrum that, although PcG components are bound at many transcriptionally silent differentiation genes in mammalian ESCs, loss of function of PcG components does not cause loss of pluripotency but instead causes defects during early embryonic differentiation. In Drosophila male germ cells, events during the switch from precursor cell proliferation to differentiation are required to recruit Pol II to the promoters of differentiation genes. Without this differentiation-dependent recruitment of Pol II, loss of Polycomb is not sufficient to precociously turn on terminal differentiation genes in precursor cells. Rather, Polycomb that is pre-bound at the differentiation gene promoters might serve to delay the onset of their transcription after the mitosis-to-differentiation switch. Robust transcription must await the expression of cell type- and stage-specific components of the transcription machinery. These might in turn guide gene-selective reversal of Polycomb repression to facilitate appropriate differentiation gene expression in specific cell types (Chen, 2011).
Several of the aly mutant alleles have sequence alterations that alter the predicted protein, confirming that the transcription unit identified from the aly region is indeed the aly gene. In addition to the eight published alleles (Lin, 1996), four new EMS-induced aly alleles that fail to complement aly5 and one another, were generated in a large scale mutagenesis screen. The P-element hybrid dysgenesis-induced allele aly5 has an insertion of the transposon Hobo associated with a 10 bp duplication of the genomic DNA within the predicted open reading frame. The EMS induced alleles aly2 and alyz3-3504 have nonsense mutations that would truncate the predicted Aly protein at amino acid positions 414 and 189, respectively. In both alyz3-4302 and alyz3-4307 the final eight bases of the second intron were replaced with a different sequence of six bases, deleting the splice acceptor site. If this lesion results in failure to splice out the intron, the resulting mRNA would encode a truncated protein containing 92 amino acids of the normal protein, followed by a novel sequence of 36 amino acids. The EMS induced alyz3-1393 allele has a missense mutation such that Val150 is changed to glutamic acid. The four new EMS alleles were induced on a different background chromosome from the previously described alleles. They shared several silent polymorphisms and four polymorphisms that lead to conservative amino acid changes compared with the previously determined genomic sequence and the other alleles. These were N62K, D66E, N220K and E518D (White-Cooper, 2000).
The C. elegans homolog of aly, lin-9, antagonizes induction of vulval differentiation by the EGFR/Ras/MAPK pathway. To test whether aly might act to directly downregulate Ras pathway signalling in the Drosophila testis, the phosphorylation (and thus the activation) state of MAP kinase was examined in wild-type and aly mutant testes. Drosophila has a single homolog of the ERK MAP kinase, encoded by the rolled gene. The di-phosophorylated active form of this 44 kDa protein is recognized by the dp-ERK antibody raised against the phosphorylated activation loop of vertebrate ERK1 and ERK2. No differences in the overall level or phosphorylation state of ERK/Rolled protein were detected by Western blot analysis of testis extracts from aly mutant males compared with wild type. This suggests that aly, and by analogy lin-9, acts downstream of MAP kinase activation, or in a separate pathway, to control target gene transcription (White-Cooper, 2000).
aly is required for cyclin B and twine expression in primary spermatocytes. The level of cyclin B protein is reduced significantly in aly mutant testes compared to wild type. Although low levels are still detected by Western blotting of testis extracts, the three different aly alleles tested all cause a similar marked reduction in cyclin B protein level when compared to wild-type testis extracts. Cyclin B protein appears to be expressed at normal levels in can, mia and sa mutant testes, indicating that these genes might influence cell cycle progression through a biochemically distinct pathway from aly. aly is not required for the expression of all cell cycle genes, since both Cyclin A and Cdc2 proteins are expressed at high levels in aly testes (White-Cooper, 1998).
The wild-type function of aly is required for the transcription or accumulation of both cyclin B and twine mRNAs. In wild-type testes, cyclin B transcripts are detected by in situ hybridization at low levels in the mitotic cells at the apical tip, but are not detected at the position where cells undergo pre-meiotic S-phase. Cyclin B message is abundant throughout the primary spermatocyte stage, accumulating to very high levels in mature primary spermatocytes. Cyclin B mRNA is detected in meiotically dividing cells, but is absent from the post-meiotic stages. In aly mutant testes cyclin B transcript is detected in the mitotic cells at a level comparable to wild type. However cyclin B mRNA is not detectable in aly mutant primary spermatocytes. The expression pattern of twine mRNA in wild-type testes is similar to that of cyclin B, except that twine mRNA is not detected in the mitotic cells at the apical tip of the testis. In aly mutant testes twine mRNA is not detected by in situ hybridization. can, mia and sa mutant testes express both twine and cyclin B mRNA at normal levels (White-Cooper, 1998).
The lack of cyclin B and twine mRNAs in aly mutant spermatocytes is not due to a general defect in transcription, since cyclin A and other messages are abundant in the mutant spermatocytes. In wild-type testes cyclin A mRNA is detected at low levels in both mitotic and S-phase cells at the apical tip of the testis. Cyclin A shows high levels of expression throughout the primary spermatocyte stage, with the message disappearing during meiosis. In aly, can, mia and sa mutant testes cyclin A mRNA is expressed in mitotic and S-phase cells and spermatocytes as in wild type. However cyclin A transcript levels remain high in the arrested mature primary spermatocytes, only disappearing at the base of the testes where the cells finally degenerate, suggesting that the wild-type function of aly, can, mia and sa is required directly or indirectly for the normal shut down of transcription and/or turnover of cyclin A message at meiosis (White-Cooper, 1998).
The timing of entry into the meiotic divisions in wild type may be controlled by post-transcriptionally regulated accumulation of Cyclin B and Twine protein. Although Cyclin B mRNA is expressed at high levels in early spermatocytes, the accumulation of Cyclin B protein is delayed in wild-type testis until the late primary spermatocyte stage. Cyclin B protein begins to accumulate in the cytoplasm of late primary spermatocytes as chromosome condensation is initiated just before the entry into the first meiotic division and is present at high levels in pro-metaphase I cells. Cyclin B protein is degraded at the metaphase to anaphase transition of meiosis I, and reaccumulates in preparation for the second meiotic division. In aly mutant testis Cyclin B protein is detected in the mitotic cells at the apical tip, but does not accumulate in the mutant spermatocytes. Twine protein is likewise delayed until just before the entry into the first meiotic division, days after the transcript is first detected. Neither protein nor mRNA is detected in an aly mutant background (White-Cooper, 1998).
aly, can, mia and sa are required for accumulation of Twine protein but not twine transcript in late primary spermatocytes. These three meiotic arrest genes are required for the expression of fuzzy onions, whose product is required for mitochondrial fusion in early spermatids. Similarly, severe reductions in message level are observed for Male-specific RNA 87F (Mst87F), a gene normally transcribed in primary spermatocytes but not translated until mid- to late-spermatid stages, days after the completion of meiosis. gonadal, which is expressed as two differentially terminated variants in the testis, shows dramatic reduction of both variants in can, mia and sa mutant testis. It is proposed that the can, mia and sa gene products act together or in a pathway to turn on transcription of spermatid differentiation genes, and that aly acts upstream of can, mia and sa to regulate spermatid differentiation. It is also proposed that control of translation or protein stability regulates entry into the first meiotic division. It is suggested that a gene or genes transcribed under the control of can, mia and sa allow(s) accumulation of Twine protein, thus coordinating meiotic division with onset of spermatid differentiation (White-Cooper, 1998).
aly, can, mia and sa are required for the transcription in primary spermatocytes of several genes involved in postmeiotic spermatid differentiation. The fuzzy onions (fzo) gene product is required for mitochondrial fusion in early haploid spermatids. fzo transcription initiates in early primary spermatocytes and the mRNA is present throughout the growing stages in wild type. fzo mRNA is greatly reduced in aly, can, mia and sa testes, despite the presence of primary spermatocytes in the mutant tissue. Message levels in mutant testes ranged from undetectable to low levels under conditions in which the in situ hybridization signal in wild type was strong, indicating that transcription may be reduced to a low basal level, but not entirely turned off. Similarly severe reductions in message level were observed for Mst87F, a gene normally transcribed in primary spermatocytes but not translated until mid- to late-spermatid stages, days after the completion of meiosis. Several other genes also showed dramatic reductions in transcript levels in meiotic arrest mutant testes when assayed by in situ hybridization. Reduced transcript levels in aly, can, mia and sa spermatocytes are not due to a general defect in transcription since a number of genes were transcribed at normal levels in mutant spermatocytes (White-Cooper, 1998).
Comparison of the effects of aly, can, mia and sa mutations on transcript levels suggests that genes normally transcribed in primary spermatocytes can be grouped into three classes. The transcription of the first (general) class of genes is independent of aly, can, mia and sa function. The second (meiotic) class of genes requires the normal function of aly, but not can, mia or sa. Expression of the third (spermiogenic) class of genes requires the wild-type activity of all four of the meiotic arrest genes (White-Cooper, 1998).
Although mutations in aly, can, mia and sa appear to cause arrest at the same point in the G2-M transition of meiosis I (Lin, 1996), the genes apparently control cell cycle progression by different biochemical mechanisms. aly, but not can, mia or sa, is required for the transcription of cyclin B and twine. The wild-type function of can, mia and sa instead appears to be required either to allow translation of twine message or to stabilize twine protein in mature primary spermatocytes. In either case aly, can, mia or sa mutations presumably cause cell cycle arrest at the same point in the G2-M transition, due to lack of active Cdc2/Cyclin B kinase complex. Cdc2 protein resolves into two distinct isoforms in Western blots. The slower migrating form, which is enriched compared to the faster migrating form in twine mutant testes, has been identified as a hyperphosphorylated, inactive form. The slower migrating form of Cdc2 also appears to be enriched compared to the faster migrating form in aly, can and sa. Production of Twine protein, but not Cyclin B, is dependent on can, mia and sa. Thus, although both Cyclin B and Twine protein accumulation are regulated posttranscriptionally in wild-type testes, the genetic control of their expression is different (White-Cooper, 1998).
It is proposed that can, mia, and sa act together or in a pathway to activate a tissue and stage-specific transcription program in primary spermatocytes, and that failure to initiate this program results in a global block in spermatid differentiation due to the lack of an array of gene products. The wild-type functions of can, mia and sa appear to be required for transcription in primary spermatocytes of a set of genes encoding products involved in post-meiotic spermatid differentiation. Transcription of these genes is initiated early in the primary spermatocyte stage, several days before the arrest point of the meiotic arrest mutants. Therefore the lack of transcription of this set of genes is likely to be a cause of the arrest rather than merely a downstream consequence (White-Cooper, 1998).
Of the eight genes identified so far that depend on can, mia and sa for transcription, some information about the function or time of action of the gene products is available for six. The product of the fzo gene is required for mitochondrial fusion, a post-meiotic event. Although fzo is transcribed in primary spermatocytes, the protein is not detected by immunofluorescence staining of testes until late in meiosis II. Expression of Mst87F, of four related genes at 84D and two related genes at 98C is regulated translationally. Although mRNAs are transcribed in primary spermatocytes, the proteins do not accumulate until days after the meiotic divisions. All of these genes encode proteins that are components of a structure in the sperm tail. Similarly the translation of janB and dj mRNAs is delayed until several days after the completion of meiosis. While the function of LanB is unknown, Dj is thought to serve a dual function; it is found in the sperm tail, but sequence comparisons suggest a possible role as a chromatin component (White-Cooper, 1998).
It is proposed that aly acts upstream of can, mia and sa, possibly to control expression or activation of components of the transcription machinery that drives expression of the spermatid differentiation genes. Wild-type function of aly is required for accumulation of at least three different mRNAs in primary spermatocytes that are not dependent on can, mia and sa, suggesting that aly is able to act independently of can, mia and sa. However aly mutations cause the same phenotype, and fail to express the same set of spermatid differentiation genes, as can, mia and sa mutations. This strongly suggests that aly might affect spermatid differentiation through an effect on expression or activity of either can, mia or sa. The block in meiotic cell cycle progression in can, mia and sa mutant testes could be due to a cross-regulatory mechanism that serves to coordinate meiosis and the spermatid differentiation program. It is proposed that a gene or genes transcribed in primary spermatocytes under the control of can, mia and sa encode(s) product(s) required either directly or indirectly to relieve the translational repression of twine message or to stabilise the Twine protein. Such a cross-regulatory mechanism between the pathways leading to spermatid differentiation and meiosis could serve in wild type to ensure that spermatocytes do not enter meiotic division until the proposed transcription program for post-meiotic spermatid differentiation genes has been successfully initiated. A late cross-regulatory mechanism may also explain why mutations that block spermatid differentiation but not meiotic cell cycle progression have not yet been isolated (White-Cooper, 1998).
The signal that activates the G2/M transition in male meiosis could be accumulation of the product of the proposed crossregulatory gene to a threshold sufficient to allow expression of twine protein. Alternatively, timing of the G2/M transition for meiosis I could be set via a less direct mechanism, involving the proposed cross regulatory gene, but not set directly by its level. For example accumulation of Twine protein may require an extrinsic signal received or transduced by a gene or genes controlled by the can, mia and sa transcription program. The degenerative spermatocyte (des) gene, encoding a novel protein that may be membrane associated, is a possible candidate for a component of such a signalling pathway (White-Cooper, 1998).
Mutations in des, like aly, can, mia and sa, cause a block in both meiotic cell cycle progression and the onset of spermatid differentiation. des mutations are also semi-lethal, suggesting a role for this gene outside the testis. Pole cell transplantation experiments also implicate extracellular signals in the regulation of meiotic progression and spermatid differentiation. Male (XY) germ cells transplanted into a female (XX) host initiate spermatogenesis in the host ovary. However the transplanted cells arrest as primary spermatocytes and fail to undergo the meiotic divisions or initiate spermatid differentiation. Part of the program of spermatid differentiation regulated by can, mia and sa could act to destabilize or turn off transcription of certain messages expressed in primary spermatocytes but not needed or deleterious after meiosis. In wild-type testes, cyclin A mRNA is present in primary spermatocytes but not detectable in post-meiotic cells. Loss of cyclin A mRNA could be an important mechanism to prevent DNA replication during meiosis II or in haploid spermatids. In wild-type testes Cyclin A protein is degraded at metaphase I and is not resynthesised for the second meiotic division. In males mutant for aly, can, mia or sa, cyclin A message and Cyclin A protein (Lin, 1996) persist in the arrested primary spermatocytes, suggesting that the wild-type function of the meiotic arrest genes and/or the transcription program they control is required directly or indirectly for disappearance of cyclin A message midway through spermatogenesis. A similar effect on message stability was seen for all of the other pre-meiotic genes tested (White-Cooper, 1998).
Yeast meiosis bears striking similarities to Drosophila spermatogenesis. In both cases S phase is followed by an extended G2 phase, characterized by high levels of transcription of genes required for meiosis and subsequent differentiation into spores or sperm. Many yeast mutants, including certain alleles of cdc2 in S. pombe, are analogous to twine, in that the mutant cells fail to complete one or both of the meiotic divisions, but still differentiate into spores. However meiosis and differentiation are coordinated, since mutations in some genes, mei4 in S. pombe or NDT80 in S. cerevisiae, like the meiotic arrest mutants of Drosophila, block both the meiotic division cycle and subsequent differentiation. The failure to accumulate both cell cycle and spermiogenesis mRNAs in aly mutants suggests that there may be parallels in the genetic control of animal spermatogenesis and yeast sporulation (White-Cooper, 1998 amd references therein).
Hybrid males resulting from crosses between closely related species of Drosophila are sterile. The F1 hybrid sterility phenotype is mainly due to defects occurring during late stages of development that relate to sperm individualization, and so genes controlling sperm development may have been subjected to selective diversification between species. It is also possible that genes of spermatogenesis experience selective constraints given their role in a developmental pathway. The molecular evolution was examined of three genes playing a role during the sperm developmental pathway in Drosophila at an early (bam), a mid (aly), and a late (don juan: dj) stage. The complete coding region of these genes was sequenced in different strains of Drosophila melanogaster and Drosophila simulans. All three genes showed rapid divergence between species, with larger numbers of nonsynonymous to synonymous differences between species than polymorphisms. Although this could be interpreted as evidence for positive selection at all three genes, formal tests of selection do not support such a conclusion. Departures from neutrality were detected only for dj and bam but not aly. The role played by selection is unique and determined by gene-specific characteristics rather than site of expression. In dj, the departure was due to a high proportion of neutral synonymous polymorphisms in D. simulans, and there was evidence of purifying selection maintaining a high lysine amino acid protein content that is characteristic of other DNA-binding proteins. The earliest spermatogenesis gene surveyed, which plays a role in both male and female gametogenesis, was bam, and its significant departure from neutrality was due to an excess of nonsynonymous substitutions between species. Bam is degraded at the end of mitosis, and rapid evolutionary changes among species might be a characteristic shared with other degradable transient proteins. However, the large number of nonsynonymous changes between D. melanogaster and D. simulans and a phylogenetic comparative analysis among species confirms evidence of positive selection driving the evolution of Bam and suggests an yet unknown germ cell line developmental adaptive change between these two species (Civetta, 2006).
Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported in this study aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. A collection of 97 recessive lethal P-lacZ enhancer trap lines were screened for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, six genes (Krüppelhomolog- 1, rpd3, jing, combgap, Aly and S6 kinase) were identified that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration (McClure, 2008).
There are three different mechanisms that organisms use to re-grow and replace lost or damaged body parts, and often, more than one mechanism can function within different tissues of the same organism. Muscle and bone, for example, repair themselves by activating a resident stem cell population, while the liver regenerates by compensatory proliferation of normally quiescent differentiated cells. Appendage/fin regeneration in lower vertebrates occurs by a process termed epimorphic regeneration, which proceeds in three distinct stages: (1) wound healing and migration of the surrounding epithelial cells to form the wound epidermis, (2) formation of the regeneration blastema -- a mass of undifferentiated and proliferating cells of mesenchymal origin and (3) regenerative outgrowth and pattern re-formation. Whether these diverse modes of regeneration share a common molecular and genetic basis is not known (McClure, 2008).
Regeneration in the Drosophila imaginal discs, the primordia of the adult fly appendages, closely parallels epimorphic limb/fin regeneration in lower vertebrates. Cells in the imaginal discs are rigidly determined to form specific adult structures (e.g., legs and wings) by the third larval instar. If the discs are fragmented at this time and cultured in vivo, they will regenerate. Disc regeneration begins 12 h after wounding, when transient heterotypic contacts are made between peripodial (squamous epithelium) and columnar cells (disc proper) near the cut edges of the wound. These initial contacts involve microvilli-like extensions and provide temporary wound closure. Then, approximately 24 h after wounding, homotypic cell contacts (between columnar or between squamous cells) are made involving the close apposition of cell membranes and cellular bridges, which eventually (48 h after wounding) restore the physical continuity of the disc. Before and during wound healing, cell division is randomly distributed throughout the disc. However, once completed (36-48 h after wounding), division is observed only in cells near the wound site. These cells are known as the regeneration blastema. Thus, like appendage regeneration in lower vertebrates, disc regeneration involves wound healing followed by blastema formation (McClure, 2008).
Blastema cells are responsible for the regeneration and repatterning of the entire missing disc fragment. Thus, these cells exhibit remarkable developmental plasticity. For example, in anterior- only leg disc fragments, some blastema cells will switch to posterior identity and establish a novel posterior compartment in the regenerate. This anterior/posterior conversion occurs during heterotypic wound healing, when hedgehog (hh)- expressing peripodial cells induce ectopic engrailed (en) expression in the apposing anterior columnar cells. In addition, the disc blastema, like its vertebrate counterpart, is able to form a normal regenerate (complete leg disc and adult leg) when isolated from the remaining disc fragment. Regenerative plasticity is also observed when a few blastema cells switch fate to that of another disc type (e.g., leg-to-wing), in a phenomenon known as transdetermination. Transdetermination events are closely associated with regenerative disc growth. Clonal analysis, for example, has shown that blastema cells first regenerate the missing disc structures, and only then, are they competent to transdetermine (McClure, 2008).
Little is known about how the regeneration blastema forms in the fragmented leg disc, although ectopic Wingless (Wg/Wnt1) expression is detected along the cut site, both prior to and during blastema formation. Wg is a developmental signal in many different tissues and animals; in flies Wg patterns all of the imaginal discs, functioning as both a morphogen and mitogen to regulate disc cell fate and growth. In lower vertebrates, Wnt ligands are key regulators of blastema formation during epimorphic regeneration. Thus, activation of Wg within the disc blastema is potentially important for regeneration. This idea is consistent with the observation that ubiquitous expression of wg during the second or third larval instars, in unfragmented leg discs, is sufficient to induce a regeneration blastema in the proximodorsal region of the disc, known as the weak point. Moreover, ubiquitous expression of wg mimics the pattern deviations associated with leg disc fragmentation and subsequent regeneration, including the duplication of ventral with concomitant loss of dorsal pattern elements and leg-to-wing transdetermination events. Thus, leg disc regeneration can be examined using two experimental protocols: fragmentation or ubiquitous wg expression. However, it is important to point out that only fragmentation-induced regeneration involves wound healing (McClure, 2008).
Precisely which molecules and signaling pathways are required for the process of regeneration remain poorly understood, partly because the organisms historically used to study regeneration (e.g., newts and salamanders) have been refractory to genetics and molecular manipulations. Recently, however, the use of new genetic techniques together with 'regeneration' model systems -- such as planarians, hydra and zebrafish have given researchers the opportunity to examine the mechanisms of regeneration and to identify the genes, proteins and signaling pathways that regulate different regenerative processes. For example, a large scale RNAi-based screen was performed to survey gene function in planarian tissue homeostasis and regeneration. Out of ~1000 genes examined, RNAi knock-down of 240 displayed regeneration-related phenotypes, including defects in wound healing, blastema formation and blastema cell differentiation. Despite these studies, however, it remains unclear whether regeneration requires only the modulation of genes expressed at the time of injury, the reactivation of earlier developmental genes and/or signaling pathways, or the activation of novel genes specific to the process of regeneration. Thus, a major interest in the field of regenerative biology is the identification of gene products that regulate blastema formation, blastema growth and regenerative cellular plasticity. A genetic screen, using wg-induced leg disc regeneration, aimed at identifying genes that regulate cellular plasticity and regeneration using Drosophila was carried out prothoracic leg discs. A collection of 97 recessive lethal P-element lacZ (PZ) insertion lines were screened for ectopic lacZ expression during wg-induced leg disc regeneration, and six genes were identified that function in wg-induced leg disc regeneration, including genes with functional ties to Wg signaling as well as chromatin remodeling proteins (McClure, 2008).
This study consisted of an enhancer trap screen designed to identify genes with changed gene expression during leg disc regeneration as well as required for regenerative proliferation and growth. The screen identified 19 genes that when heterozygous mutant (PZ/+), dominantly modify wg-induced leg-to-wing transdetermination, which serves as a functional assay for disc regeneration. Of the 19 genes, 37% are transcription factors or involved in transcriptional regulation (tai, Krh1, ken, jing, combgap (cg), rpd3 and Aly), 21% function in cell cycle regulation and growth (oho23B, S6k, polo and cycA), 10.5% play a role in protein secretion (Secβ61 and Syx13), and 31% are of other or unknown function [l(3)01629, CG30947, l(2)00248, l(3)05203, l(3)01344, Nup154]. The identification of transcription factors as the most frequent class of genes that modify wg-induced leg disc regeneration was similarly observed in a DNA microarray screen designed to identify genes enriched in leg disc cells that transdetermine to wing (Klebes, 2005). Together, these findings strongly suggest that transcription factors and their downstream targets play a prominent role in disc cell plasticity (McClure, 2008).
Using lacZ expression analyses, together with whole mount in situ hybridization experiments, the expression patterns of the 19 genes that modified wg-induced leg-to-wing transdetermination were verified. This analysis identified several different expression patterns upon wg-induced regeneration, including a loss of gene expression, ubiquitous expression and genes with expression limited to the regeneration blastema. Such observations indicate that a complex change of gene expression, both negative and positive, mediates the process of epimorphic regeneration. Six (jing, Alyi cg, rpd3, Kr-h1 and S6k) of the 19 modifiers displayed expression limited to the regeneration blastema, indicating that novel markers of regeneration and transdetermination have been identified. The blastema-specific expression patterns of jing, Aly, cg, Kr-h1, rpd3 and S6k raised the intriguing possibility that these genes may be functionally involved in the formation, cell proliferation or maintenance of the blastema during disc regeneration. Indeed, upon ubiquitous wg expression jing/+ animals rarely formed a regeneration blastema, indicating that two wild-type copies of jing are required for the initiation of the regenerative process. In contrast, Aly/+ and cg/+ animals formed a normal blastema, but only after a one-day delay. Therefore, two wild-type copies of the Aly and cg genes are required for the proper timing of regeneration. In addition, it was found that the frequency of blastema formation was reduced in rpd3/+ animals, implicating this gene in the process of regeneration. Interestingly, heterozygous mutations in all four of these genes (jing, Aly, cg and rpd3) strongly suppress wg-induced leg-to-wing transdetermination. It is speculated that the transdetermination frequency declines in these mutant animals because the initiation and/or timing of blastema formation is delayed. This idea is consistent with all previous work which has shown that blastema cells are only competent to transdetermine after they have regenerated the missing disc structures. Heterozygous mutations in Kr-h1 and S6k did not significantly alter the formation of the wg-induced regeneration blastema, however, these genes did affect regeneration-induced transdetermination. Such results suggest that Kr-h1 and S6k specifically function to modulate the cell fate changes that occur as a consequence of regeneration (McClure, 2008).
Investigations into the molecular basis of transdetermination have shown that inputs from the Wg, Decapentapelagic (Dpp) and Hedgehog (Hh) signaling pathways activate key selector genes out of their normal developmental context, such as ectopic Vg activation in the leg disc, which then drives cell-fate switches. Several of the genes identified in this screen have functional ties to Wg, Dpp and Hh signaling pathways. For example, Cg is a zinc-finger transcription factor that is required for proper transcriptional regulation of the Hh signaling effector gene Cubitus interruptus (Ci). In cg mutant wing and leg discs, Ci expression is lowered in the anterior compartment, resulting in the ectopic activation of wg and dpp and significant disc overgrowth. Another gene identified in this screen -- ken, functions in concert with Dpp to direct the development of the Drosophila terminalia. Further characterizations of whether these genes and other modifiers of transdetermination and regeneration affect Wg, Dpp and Hh expression and/or signaling may shed light on the regulation of regeneration and regeneration-induced proliferation and cell fate plasticity (McClure, 2008).
Muscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene. This study used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematodes to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors leads to an externally rough eye morphology. This phenotype has been used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog arrest, exon junction complex components tsunagi and always early, and pro-apoptotic genes Traf1 and reaper. This study further investigated Muscleblind implication in apoptosis and splicing regulation. Missplicing of troponin T was found in muscleblind mutant pupae, and Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing was confirmed in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression. Taken together these genetic approaches identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell (Vicente-Crespo, 2008).
Using Drosophila as a model organism, this study reports the first screen specifically addressed to identify gene functions related to the biomedically important protein Muscleblind. In support of the relevance of the results, the strong functional conservation between fly and vertebrate Muscleblind proteins is shown. Furthermore, data is presented supporting that Muscleblind can induce apoptosis in vivo in imaginal disc tissue, and a conserved motif in the MblC protein isoform was identified that conferred pro-apoptotic activity in Drosophila cell culture when mutated. Noteworthy, this is the first conserved motif (besides CCCH zinc fingers) that is associated with a particular function in Muscleblind proteins (Vicente-Crespo, 2008).
Whereas most vertebrates include three muscleblind paralogues in their genomes, a single muscleblind gene carries out all muscleblind-related functions in Drosophila. These functions are probably accomplished through alternative splicing, which generates four Muscleblind protein isoforms with different carboxy-terminal regions. An evolutionary analysis was performed with isoform-specific protein sequences in order to assess conservation of alternative splicing within protostomes. MblC-like isoforms have been detected even in the nematodes C. elegans and Ascaris suum but not MblA, B or D, that were only consistently found within Drosophilidae. Interestingly, also vertebrate Mbnl1 genes included MblC-like sequences. This finding, together with previous studies that shown that mblC is the isoform with the strongest activity in a muscleblind mutant rescue experiment and α-actinin minigene splicing assay point to mblC as the isoform performing most of muscleblind functions in the fly. Despite this, Muscleblind isoforms are partially redundant. Both mblA and B partially rescue the embryonic lethality of muscleblind mutant embryos and were able to similarly promote foetal exon exclusion in murine TnnT3 minigene splicing assays. MblD showed no activity in splicing assays or in vivo overexpression experiments. However, we show a marginal increase in cell viability in cell death assays. Using isoform-specific RNAi constructs we plan to re-evaluate the function of Muscleblind isoforms both in vivo and in cell culture (Vicente-Crespo, 2008).
Although the regulation of alternative splicing by Muscleblind proteins is an established fact, the cellular processes in which the protein participates are largely unknown. Genetic screens provide a way to approach those processes as they interrogate a biological system as a whole. Overexpression of MblC in the Drosophila eye originated an externally rough eye phenotype that is temperature sensitive, thus indicating sensitization to the muscleblind dose. A deficiency screen was performed, and several candidate mutations were tested for dominant modification of the phenotype. Nineteen were identifed genes of which more that half can be broadly classified as involved in apoptosis regulation (rpr, th and Traf1), RNA metabolism (Aly, tsu, aret and nonA) or transcription regulation (jumu, amos, Dp, CG15435 and CG15433), whereas the rest do not easily fall into defined classes. muscleblind has been shown to regulate α-actinin and troponinT alternative splicing both in vivo and in cell culture. The genetic interaction with the Drosophila homolog of human splicing factor CUG-BP1 (aret) and nonA supports a functional relationship in flies. The antagonism between MBNL1 and CUG-BP1 has actually been shown in humans, whereas RNA-binding protein NonA might be relevant to Muscleblind sequestration by CUG repeat RNA in flies (Vicente-Crespo, 2008 and references therein).
Reduction of dose of exon junction complex (EJC) components tsunagi and Aly also modify MblC overexpression phenotype. EJC provides a binding platform for factors involved in mRNA splicing, export and non-sense mediated decay (NMD). This suggests a previously unforeseen relationship between Muscleblind and EJC, perhaps helping to couple splicing to mRNA export. Consistently, Aly mutations enhanced a CUG repeat RNA phenotype in the Drosophila eye. A similar coupling between transcription and splicing might explain the identification of a number of transcription factors in the screen. Of these, the effect of jumu alleles in the eye and wing MblC overexpression phenotypes were studied in some detail. Loss of function jumu mutations suppress both wing defects and rough eye, whereas they have no effect on unrelated overexpression phenotypes thus suggesting that the interaction is specific (Vicente-Crespo, 2008).
Mutations in the Drosophila homolog of vertebrate Inhibitor of Apoptosis (Diap1 or thread) dominantly enhanced the rough eye phenotype. Consistently with the specificity of the interaction, a second Drosophila paralog, Diap2, did not interact. Also, a deficiency that removes the Drosophila proapoptotic genes hid, reaper and grim (which inhibit thread) was a dominant suppressor while reaper overexpression in eye disc enhanced the phenotype. Interestingly the human homolog of Drosophila Hsp70Ab, Hsp70, has been related to apoptosis as it directly interacts with Apaf-1 and Apoptosis Inducing Factor (AIF) resulting in the inhibition of caspase-dependent and caspase-independent apoptosis. All these genetic data are consistent with MblC overexpressing eye discs being sensitized to enter apoptosis, although no increase in caspase-3 activation was detected in third instar eye imaginal disc overexpressing MblC (Vicente-Crespo, 2008).
Human MBNL1 and CUB-BP1 cooperate to regulate the splicing of cardiac TroponinT (cTNT). The current study detected splicing defects in Drosophila troponinT mRNA in muscleblind mutant pupae. Interestingly, an abnormal exclusion of exon 3 was detected in muscleblind mutant pupae, encoding a glutamic acid-rich domain homologous to the foetal exon of cTNT regulated by human MBNL1. Drosophila exon 3 is only absent in the troponinT isoform expressed in TDT and IFM muscles and probably confers specific functional properties much like the foetal exon does in humans. This identifies troponinT as a new target of Muscleblind activity in flies (Vicente-Crespo, 2008).
CUG-BP1 protein antagonizes MBNL1 exon choice activity in IR and cTNT pre-mRNAs. Moreover, a genetic interaction has been detected between MblC overexpression and aret loss of function mutations. In order to further characterize the functional interaction between Muscleblind and Bruno proteins, their ability to regulate murine TnnT3 was examined in human cell culture. MblA, B and C showed strong activity on TnnT3 mRNA but no significant activity was detected for any Bruno protein. This shows a strong functional conservation between fly and vertebrate Muscleblind proteins as Drosophila isoforms can act over a murine target in a human environment. In contrast, Bruno proteins might not conserve the regulatory activity over troponinT mRNA described for their vertebrate homologues or at least they were not functional in the cellular environment used in this assay. Because GFP-tagged Bruno proteins were only weakly expressed in HEK cells under the experimental conditions used, the level of expression might be insufficient to overcome endogenous Muscleblind activity in cell culture. Furthermore, Bruno proteins might antagonize Muscleblind on a different subset of RNA targets. Although bruno1 has been shown to regulate splicing of some transcripts in S2 cell culture and Bruno3 binds the same EDEN sequence than human CUG-BP, no in vivo experiments have addressed the functional conservation between fly and vertebrate Brunos. Bruno1 is expressed in the germ line where it acts as translational repressor of oskar and gurken mRNAs (Vicente-Crespo, 2008).
Wing imaginal discs stained with anti-caspase-3 and with TUNEL showed that activation of apoptosis was not general in cells expressing MblC but restricted to defined regions within the disc, in particular the wing blade. The spatial constraints that were observed within the imaginal disc might explain the small effect detected when expressing Muscleblind proteins in S2 cells. MblC might require the presence of other factors to be able to unleash programmed cell death. Alternatively, the level of overexpression may be critical and transfected Muscleblind proteins may not reach a critical threshold in Drosophila S2 cells. MblC activation of apoptosis could reveal a direct regulation of apoptotic genes at RNA level or be an indirect effect. Several apoptotic genes produce pro-apoptotic or anti-apoptotic isoforms depending on the regulation of their alternative splicing. MblC could be similarly regulating protein isoforms originating from one or a number of key apoptotic genes at the level of pre-mRNA splicing. Alternatively, MblC could be regulating isoform ratio of a molecule indirectly related to programmed cell death, for example a cell adhesion molecule causing apoptosis by inefficient cell attachment to the substrate. Furthermore, human MBNL proteins are implicated not only in splicing but also in RNA localization, a process that if conserved in flies can potentially impinge in apoptosis regulation (Vicente-Crespo, 2008).
The analysis of MblC-specific sequence revealed a region conserved in Muscleblind proteins from nematodes to humans. Post-translational prediction programs found a motif (FKRP) weakly resembling a sumoylation target site. However, results in S2 cells suggest that sumoylation, if actually taking place, modifies only a small fraction of MblC proteins. FKRP may alternatively participate in an interaction with a Muscleblind partner potentially regulating activity or location in cell compartments, assist in protein dimerization, or others functions. The FKRP site was mutated and a number of functional assays were performed using the mutant MblC. Whereas MblCK202I excluded foetal exon in TnnT3 minigene splicing assays and bound CUG repeat RNA like its wild type counterpart, the mutant protein showed a different preferential distribution in human cells and significantly increased cell death activation upon overexpression. The mechanism by which the FKRP site influences subcellular distribution and cell death-inducing activities is currently unknown, but nevertheless constitutes the first motif, other than zinc fingers, that is associated with a function within Muscleblind proteins (Vicente-Crespo, 2008).
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date revised: 10 February 2013
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