Knock-down of 25 kDa subunit of cleavage factor Im in Hela cells alters alternative polyadenylation within 3′-UTRs

Abstract

Alternative polyadenylation leads to mRNAs with variable 3′ ends. Since a 3′-untranslated region (3′-UTR) often contains cis elements that impact stability or localization of mRNA or translation, selection of poly(A) sites in a 3′-UTR is regulated in mammalian cells. However, the molecular basis for alternative poly(A) site selection within a 3′-UTR has been unclear. Here we show involvement of cleavage factor Im (CFIm) in poly(A) site selection within a 3′-UTR. CFIm is a heterodimeric 3′ end-processing complex, which functions to assemble other processing factors on pre-mRNA in vitro. We knocked down 25 kDa subunit of CFIm (CFIm25) in HeLa cells and analyzed alternative poly(A) site selection of TIMP-2, syndecan2, ERCC6 and DHFR genes by northern blotting. We observed changes in the distribution of mRNAs in CFIm25 depleted cells, suggesting a role for CFIm in alternative poly(A) site selection. Furthermore, tissue specific analysis demonstrated that the CFIm25 gene gave rise to 1.1, 2.0 and 4.6 kb mRNAs. The 4.6 kb mRNA was ubiquitously expressed, while the 1.1 and 2.0 kb mRNAs were expressed in a tissue specific manner. We found three likely poly(A) sites in the CFIm25 3′-UTR, suggesting alternative polyadenylation. Our results indicate that alternative poly(A) site selection is a well-regulated process in vivo.

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns

“…TIMP-2, an inhibitor of matrix metalloproteinases, contains 2 functional poly(A) sites, producing mRNAs of 1.2 kb and 3.8 kb. In agreement with previous results, 34 PCR-mediated 3 0 end amplifications demonstrate that CF1m25 KD significantly induced proximal poly(A) site selection (Fig. 2C).…”

“…To test the hypothesis that altering polyadenylation site selection induces AS changes, we induced APA through RNAi mediated knockdown of CF1m25 in HeLa cells, which was shown to induce proximal poly(A) site selection in TIMP-2, SDC2, ERCC6 and DHFR. 34 At efficient CF1m25 KD conditions (60-80%) ( Fig. 2A), reduced CF1m25 levels resulted in alternative TIMP-2 poly(A) site selection as demonstrated by 3 0 RACE analysis (Fig.…”

“…Changing the length of the 3 0 UTR is believed to affect the stability, localization, transport, and even translation of the mRNA through altered interactions with microRNAs or other regulatory RNA binding proteins. [28][29][30][31][32] Several polyadenylation factors have been implicated in APA including CF1m 33,34 and CstF. 35,36 For example, knockdown of CF1m25 (CF1m25 KD) or CstF64 (CstF64 KD) has been shown to result in the activation of alternative poly(A) sites.…”

“…35,36 For example, knockdown of CF1m25 (CF1m25 KD) or CstF64 (CstF64 KD) has been shown to result in the activation of alternative poly(A) sites. 34,36 Thus, APA events are mediated through factors that are also engaged in interactions with spliceosomal components.…”

Trypanosomatid EST: a neglected information resource regarding flagellated protozoa?

“…Variant mRNAs can be found in trypanosomes due to different transcription initiation sites that generate distinct 5' UTRs lengths (alternative transsplicing) (Nepomuceno-Silva et al 2001). Conversely, alternative polyadenylation is also a mechanism for the generation of transcriptional diversity (different 3' UTR lengths) from the same gene (Kubo et al 2006). Ten years ago, the first T. cruzi ESTs were publicly available (Brandão et al 1997).…”