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• W2009605610 abstract "In adult stem cell lineages, progenitor cells commonly undergo mitotic transit amplifying (TA) divisions before terminal differentiation, allowing production of many differentiated progeny per stem cell division. Mechanisms that limit TA divisions and trigger the switch to differentiation may protect against cancer by preventing accumulation of oncogenic mutations in the proliferating population. Here we show that the switch from TA proliferation to differentiation in the Drosophila male germline stem cell lineage is mediated by translational control. The TRIM-NHL tumor suppressor homolog Mei-P26 facilitates accumulation of the differentiation regulator Bam in TA cells. In turn, Bam and its partner Bgcn bind the mei-P26 3′ untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through distinct, sequential regulatory states, from Mei-P26 on/Bam off to Bam on/Mei-P26 off. TRIM-NHL homologs across species facilitate the switch from proliferation to differentiation, suggesting a conserved developmentally programmed tumor suppressor mechanism. In adult stem cell lineages, progenitor cells commonly undergo mitotic transit amplifying (TA) divisions before terminal differentiation, allowing production of many differentiated progeny per stem cell division. Mechanisms that limit TA divisions and trigger the switch to differentiation may protect against cancer by preventing accumulation of oncogenic mutations in the proliferating population. Here we show that the switch from TA proliferation to differentiation in the Drosophila male germline stem cell lineage is mediated by translational control. The TRIM-NHL tumor suppressor homolog Mei-P26 facilitates accumulation of the differentiation regulator Bam in TA cells. In turn, Bam and its partner Bgcn bind the mei-P26 3′ untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through distinct, sequential regulatory states, from Mei-P26 on/Bam off to Bam on/Mei-P26 off. TRIM-NHL homologs across species facilitate the switch from proliferation to differentiation, suggesting a conserved developmentally programmed tumor suppressor mechanism. The TRIM protein Mei-P26 allows accumulation of the key differentiation factor Bam Bam and Bgcn repress mei-P26 translation in late TA cells Bam specifically binds the mei-P26 3′ UTR Translational control mechanisms may underlie tumor suppression in stem cell lineages Adult stem cells act throughout life to replenish differentiated cells lost to turnover or injury. In many adult stem cell lineages, stem cell daughters destined for differentiation first undergo a limited number of mitotic divisions to amplify cell number prior to terminal differentiation. This transit amplifying (TA) division strategy may protect large long-lived animals from tumorigenesis by minimizing the number of stem cell divisions required for tissue homeostasis and preventing accumulation of oncogenic mutations in progenitor cells due to programmed differentiation. The mechanisms that limit the number of TA divisions and initiate terminal differentiation thus may provide tumor suppressor function, and defects may contribute to progression toward cancer in adult stem cell lineages (Clarke and Fuller, 2006Clarke M.F. Fuller M. Stem cells and cancer: two faces of eve.Cell. 2006; 124: 1111-1115Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar; Jamieson et al., 2004Jamieson C.H. Ailles L.E. Dylla S.J. Muijtjens M. Jones C. Zehnder J.L. Gotlib J. Li K. Manz M.G. Keating A. et al.Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML.N. Engl. J. Med. 2004; 351: 657-667Crossref PubMed Scopus (1263) Google Scholar; Krivtsov et al., 2006Krivtsov A.V. Twomey D. Feng Z. Stubbs M.C. Wang Y. Faber J. Levine J.E. Wang J. Hahn W.C. Gilliland D.G. et al.Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9.Nature. 2006; 442: 818-822Crossref PubMed Scopus (1163) Google Scholar). Here we investigate the mechanisms that force TA cells to stop proliferating and initiate terminal differentiation in the Drosophila male germline adult stem cell lineage. Drosophila male germline stem cells (GSCs) reside in a niche at the tip of the testis, attached to somatic hub cells and flanked by somatic cyst stem cells (CySCs) (Figure 1A) (Fuller and Spradling, 2007Fuller M.T. Spradling A.C. Male and female Drosophila germline stem cells: two versions of immortality.Science. 2007; 316: 402-404Crossref PubMed Scopus (332) Google Scholar). When a GSC divides, one daughter remains in the niche and self-renews, while the other is displaced away and initiates differentiation. The resulting gonialblast, which is enveloped by a pair of CySCs, proceeds through four synchronous TA divisions with incomplete cytokinesis, producing a clone of 16 interconnected germ cells. These 16 mitotic sisters normally stop proliferating, undergo premeiotic DNA synthesis in synchrony, and switch to the spermatocyte program of cell growth, meiosis, and terminal differentiation (Figure 1A) (Fuller, 1993Fuller M.T. Spermatogenesis.in: Martinez-Arias M.B.A. The Development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, New York1993: 71-147Google Scholar). Because TA sister cells are contained within a common somatic cell envelope, are joined by cytoplasmic bridges, and divide in synchrony, mutations that cause overproliferation of TA cells can be easily identified. The bag of marbles (bam) gene is required cell autonomously for TA spermatogonia to stop proliferating and enter the spermatocyte differentiation program. Male germ cells mutant for bam undergo several extra rounds of mitotic TA division, fail to differentiate, and eventually die (Gönczy et al., 1997Gönczy P. Matunis E. DiNardo S. bag-of-marbles and benign gonial cell neoplasm act in the germline to restrict proliferation during Drosophila spermatogenesis.Development. 1997; 124: 4361-4371PubMed Google Scholar; McKearin and Spradling, 1990McKearin D.M. Spradling A.C. bag-of-marbles: a Drosophila gene required to initiate both male and female gametogenesis.Genes Dev. 1990; 4: 2242-2251Crossref PubMed Scopus (271) Google Scholar). The number of TA divisions appears to be set by the time required for Bam protein to accumulate to a critical threshold. Bam protein is normally first detected in 4-cell cysts, increases to a peak in 8-cell cysts, and is degraded in early 16-cell cysts immediately after premeiotic DNA replication. Lowering the bam dosage slowed Bam protein accumulation and delayed the transition to differentiation, whereas early accumulation of Bam protein caused a premature switch to differentiation (Insco et al., 2009Insco M.L. Leon A. Tam C.H. McKearin D.M. Fuller M.T. Accumulation of a differentiation regulator specifies transit amplifying division number in an adult stem cell lineage.Proc. Natl. Acad. Sci. USA. 2009; 106: 22311-22316Crossref PubMed Scopus (78) Google Scholar). Bam, a protein with no recognizable domains, acts with a partner, benign gonial cell neoplasm (Bgcn), discovered in a genetic screen for Drosophila tumor suppressors (Gateff, 1994Gateff E. Tumor suppressor and overgrowth suppressor genes of Drosophila melanogaster: developmental aspects.Int. J. Dev. Biol. 1994; 38: 565-590PubMed Google Scholar). bam and bgcn have similar mutant phenotypes (Gönczy et al., 1997Gönczy P. Matunis E. DiNardo S. bag-of-marbles and benign gonial cell neoplasm act in the germline to restrict proliferation during Drosophila spermatogenesis.Development. 1997; 124: 4361-4371PubMed Google Scholar), and Bam protein directly interacts with Bgcn in Drosophila ovaries or when coexpressed in cultured cells or yeast (Li et al., 2009Li Y. Minor N.T. Park J.K. McKearin D.M. Maines J.Z. Bam and Bgcn antagonize Nanos-dependent germ-line stem cell maintenance.Proc. Natl. Acad. Sci. USA. 2009; 106: 9304-9309Crossref PubMed Scopus (111) Google Scholar; Shen et al., 2009Shen R. Weng C. Yu J. Xie T. eIF4A controls germline stem cell self-renewal by directly inhibiting BAM function in the Drosophila ovary.Proc. Natl. Acad. Sci. USA. 2009; 106: 11623-11628Crossref PubMed Scopus (75) Google Scholar). Bgcn is related to the DExH-box family of RNA-dependent helicases, indicating that Bgcn, and with it Bam, may regulate RNA. Consistent with a role in translational repression, Bam protein binds the translation initiation factor eIF4A. Furthermore, expression of Bam and Bgcn in Drosophila cultured cells resulted in a 4-fold reduction in expression of a luciferase reporter coupled to the 3′ untranslated region (UTR) of e-cadherin messenger RNA (mRNA), and tethering Bam to the 3′ UTR induced translational repression of the attached reporter (Shen et al., 2009Shen R. Weng C. Yu J. Xie T. eIF4A controls germline stem cell self-renewal by directly inhibiting BAM function in the Drosophila ovary.Proc. Natl. Acad. Sci. USA. 2009; 106: 11623-11628Crossref PubMed Scopus (75) Google Scholar). In female germ cells, Bam and Bgcn allow the onset of differentiation through translational repression of nanos (nos) via the nos 3′ UTR (Li et al., 2009Li Y. Minor N.T. Park J.K. McKearin D.M. Maines J.Z. Bam and Bgcn antagonize Nanos-dependent germ-line stem cell maintenance.Proc. Natl. Acad. Sci. USA. 2009; 106: 9304-9309Crossref PubMed Scopus (111) Google Scholar). However, direct interaction of Bam or Bgcn protein with e-cadherin or nos mRNAs has not been demonstrated. Here we identify the microRNA (miRNA) regulator and TRIM-NHL (tripartite motif and Ncl-1, HT2a, and Lin-41 domain) family member Mei-P26 (Neumüller et al., 2008Neumüller R.A. Betschinger J. Fischer A. Bushati N. Poernbacher I. Mechtler K. Cohen S.M. Knoblich J.A. Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage.Nature. 2008; 454: 241-245Crossref PubMed Scopus (187) Google Scholar; Page et al., 2000Page S.L. McKim K.S. Deneen B. Van Hook T.L. Hawley R.S. Genetic studies of mei-P26 reveal a link between the processes that control germ cell proliferation in both sexes and those that control meiotic exchange in Drosophila.Genetics. 2000; 155: 1757-1772PubMed Google Scholar) both as a regulator of Bam protein accumulation and, subsequently, as a direct target of translational repression by Bam and Bgcn in male germ cells. Mei-P26 function facilitates both the switch from mitosis to meiosis and spermatocyte differentiation. In mei-P26 mutant males, Bam protein failed to accumulate to its normal peak levels. The overproliferation of TA cells in mei-P26 mutant testes was suppressed by expression of additional Bam, suggesting that the continued TA cell proliferation in mei-P26 mutant males is due to the failure of Bam protein to reach the threshold required for the switch to the spermatocyte state. In turn, Bam specifically binds the mei-P26 3′ UTR, and Bam and Bgcn function are required for translational repression of mei-P26 via its 3′ UTR in vivo. Mutating two potential let-7 target sites within the mei-P26 3′ UTR derepressed reporter expression in vivo and disrupted Bam binding in vitro. Our data suggest that a stepwise progression in regulatory states from [Mei-P26 on/Bam off] to [Mei-P26 on/Bam on] to [Bam on/Mei-P26 off], choreographed by translational regulation, accompanies the switch from TA cell proliferation to terminal differentiation in the Drosophila male GSC lineage. Loss of mei-P26 function in males led to overproliferation of spermatogonial TA cysts. Whereas the apical third of wild-type testes contains many large spermatocytes easily visible by phase-contrast microscopy (Figure 1B, arrows), testes from flies hemizygous for the null allele mei-P26mfs1 (Page et al., 2000Page S.L. McKim K.S. Deneen B. Van Hook T.L. Hawley R.S. Genetic studies of mei-P26 reveal a link between the processes that control germ cell proliferation in both sexes and those that control meiotic exchange in Drosophila.Genetics. 2000; 155: 1757-1772PubMed Google Scholar) showed cysts full of many small cells (Figure 1C), resembling testes from bam mutant males (Gönczy et al., 1997Gönczy P. Matunis E. DiNardo S. bag-of-marbles and benign gonial cell neoplasm act in the germline to restrict proliferation during Drosophila spermatogenesis.Development. 1997; 124: 4361-4371PubMed Google Scholar; McKearin and Spradling, 1990McKearin D.M. Spradling A.C. bag-of-marbles: a Drosophila gene required to initiate both male and female gametogenesis.Genes Dev. 1990; 4: 2242-2251Crossref PubMed Scopus (271) Google Scholar). The clusters of small cells were Vasa positive, indicating germ cell identity, and had small nuclei (Figure 1E), similar to early germ cells at the apical tip of wild-type testes (Figure 1D). A short pulse of the nucleotide analog 5-ethynyl-2′-deoxyuridine (EdU) showed that early germ cells in mei-P26 mutant testes underwent several extra rounds of mitotic division and behaved as TA cells rather than as stem cells, with all cells within a cyst going through S phase synchronously (Figure 1G, arrow). Whereas wild-type testes briefly pulsed with EdU showed cysts in S phase with 2, 4, 8, or 16 cells, but none with >16 cells (Figure 1F, arrow) (n = 7 testes: 14 S phase cysts with 8 or 16 cells, and zero with >16 cells), mei-P26 mutant testes had many cysts with more than 16 cells undergoing S phase (Figure 1G, arrow) (n = 9 testes: 21 S phase cysts with 8 or 16 cells, and 52 with >16 cells). Overproliferating spermatogonial cysts in mei-P26 mutants eventually died, as indicated by the refractile appearance in phase-contrast images (Figure 1C, arrowheads) and by TUNEL staining (Figure S1B available online), similar to the eventual death of overproliferating germ cell cysts in bam mutant testes (Insco et al., 2009Insco M.L. Leon A. Tam C.H. McKearin D.M. Fuller M.T. Accumulation of a differentiation regulator specifies transit amplifying division number in an adult stem cell lineage.Proc. Natl. Acad. Sci. USA. 2009; 106: 22311-22316Crossref PubMed Scopus (78) Google Scholar; McKearin and Spradling, 1990McKearin D.M. Spradling A.C. bag-of-marbles: a Drosophila gene required to initiate both male and female gametogenesis.Genes Dev. 1990; 4: 2242-2251Crossref PubMed Scopus (271) Google Scholar). Early germ cells in mei-P26 mutant testes had enlarged nucleoli compared to their wild-type counterparts (Figures 1H–1J). Similar increases in nucleolar size were observed in mei-P26 mutant female germ cells (Neumüller et al., 2008Neumüller R.A. Betschinger J. Fischer A. Bushati N. Poernbacher I. Mechtler K. Cohen S.M. Knoblich J.A. Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage.Nature. 2008; 454: 241-245Crossref PubMed Scopus (187) Google Scholar) and in C. elegans cells mutant for the worm mei-P26 homolog ncl-1 (Hedgecock and Herman, 1995Hedgecock E.M. Herman R.K. The ncl-1 gene and genetic mosaics of Caenorhabditis elegans.Genetics. 1995; 141: 989-1006PubMed Google Scholar). The enlarged nucleoli observed in mei-P26 mutant male germ cells (Figures 1I and 1J) may correlate with accumulation of abnormally high levels of dMyc (Grewal et al., 2005Grewal S.S. Li L. Orian A. Eisenman R.N. Edgar B.A. Myc-dependent regulation of ribosomal RNA synthesis during Drosophila development.Nat. Cell Biol. 2005; 7: 295-302Crossref PubMed Scopus (304) Google Scholar). In wild-type testes, dMyc levels were relatively low in GSCs and early TA cells and then increased in 8- and early 16-cell cysts (Figures S1C and S1E). However, dMyc protein levels were uniformly high in GSCs and early TA cells in mei-P26 mutant testes (Figures S1D and S1E). Mei-P26 function was not absolutely required for male germ cells to become spermatocytes, but rather appeared to make the switch to differentiation accurate and timely. Some germ cell cysts in mei-P26 mutant testes did switch to the spermatocyte state, exhibiting the characteristic growth in cell size and expression of the spermatocyte marker Sa (Figure 2B versus 2A). In wild-type testes, 95% of spermatocyte cysts located in the apical third of the testis contained 16 spermatocytes per cyst (Figure 2C, black bars, and 2D). However, in a comparable region of mei-P26 mutant testes, 68% of cysts had many small germ cells, consistent with overproliferating TA cells (20% >64 small cells [Figure 2E]; 48% refractile dying cysts), and 32% of cysts progressed to the spermatocyte state (Figure 2C, gray bars). Of the 32% of cysts that did progress to the spermatocyte state, only a third (10% of total cysts) had the correct 16 spermatocytes per cyst. Another third (9% of total cysts) had >16 spermatocytes per cyst, indicating that too many TA divisions occurred prior to the switch. Mei-P26 function was also required for proper differentiation of spermatocytes. Many cysts (13% of total cysts) had differentiating cells with grossly abnormal morphology, including elongating spermatids with 16 large nuclei instead of 64 small nuclei, consistent with progression to spermatid differentiation without meiotic division (Figure 2F). The mei-P26 mutant overproliferation and differentiation defects were rescued by a chromosomal duplication or a genomic transgene containing mei-P26 (Experimental Procedures). The inability of many mei-P26 mutant germ cell cysts to properly exit the TA divisions appears to be due to the failure to accumulate Bam protein to levels required for triggering the switch from proliferation to differentiation. The level of Bam protein detected by immunofluorescence staining was lower in 4-, 8-, and 16-cell TA cysts from mei-P26 mutant testes compared to wild-type testes stained on the same slides (Figures 3A–3C). In addition, Bam protein expression perdured at low levels in late overproliferating cysts in the mei-P26 mutant (Figures 3B and 3C). Strikingly, increasing the gene dosage of Bam by introducing a genomic transgene rescued the TA cell overproliferation defects observed in mei-P26 mutant testes, indicating that overproliferation of TA cells in mei-P26 mutant males is due to the failure to accumulate normal levels of Bam protein. Whereas testes from mei-P26mfs1 sibling controls showed overproliferation of early germ cell cysts (Figures 3D and 3G, light gray bars), introducing a single copy of a genomic transgene expressing either wild-type Bam (alpha-Bam, Figures 3E and 3G, dark gray bars) or Bam protein lacking the C-terminal PEST degradation sequence (Figures 3F and 3G, black bars) into the mei-P26 mutant background eliminated TA cell overproliferation. Testes from mei-P26mfs1;alpha-Bam/+ males had no cysts full of small cells or refractile cysts in the corresponding region (Figure 3E). Rather, 72% of the cysts in the spermatocyte region had 16 spermatocytes per cyst, and <1% had 8 spermatocytes per cyst (Figure 3G, dark gray bars). Likewise, in mei-P26mfs1;BamΔPEST/+ testes, none of the cysts in the spermatocyte region had overproliferating small cells (Figure 3F), 43% had 16 spermatocytes per cyst, and 37% had 8 spermatocytes per cyst (Figure 3G, black bars), similar to BamΔPEST alone (Insco et al., 2009Insco M.L. Leon A. Tam C.H. McKearin D.M. Fuller M.T. Accumulation of a differentiation regulator specifies transit amplifying division number in an adult stem cell lineage.Proc. Natl. Acad. Sci. USA. 2009; 106: 22311-22316Crossref PubMed Scopus (78) Google Scholar). Similar suppression of the mei-P26 mutant overproliferation phenotype was observed after forced expression of Bam under the control of the heat shock promoter (Figure S2A). In addition to rescuing TA cell overproliferation, introducing an extra copy of bam also normalized the enlarged nucleoli observed in mei-P26 mutant testes. Whereas mei-P26 mutant germ cells in 16-cell cysts had enlarged nucleoli, both mei-P26mfs1;alpha-Bam/+ and mei-P26mfs1;BamΔPEST/+ germ cells had smaller nucleoli that were similar in size to wild-type nucleoli (Figure S2B). Introducing an extra copy of bam did not, however, rescue the meiotic and spermatid differentiation defects characteristic of mei-P26 mutant males. In mei-P26mfs1;BamΔPEST/+ testes, 30% of cysts from the spermatocyte region had differentiation defects, including abnormal mitochondrial derivatives and cysts of elongating spermatids with 16 large nuclei, indicating spermatid differentiation without meiosis (Figure 3I). Likewise, 27% of cysts from mei-P26mfs1;alpha-Bam/+ males showed spermatocyte or spermatid differentiation defects (Figure 3G, dark gray bars), indicating a late requirement for Mei-P26 function independent of Bam. Taken together, the results suggest that Mei-P26 first functions in early TA germ cells to facilitate timely accumulation of Bam protein. Mei-P26 also acts later, after the switch to spermatocyte state, facilitating the normal progression of meiotic divisions and spermatid differentiation. Mei-P26 protein levels detected by immunofluorescence changed dynamically in TA cells, starting high in early TA cells, consistent with a role in Bam accumulation, and dropping to background levels as Bam protein expression peaked (Figures 4A–4A″ and 4 C). Mei-P26 protein levels rose again in spermatocytes, consistent with the later requirement of Mei-P26 for meiosis and differentiation. Analysis of Mei-P26 and Bam protein in testes pulsed briefly with EdU revealed that Mei-P26 expression was at high levels in GSCs and early TA cells, gradually decreased in level in 4- through 8-cell cysts, and was below the level of detection by our immunofluorescence methods in 16-cell cysts in S phase. Mei-P26 reappeared in early spermatocytes after Bam disappeared (Figures 4B–4C). Mei-P26 protein appeared to concentrate in puncta in the cytoplasm, especially in early germ cells (Figure 4D, arrows). Many of the Mei-P26 puncta in early germ cells colocalized with the miRNA-induced silencing complex (RISC) component GW182 (Figures 4E–4G). Drosophila GW182 interacts with argonaute-1 (Ago-1) (Behm-Ansmant et al., 2006Behm-Ansmant I. Rehwinkel J. Doerks T. Stark A. Bork P. Izaurralde E. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.Genes Dev. 2006; 20: 1885-1898Crossref PubMed Scopus (733) Google Scholar), and both GW182 and Ago-1 have recently been shown to coimmunoprecipitate with Mei-P26 in the Drosophila ovary (Li et al., 2012Li Y. Maines J.Z. Tastan O.Y. McKearin D.M. Buszczak M. Mei-P26 regulates the maintenance of ovarian germline stem cells by promoting BMP signaling.Development. 2012; 139: 1547-1556Crossref PubMed Scopus (50) Google Scholar). The drop in Mei-P26 protein expression in late TA cells was dependent on the function of Bam and Bgcn. In wild-type testes, Mei-P26 protein levels decreased in 8-cell cysts (Figure 4H, white outlines), and Mei-P26 was not detected in later 8-cell (Figure 4H′, arrow) or early 16-cell cysts (Figure 4B′, white outline). However, in bam or bgcn mutant testes, Mei-P26 protein levels remained high throughout the region of overproliferating early germ cells, including in 8-cell (Figures 4I, upper outline, and 4J, left outline) and 16-cell cysts in S phase (Figures 4I, lower outline, and 4J, right outline). Downregulation of Mei-P26 expression in late TA and early 16-cell cysts may be important for correct timing of the switch from spermatogonial proliferation to differentiation in the male germline. Early forced re-expression of Mei-P26 protein in late TA cells using the bamGal4 driver and a UAS-mei-P26 construct with a SV40 termination signal in place of the mei-P26 3′ UTR was sufficient for driving 23% of spermatocyte cysts to differentiate early, with fewer than 16 cells (Figure S3C). In contrast, <1% of spermatocyte cysts in wild-type testes had fewer than 16 cells per cyst. The decrease in Mei-P26 protein expression in late TA cells and early 16-cell cysts raised the possibility that mei-P26 might be translationally repressed. Although Mei-P26 protein levels dropped, in situ hybridization showed no break in mei-P26 mRNA expression in late TA or early 16-cell cysts (Figure S4A). Consistent with translational control, the 3′ UTR of mei-P26 was sufficient to reduce expression of an enhanced yellow fluorescent protein (eYFP) reporter in TA cells until Bam was downregulated in vivo (Figures 5B–5B″ , white outline). Reporters were transcribed specifically in TA cells with the use of bamGal4 for driving upstream activating sequence (UAS) constructs. A control reporter expressing eYFP with the SV40 termination sequence revealed eYFP expression in 79% of Bam-positive cysts (Figures 5A–5A″, 5C, and 5D, reporter 1). The control reporter was routinely first detected in cysts after Bam expression was initiated, presumably due to the time delay caused by the bipartite Gal4/UAS system. Consistent with this, the 20% of Bam-positive cysts that lacked eYFP with the control reporter were commonly the most apical (youngest) cysts (Figures 5A–5A″). To generate a reporter containing mei-P26 sequences, the mei-P26 3′ UTR expressed in wild-type testes was determined by 3′ rapid amplification of complementary DNA ends and then inserted between eYFP and the SV40 termination sequence. The mei-P26 3′ UTR expressed in the testes was shorter than the form expressed in embryos (549 versus 640 nt), terminating before the site bound and regulated by Vasa in female germ cells (Liu et al., 2009Liu N. Han H. Lasko P. Vasa promotes Drosophila germline stem cell differentiation by activating mei-P26 translation by directly interacting with a (U)-rich motif in its 3′ UTR.Genes Dev. 2009; 23: 2742-2752Crossref PubMed Scopus (85) Google Scholar) (Figure S5A). When transcription of the eYFP reporter containing the full-length mei-P26 3′ UTR was driven by bamGal4, the reporter was expressed in only 28% of Bam-positive cysts (Figures 5B–5D, reporter 2), in contrast to the 79% observed with the control reporter (Figures 5A–5A″, 5C, and 5D, reporter 1). Analysis of smaller regions of the mei-P26 3′ UTR in reporter constructs in vivo mapped the sequence sufficient for translational repression to 247 nucleotides (94–341) of the mei-P26 3′ UTR (Figures 5C and 5D, reporters 3, 5, and 6). Further deletion from the 3′ end of the 3′ UTR appeared to abolish translational repression in vivo, as reporter constructs carrying nucleotides 94 to 292 or 94 to 245 showed detectable eYFP expression in 60% of Bam-positive cysts (Figures 5C and 5D, reporters 10 and 11). Nucleotide substitutions in a predicted Pumilio binding site (482–489 nt) failed to disrupt repression in TA cells (Figures 5C and 5D, reporter 4). Known homologs of mei-P26, such as C.elegans lin-41 and Drosophila dappled, are translationally repressed via their 3′ UTR by the miRNA let-7 (O’Farrell et al., 2008O’Farrell F. Esfahani S.S. Engström Y. Kylsten P. Regulation of the Drosophila lin-41 homologue dappled by let-7 reveals conservation of a regulatory mechanism within the LIN-41 subclade.Dev. Dyn. 2008; 237: 196-208Crossref PubMed Scopus (33) Google Scholar; Reinhart et al., 2000Reinhart B.J. Slack F.J. Basson M. Pasquinelli A.E. Bettinger J.C. Rougvie A.E. Horvitz H.R. Ruvkun G. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans.Nature. 2000; 403: 901-906Crossref PubMed Scopus (3764) Google Scholar; Slack et al., 2000Slack F.J. Basson M. Liu Z. Ambros V. Horvitz H.R. Ruvkun G. The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor.Mol. Cell. 2000; 5: 659-669Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar). Given this and recent reports of atypical miRNA target sites found through biochemical methods (Chi et al., 2012Chi S.W. Hannon G.J. Darnell R.B. An alternative mode of microRNA target recognition.Nat. Struct. Mol. Biol. 2012; 19: 321-327Crossref PubMed Scopus (274) Google Scholar; Grimson et al., 2007Grimson A. Farh K.K. Johnston W.K. Garrett-Engele P. Lim L.P. Bartel D.P. MicroRNA targeting specificity in mammals: determinants beyond seed pairing.Mol. Cell. 2007; 27: 91-105Abstract Full Text Full Text PDF PubMed Scopus (3033) Google Scholar), we searched by eye for possible let-7 seed sequences and found two sites within the region of the mei-P26 3′ UTR sufficient for translational repression (185–190 nt and 285–292 nt). RNAhybrid verified that the putative target sites were the areas most likely to exhibit let-7 binding in the entire mei-P26 3′ UTR (Figures 5E, S5C, and S5D) (Rehmsmeier et al., 2004Rehmsmeier M. Steffen P. Hochsmann M. Giegerich R. Fast and effective prediction of microRNA/target duplexes.RNA. 2004; 10: 1507-1517Crossref PubMed Scopus (1840) Google Scholar). The 5′ end of one of the predicted let-7 target sites contributed to a PhastCons conserved element (Figure S5B, asterisk 2). Strikingly, introducing nucleotide substitutions (from CTCC to AGAA) in either seed sequence into the reporter carrying the mei-P26 3′ UTR disrupted repression, restoring eYFP expression to 79% of the Bam-positive cysts (Figures 5C and 5D, reporters 7–9). Consistent with the possibility that let-7 might contribute to translational repression of endogenous Mei-P26 in early male germ cells in vivo, varying the level of let-7 expression led to changes in Mei-P26 protein expression. In let-7-C homozygous mutant testes, the level of Mei-P26 protein detected by immunofluorescence was higher in GSCs and in 2-, 4-, 8-, and 16-cell cysts compared to wild-type testes (Figures 5F–5H). Forced expression of let-7-C in early germ cells using nanosGal4 to drive UAS-let-7-C led to decreased levels of Mei-P26 protein expression at all cell stages compared to wild-type testes (Figures S5E–S5G). The action of Bam and Bgcn was required for repression of the mei-P26 3′ UTR reporter in TA cells in vivo. We compared the onset of" @default.
• W2009605610 created "2016-06-24" @default.
• W2009605610 creator A5001407100 @default.
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• W2009605610 date "2012-11-01" @default.
• W2009605610 modified "2023-10-16" @default.
• W2009605610 title "A Self-Limiting Switch Based on Translational Control Regulates the Transition from Proliferation to Differentiation in an Adult Stem Cell Lineage" @default.
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