REF2 ENCODES AN RNA-BINDING PROTEIN DIRECTLY INVOLVED IN YEAST MESSENGER-RNA 3'-END FORMATION

The Saccharomyces cerevisiae mutant ref2-1 (REF = RNA end formation) w as originally identified by a genetic strategy predicted to detect dec reases in the use of a CYC1 poly(A) site interposed within the intron of an ACT1-HIS4 fusion reporter gene, Direct RNA analysis now proves t his effect and also demonstrates the trans action of the REF2 gene pro duct on cryptic poly(A) sites located within the coding region of a pl asmid-borne ACT1-lacZ gene. Despite impaired growth of ref2 strains, p ossibly because of a general defect in the efficiency of mRNA 3'-end p rocessing, the steady-state characteristics of a variety of normal cel lular mRNAs remain unaffected, Sequencing of the complementing gene pr edicts the Reap product to be a novel, basic protein of 429 amino acid s (M(r), 48,000) with a high-level lysine/serine content and some unus ual features, Analysis in vitro, with a number of defined RNA substrat es, confirms that efficient use of weak poly(A) sites requires Reap: e ndonucleolytic cleavage is carried out accurately but at significantly lower rates in extracts prepared from Delta ref2 cells, The addition of purified, epitope-tagged Reap (Ref2pF) reestablishes wild-type leve ls of activity in these extracts, demonstrating direct involvement of this protein in the cleavage step of 3' mRNA processing, Together with the RNA-binding characteristics of Ref2pF in vitro, our results suppo rt an important contributing role for the REF2 locus in 3'-end process ing, As the first gene genetically identified to participate in mRNA 3 ' end maturation prior to the final polyadenylation step, REF2 provide s an ideal starting point for identifying related genes in this event.