EXONUCLEOLYTIC PROCESSING OF SMALL NUCLEOLAR RNAS FROM PRE-MESSENGER-RNA INTRONS

Many small nucleolar RNAs (snoRNAs) in vertebrates are encoded within introns of protein genes. We have reported previously that two isoform s of human U17 snoRNA are encoded in introns of the cell-cycle regulat ory gene, RCC1. We have now investigated the mechanism of processing o f U17 RNAs and of another intron-encoded snoRNA, U19. Experiments in w hich the processing of intronic RNA substrates was tested in HeLa cell extracts suggest that exonucleases rather than endonucleases are invo lved in the excision of U17 and U19 RNAs: (1) Cutoff products that wou ld be expected from endonucleolytic cleavages were not detected; (2) c apping or circularization of substrates inhibited formation of snoRNAs ; and (3) U17 RNA was faithfully processed from a substrate carrying u nrelated flanking sequences. To study in vivo processing, the coding r egions of snoRNAs were inserted into intron 2 of the human beta-globin gene. Expression of resulting pre-mRNAs in simian COS cells resulted in formation of correctly processed snoRNAs and of the spliced globin mRNA, demonstrating that snoRNAs can be excised from a nonhost intron and that their sequences contain all the signals essential for accurat e processing. When the U17 sequence was placed in a beta-globin exon, no formation of U17 RNA took place, and when two U17 RNA-coding region s were placed in a single intron, doublet U17 RNA molecules accumulate d. The results support a model according to which 5' --> 3' and 3' --> 5' exonucleases are involved in maturation of U17 and U19 RNAs and th at excised and debranched introns are the substrates of the processing reaction.