The role of the Brr5/Ysh1 C-terminal domain and its homolog Syc1 in mRNA 3′-end processing in Saccharomyces cerevisiae

Abstract

The cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae is thought to provide the catalytic activities of the mRNA 3¢-end processing machinery, which include endonucleolytic cleavage at the poly(A) site, followed by synthesis of an adenosine polymer onto the new 3¢-end by the CPF subunit Pap1. Because of similarity to other nucleases in the metallo-blactamase family, the Brr5/Ysh1 subunit has been proposed to be the endonuclease. The C-terminal domain of Brr5 lies outside of b-lactamase homology, and its function has not been elucidated. We show here that this region of Brr5 is necessary for cell viability and mRNA 3¢-end processing. It is highly homologous to another CPF subunit, Syc1. Syc1 is not essential, but its removal improves the growth of other processing mutants at restrictive temperatures and restores in vitro processing activity to cleavage/ polyadenylation-defective brr5-1 extract. Our findings suggest that Syc1, by mimicking the essential Brr5 C-terminus, serves as a negative regulator of mRNA 3¢-end formation.

Protein factors in pre-mRNA 3′-end processing

“…The C-terminal residues of Brr5p/Ysh1p have strong sequence homology to another member of the CPF in the yeast 3′-end processing complex, Syc1p (38% identity) [111]. This will be discussed in more detail in the section on Syc1p later.…”

“…While the C-terminal region of CPSF-73 is not as conserved as the N-terminal region, removal of the C-terminus of Brr5p/Ysh1p, by as little as the last 30 residues, results in cell death [111]. On the other hand, removal of the last 10 or 19 residues do not affect cell viability.…”

“…Disruption of these putative metal binding residues results in 3′-end processing defects and cell death [109,111]. Dialysis of the nuclear extract leads to partial loss of cleavage activity, which can be rescued by the addition of ZnCl 2 .…”

“…Sequence analysis suggests that a leucine zipper, which is usually involved in protein-protein interactions, may be present in the final 30 residues of Brr5p/Ysh1p. The C-terminus of CPSF-73 does interact with other proteins in the 3′-end processing complex, including CPSF-100 [117] and Pta1p [111]. Brr5p/Ysh1p has also been shown to interact strongly with Clp1p, but the binding region is unknown [118].…”

“…Pta1p purifies with CPF and can be further purified into CF II. It interacts directly with the C-terminus of Brr5p/Ysh1p [111], Cft2p/Ydh1p [118], Syc1p, Glc7p, Pti1p, Ssu72p and Swd2p [81,202,203]. Symplekin was identified through its interaction with the hinge region of CstF-64 [141], and forms a stable complex with CPSF and CstF.…”