Direct Interactions between Subunits of CPSF and the U2 snRNP Contribute to the Coupling of Pre-mRNA 3′ End Processing and Splicing

Abstract

Eukaryotic pre-mRNAs are capped at their 5' ends, polyadenylated at their 3' ends, and spliced before being exported from the nucleus to the cytoplasm. Although the three processing reactions can be studied separately in vitro, they are coupled in vivo. We identified subunits of the U2 snRNP in highly purified CPSF and showed that the two complexes physically interact. We therefore tested whether this interaction contributes to the coupling of 3' end processing and splicing. We found that CPSF is necessary for efficient splicing activity in coupled assays and that mutations in the pre-mRNA binding site of the U2 snRNP resulted in impaired splicing and in much reduced cleavage efficiency. Moreover, we showed that efficient cleavage required the presence of the U2 snRNA in coupled assays. We therefore propose that the interaction between CPSF and the U2 snRNP contributes to the coupling of splicing and 3' end formation.

Ending the message: poly(A) signals then and now

“…In particular, the 39SS-associated factor U2AF was shown to enhance PAS function by direct molecular contacts with poly(A) polymerase (Vagner et al 2000). Similarly, protein components of U2snRNP that associate with the 39SS and nearby lariat branch point help enhance downstream 39-end processing through interactions with CPSF (Kyburz et al 2006). This enhancement of terminal exon definition has several other consequences, as PAS recognition also enhances terminal intron splicing, and, furthermore, 39SS recognition is required for Pol II termination, as this in turn depends on PAS recognition.…”

Control of alternative pre-mRNA splicing by Ca++ signals

“…In another report, polyadenylation specificity factor 30 (CPSF30) of Arabidopsis thaliana was found to bind calmodulin [192], which inhibits the RNA binding activity of CPSF30. In HeLa nuclear extracts, CPSF couples transcription with splicing [193]. Therefore, it will be interesting to know whether varying Ca ++ concentrations and CaM can control any specific alternative splicing event through direct interaction with the factors that regulate splicing.…”

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

“…Terminal exon definition has been shown to be aided by the polyadenylation machinery 14,15 through interactions between U2AF and the polyadenylation polymerase (PAP) 16 or cleavage factor 1m (CF1m), 17 or through interactions between splicing factor 3b (SF3b), a component of U2 snRNP, and the cleavage and polyadenylation specificity factor (CPSF). 18 Furthermore, the U1 snRNP component U1A has been shown to stimulate polyadenylation through interaction with CPSF160, 19 however, U1 snRNP also plays a role in preventing premature cleavage and polyadenylation through binding of U1 snRNA to cryptic poly(A) sites 20 as well as through direct interactions between U1-70K and the PAP. 21 More recent findings have demonstrated that a terminal splice acceptor site 22 and poly(A) tail may be necessary for terminal intron removal with splicing promoted through the coordinated efforts of PAP and nuclear poly(A) binding protein, PABPN1.…”