Control of cleavage site selection during mRNA 3' end formation by a yeast hnRNP

Abstract

Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3Ј end formation. It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3Ј end cleavage in yeast. CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5Ј splice site selection. Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3Ј end endonucleolytic cleavage. We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites. Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection.

Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I

“…In contrast to the results of Minvielle-Sebastia et al (19), but in agreement with Kessler et al (18), we find that CF IB is absolutely required not only for proper selection of the cleavage site, but also for reconstitution of even minimal cleavage activity upon the GAL7 3Ј untranslated region. We confirmed that CF IB was required for reconstitution of polyadenylation activity upon this substrate, in agreement with published reports (18,19).…”

“…3, lane 20). Although Minvielle-Sebastia et al (19) observed cleavage in the absence of Hrp1 within 3 min of starting the reaction, we observed no cleavage without Hrp1, even if the reaction was carried out to 60 min (data not shown).…”

“…A fifth, the major yeast poly(A) binding protein Pab1, also copurified with the CF I activity and was shown to participate in the control of poly(A) tail length (16)(17)(18). In contrast to Kessler et al, Hrp1 was found to be required for polyadenylation, but only to have a stimulatory effect on the accuracy and efficiency of CF I-mediated cleavage (19).…”

3′-End processing of pre-mRNA in eukaryotes

“…More recently, however, it has been demonstrated that Hrp1p/Nab4p is not required for cleavage per se, but for the selection of the correct cleavage site: in the absence of the protein, several cleavage sites upstream of the correct one were used [114]. Although an e¡ect on polyadenylation was found in reconstitution experiments in vitro, no polyadenylation defect was seen in vivo [114]. Genetic experiments showed direct interactions of Hrp1/Nab4 with subunits of CF IA.…”

“…UV cross-linking of puri¢ed recombinant Hrp1p to RNA was dependent on the presence of the e¤ciency element (see Section 3.1). Hrp1p was thus suggested to be responsible for recognizing this sequence [113] (but see [114]). …”