X‐ray structure and activity of the yeast Pop2 protein: a nuclease subunit of the mRNA deadenylase complex

Abstract

In Saccharomyces cerevisiae, a large complex, known as the Ccr4-Not complex, containing two nucleases, is responsible for mRNA deadenylation. One of these nucleases is called Pop2 and has been identified by similarity with PARN, a human poly(A) nuclease. Here, we present the crystal structure of the nuclease domain of Pop2 at 2.3 Å resolution. The domain has the fold of the DnaQ family and represents the first structure of an RNase from the DEDD superfamily. Despite the presence of two noncanonical residues in the active site, the domain displays RNase activity on a broad range of RNA substrates. Site-directed mutagenesis of active-site residues demonstrates the intrinsic ability of the Pop2 RNase D domain to digest RNA. This first structure of a nuclease involved in the 3 0 -5 0 deadenylation of mRNA in yeast provides information for the understanding of the mechanism by which the Ccr4-Not complex achieves its functions.

Mouse CAF1 Can Function As a Processive Deadenylase/3′–5′-Exonuclease in Vitro but in Yeast the Deadenylase Function of CAF1 Is Not Required for mRNA Poly(A) Removal

“…The Deadenylase Function of yCAF1 and mCAF1 Are Not Required in Yeast-Because both yCAF1 (8,19) and mCAF1 have been shown to display deadenylase functions in vitro, we determined subsequently whether the activities of yCAF1 and mCAF1 were required in vivo. LexA-yCAF1 proteins were created containing either the S199A/E201A alteration that inactivated yCAF1 deadenylase function in vitro (19), which is comparable with the D39A/E41A mutation in mCAF1 that blocked its deadenylase activity (Fig.…”

“…LexA-yCAF1 proteins were created containing either the S199A/E201A alteration that inactivated yCAF1 deadenylase function in vitro (19), which is comparable with the D39A/E41A mutation in mCAF1 that blocked its deadenylase activity (Fig. 1B), or the Y320A/D321A mutation that is comparable with the Y159A/D160A of mCAF1 that inhibited its activity.…”

“…CAF1, which binds CCR4 and links it to the remainder of the CCR4-NOT complex (11,18), is a member of the DEDDh family of RNases (16). In vitro studies have demonstrated that yeast CAF1 can display deadenylase activity (8,19), but the role of this activity in vivo is unclear. Removal of CAF1 does not impair CCR4 activity in vitro (5,7), whereas mutations in the CCR4 putative catalytic residues abolish CCR4-NOT deadenylase function in vivo (6,7).…”

The Arabidopsis homologs of CCR4-associated factor 1 show mRNA deadenylation activity and play a role in plant defence responses

“…A volume of 10 µl of the reaction mixture was incubated at 25 °C for the indicated time. Reactions were stopped by the addition of formamide/EDTA buffer and then loaded onto 7 M urea/10% acrylamide (19:1) gels [19].…”

“…CAF1 proteins belong to the DEDDh subgroup of the DEDD family of nucleases, which requires three aspartates (D), a glutamate (E), and a nearby histidine for activity (DEDDh) [17,18]. Although the yeast CAF1 shows deadenylase activity in vitro [17,19], the role of this activity in vivo is unclear. Inactivation of the predicted key catalytic active sites of yeast CAF1 did not affect in vivo deadenylation function [20].…”