Biomedical subjects

Brinda Dass

Publications and source records attributed to Brinda Dass.

4 recordsLinked to original sources

PubMed2007

Polyadenylation proteins CstF-64 and tauCstF-64 exhibit differential binding affinities for RNA polymers.

CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, tauCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between tauCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of tauCstF-64 and CstF-64 have different affinities for RNA elements. We quantified K(d) values of CstF-64 and tauCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)18, poly(A)18 or poly(C)18, with affinity for poly(C)18 being the lowest. However, CstF-64 had a higher affinity for poly(U)18 than tauCstF-64, whereas it had a lower affinity for poly(GU)9. Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)18, but changes in amino acids downstream of the C-terminal alpha-helical region decreased affinity towards poly(U)18. Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is mportant in RNA sequence recognition. This supports the hypothesis that tauCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements.

Amino Acid Sequence↗

PubMed2006

Systematic variation in mRNA 3'-processing signals during mouse spermatogenesis.

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3'-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3'-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3'-processing characteristics in the testicular samples, compared to control sets of widely used 3'-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3'-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3'-untranslated regions (3'-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3'-UTR truncation and no significant difference in 3'-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3'-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis.

3' Untranslated Regions↗

PubMed2005

The mRNA encoding tauCstF-64 is expressed ubiquitously in mouse tissues.

Polyadenylation is a process of endonucleolytic cleavage of the mRNA, followed by addition of up to 250 adenosine residues to the 3' end of the mRNA. Polyadenylation is essential for eukaryotic mRNA expression, and CstF-64 is a subunit of the CstF polyadenylation factor that is required for accurate polyadenylation. We discovered that there are two forms of the CstF-64 protein in mammalian male germ cells, one of which (CstF-64) is expressed in all tissues, the other of which (tauCstF-64) is expressed only in male germ cells and in brain (albeit at significantly lower levels in the brain). Therefore, we were surprised to find that, using reverse transcription-PCR, cDNA cloning, and RNA blot analyses, tauCstF-64 mRNA was expressed at higher levels in brain than in testis. Also, tauCstF-64 mRNA was expressed at lower but detectable levels in all tissues tested, including epididymis, heart, kidney, liver, lung, muscle, ovary, spleen, thymus, and uterus. These results suggest the hypothesis that tauCstF-64 mRNA is regulated at the translational or post-translational level.

3' Untranslated Regions↗

PubMed2002

The gene CSTF2T, encoding the human variant CstF-64 polyadenylation protein tauCstF-64, lacks introns and may be associated with male sterility.

Messenger RNA polyadenylation in male germ cells does not seem to require the AAUAAA polyadenylation signal required in all other cell types. To account for this difference, we found a variant form of the polyadenylation protein, the 64,000 Mr protein of the cleavage stimulation factor (CstF-64), in mouse meiotic and postmeiotic germ cells. This protein is a candidate to alter polyadenylation in those cells. More recently, we reported the cloning from mouse pachytene spermatocytes of mouse tauCstF-64 (gene symbol Cstf2t), which is a homolog of CstF-64 fitting the criteria we expected for the variant CstF-64 protein. Here we report the cloning and mapping of the human ortholog of mouse tauCstF-64. The human tauCstF-64 cDNA (gene symbol CSTF2T) is 2324 bp in length and encodes a protein of 616 amino acids (64,442.90 Da). Although most highly related to mouse tauCstF-64 (89.8% identity), human tauCstF-64 is also related to the human and mouse somatic CstF-64 (74.9% and 73.4% identity, respectively). Alignment of human tauCstF-64 with human genome sequence from chromosome 10 shows that CSTF2T lacks introns. Radiation hybrid mapping places the human tauCstF-64 gene at 10q22-q23, which is the site of a translocation that has been associated with human neurological problems and male infertility.

Amino Acid Sequence↗