FIG. 9.

Lack of endoribonuclease activity in extracts of yeast expressing 2.1vhs. Internally labeled pCITE-1 and SRPα RNAs were added to control RRL (RRL), RRL containing the pretranslated vhs (RRLvhs), and extracts of yeast containing (2.1vhs) and lacking (empty vector) 2.1vhs protein. RNA was extracted at the indicated time points, resolved on a 1% agarose–1.8% formaldehyde gel, and transferred to a GeneScreen Plus membrane, and the RNA signal was detected by autoradiography (panels C and D). (A and B) Diagrams of the pCITE-1 and SRPα RNA substrates, indicating the positions of the initial vhs-induced cleavage events in the RRL system. The EMCV IRES present on pCITE-1 is indicated. (C and D) Analysis of endoribonuclease activity on pCITE-1 and SRPα RNAs, respectively. The solid square and diamond indicate the previously described 5′ and 3′ degradation products of pCITE-1, respectively, while the open square and diamond indicate the additional 1,500- and 1,000-nt products, respectively, described in the text. The sizes of RNA markers (M) are indicated in nucleotides at the left.