| Summary: | Deleted in AZoospermia Associated Protein 1 (DAZAP1) is a ubiquitous hnRNP protein that has been implicated in RNA transcription, splicing, and translation. It is highly expressed in testes, predominantly in late stage spermatocytes and post-meiotic spermatids. Dazap1 deficiency in mice results in growth retardation and spermatogenic arrest. The gene produces two major transcripts of 2.4 and 1.8 kb, designated Dazap1-L and Dazap1-S, respectively. Results of our previous RNA in situ hybridization and immunostaining suggested translational regulation of the Dazap1 transcripts during spermatogenesis. The main objectives of the study were to determine the origin of the two Dazap1 transcripts and to investigate whether they were similarly translated. Our Northern and 3' RACE analyses showed that the two transcripts were generated through alternative polyadenylation. In mouse testes, the levels of both transcripts were low at postnatal day 12 (P12), increased significantly at P18, and reached maximum at P27. Sucrose gradient analyses showed that at P12 both transcripts were actively translated. Afterward, an increasing portion of Dazap1-S became associated with the translationally inactive mRNPs, and the translational repression was accompanied by an increase in the length of its poly(A) tail. A much smaller portion of Dazap1-L was also sequestered to mRNPs as testes matured, but there was no changes in its poly(A) tail length. Using RNA pull-down followed by mass spectrometry, we identified DAZL, a germ-cell specific translation regulator, as one of the proteins that bound to the 3'UTR region specific for Dazap1-L. We further showed that DAZL preferentially bound to Dazap1-L in testis lysates and stimulated the translation of a reporter gene carrying Dazap1-L 3'UTR. In summary, our study shows that the translation of the two Dazap1 transcripts is differentially regulated. It also provides a new example of translational repression associated with poly(A) tail elongation during spermatogenesis.
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