Functional analysis of Cleavage factor I (CFIm) during spermatogenesis
Messenger RNA (mRNA) processing at the 3'-end, including alternative polyadenylation, is a prevalent mechanism of regulating RNA metabolism in male germ cells. Studying processes involved in alternative polyadenylation during spermatogenesis could provide valuable insight into the molecular mechanisms of male infertility, potentially leading to more effective diagnostic tools to identify low quality sperm. Cleavage factor I (CFIm) is a core polyadenylation factor and directs alternative polyadenylation in somatic cells but the function of CFIm has not been investigated in male germ cells. To study the function of CFIm during spermatogenesis, the NUDT21 subunit of CFIm was suppressed in the male germ cell line GC-4spc as well as in somatic control line 3T3-L1 using short-interfering RNA (siRNA) and the subsequent changes in isoform expression of candidate genes Nr6a1, Cpsf6, and Timp-2, were determined. The Nr6a1 and Cpsf6 genes were chosen due to their male germ cell-specific expression while Timp-2 was chosen as a somatic cell control transcript that also undergoes alternative polyadenylation. The GC-4spc cells mimic male germ cell gene expression as the short 3' untranslated region (3'UTR) isoforms of Nudt21, Cpsf6, and Nr6a1 were amplified in GC-4spc compared to the long 3'UTR isoforms of these transcripts, consistent with normal male germ cell gene expression. As expected, transcript levels of NUDT21 were reduced after siRNA transfection (NUDT21 knock-down; 71-74% reduction in gene expression) compared to non-targeting (NT) siRNA controls. In general, reduction of Nudt21 levels in GC-4spc showed significant increases in the short 3'UTR isoform for the male germ cells genes Nr6a1 and Cpsf6 but no changes in long 3'UTR expression. In NUDT21 knock-down GC-4spc cells, the short Nr6a1 3'UTR isoform increased (Nudt21-siRNA = 0.033 ± 0.009 normalized mean copy number (NMCN) vs. NT control = 0.01 ± 0.003 NMCN; P ≤ 0.04) while the longer 3'UTR isoforms remained unchanged, demonstrating that NUDT21 has a role in the efficiency of polyadenylation site selection for the short Nr6a1 3'UTR isoforms in male germ cells. The two cell lines differed in efficiency of processing of the short and long 3'UTRs in siRNA experiments. The short 3'UTR isoform of Cpsf6 (Nudt21-siRNA = 2.2 ± 0.34 NMCN vs. NT control = 0.85 ± 0.12 NMCN; P ≤ 0.005), and Timp-2 (Nudt21-siRNA = 179.5 NMCN ± 13.5 vs. NT control = 109.8 ± 26.9 NMCN; P ≤ 0.04) increased in GC-4spc; the Nr6a1 and Cpsf6 short 3'UTR isoforms were not significantly changed in 3T3-L1, suggesting male germ cell-specific regulation of polyadenylation for specific transcripts. Preliminary data demonstrated decreased NR6A1 protein levels in NUDT21 knock-down cells, confirming a change in translational efficiency with changed isoform length. Our data indicate a role of Nudt21 in efficiency of polyadenylation site usage co-transcriptionally and not the result of a site selection (for example non-canonical vs. canonical). Further investigation of the impact of reduced CFIm on alternative polyadenylation of germ cell-specific transcripts will lead to an improved understanding of the role of mRNA metabolism in male infertility.
Molecular biology|Animal sciences
"Functional analysis of Cleavage factor I (CFIm) during spermatogenesis"
Dissertations and Master's Theses (Campus Access).