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Differential expression of Rfx1-4 during mouse spermatogenesis.

ABSTRACT: The regulatory factor X (RFX) family of transcription factors has been recently implicated in gene regulation during spermatogenesis. However, the relative expression of individual members during this developmental process is not completely characterized, particularly in the case of Rfx4, which has multiple transcript variants in the testis. We used reverse transcriptase-dependent real-time PCR, 5'-RACE cloning, and Western blotting to compare transcripts and protein levels for this family in cell populations from the three major phases of spermatogenesis (mitotic, meiotic, and haploid). Transcripts for Rfx1-4 were present at trace to low levels in spermatogonia prepared from 8-day-old mice. Transcripts for both Rfx2 and Rfx4 were elevated in mid-late pachytene spermatocytes; however, the dominant Rfx4 transcript present begins at a downstream exon and lacks the DNA binding domain. Transcripts for all four genes were elevated in early haploid cells (round spermatids). In these cells Rfx4 transcripts originate primarily from a newly described promoter with intron 1 but are expected to be translationally compromised due to a poorly situated start codon. Western blotting confirmed that RFX2 is greatly elevated beginning in meiosis and also confirmed that full-length RFX4 protein is not prevalent in mouse testis at any stage. These results imply that RFX2 is the most likely X box binding factor to influence novel gene expression during meiosis, that RFX1-3 may all play roles in haploid cells but that RFX4 is much less prevalent than implied by its high transcript levels.

SUBMITTER: Kistler WS

PROVIDER: S-EPMC2761754 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Differential expression of Rfx1-4 during mouse spermatogenesis.

Kistler W Stephen WS Horvath Gary C GC Dasgupta Anindya A Kistler Malathi K MK

Gene expression patterns : GEP 20090719 7

The regulatory factor X (RFX) family of transcription factors has been recently implicated in gene regulation during spermatogenesis. However, the relative expression of individual members during this developmental process is not completely characterized, particularly in the case of Rfx4, which has multiple transcript variants in the testis. We used reverse transcriptase-dependent real-time PCR, 5'-RACE cloning, and Western blotting to compare transcripts and protein levels for this family in ce ...[more]

PMID: 19596083

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Project description: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.

Dataset Information

Differential expression of Rfx1-4 during mouse spermatogenesis.

Publications

Differential expression of Rfx1-4 during mouse spermatogenesis.

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