Separation of Factors Required for Cleavage and Polyadenylation of Yeast Pre-mRNA

Abstract

Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.

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

“…Efficient cleavage of mutant cyc1‐512, which lacks these elements, supports this conclusion. Although GAL7 3′‐end‐forming signals belong to a different class of motifs with UA repeats as essential sequences (Abe et al ., 1990; Chen and Moore, 1992; Guo and Sherman, 1995), GAL7‐1 wild‐type transcript could also be cleaved at the normal cleavage site in the absence of Nab4p/Hrp1p. However, similarly to CYC1, additional sites were also used.…”

“…Capped and labeled RNAs were synthesized from restriction enzyme‐linearized templates by transcription with bacteriophage RNA polymerases (Stratagene, Boehringer) and purified on 6% polyacrylamide–8.3 M urea gels. CYC1 and CYC1 pre‐cleaved RNAs were transcribed from the pG4‐CYC1 and pG4‐CYC1pre plasmids, respectively (Minvielle‐Sebastia et al ., 1994; Preker et al ., 1995); GAL7 fulllength wild‐type and mutant RNAs, and GAL7 pre‐cleaved RNAs were obtained from the pJCGAL7‐1, pJCGAL7‐3 and pJCGAL7‐9 plasmids, respectively (Chen and Moore, 1992; Zhelkovsky et al ., 1995); the Ty‐derived RNAs were transcribed from the U3RU5 plasmid (Hou et al ., 1994).…”

“…It was remarkable that the abundance of CP III correlated with the purity of the CF II fractions used (Figure 2A, lanes 2-7). To test whether cleavage at additional sites occurs only with CYC1, we also performed in vitro reactions with the 295 nucleotide GAL7-1 transcript, derived from the 3Ј-UTR of the GAL7 gene (Chen and Moore, 1992). Similarly to CYC1, the normal upstream cleavage product (CP I) was produced together with two additional smaller fragments (CP V and VI; Figure 2B, lane 2).…”

Yeast cleavage factor Hrp1 is a novel guard protein that surveils pre-mRNA 3’ processing

“…The CPF subcomplex has initially been separated into the cleavage factor II (CF II) and polyadenylation factor I (PF I) subcomplexes according to the contributions of the components to the in vitro cleavage and polyadenylation reactions (Chen and Moore, 1992). Interestingly, together with the CF I complex, the CF II complex has been shown to be sufficient to proceed with cleavage of GAL7 and CYC1 precursors.…”