Exonucleolytic processing of small nucleolar RNAs from pre-mRNA introns.

Abstract

Many small nucleolar RNAs (snoRNAs) in vertebrates are encoded within introns of protein genes. We have reported previously that two isoforms of human U17 snoRNA are encoded in introns of the cell-cycle regulatory gene, RCC1. We have now investigated the mechanism of processing of U17 RNAs and of another intron-encoded snoRNA, U19. Experiments in which the processing of intronic RNA substrates was tested in HeLa cell extracts suggest that exonucleases rather than endonucleases are involved in the excision of U17 and U19 RNAs: (1) Cutoff products that would be expected from endonucleolytic cleavages were not detected; (2) capping or circularization of substrates inhibited formation of snoRNAs; and (3) U17 RNA was faithfully processed from a substrate carrying unrelated flanking sequences. To study in vivo processing, the coding regions of snoRNAs were inserted into intron 2 of the human 13-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 accurate processing. When the U17 sequence was placed in a 13-globin exon, no formation of U17 RNA took place, and when two U17 RNA-coding regions were placed in a single intron, doublet U17 RNA molecules accumulated. The results support a model according to which 5' --* 3' and 3' ~ 5' exonucleases are involved in maturation of U17 and U19 RNAs and that excised and debranched introns are the substrates of the processing reaction.

Position within the host intron is critical for efficient processing of box C/D snoRNAs in mammalian cells

“…The synthesis of U75 snoRNA in vitro depends on the splicing of its host intron, the integrity of both boxes C and D, and the length of the downstream spacer. Several intronic snoRNAs in vertebrates have been reported to be processed exclusively via a splicing-dependent pathway (18), and our in vitro system supports these observations. In contrast, in vitro processing of U15, which is located at an exceptional position within its host intron (155 nt upstream of the 3Ј splice site), did not require splicing (16); it possesses a stable internal stem structure, perhaps folding it into the correct snoRNA structure without synergy from splicing.…”

“…4B) or of two other 2Ј-O-methyl oligonucleotides, which inhibit splicing at distinct steps (data not shown). These data indicate that the in vitro synthesis of U75 depends on the splicing of the host intron, consistent with in vivo observations (18).…”

“…Most previous in vitro studies analyzing the processing of mammalian snoRNAs have focused on the exonucleolytic trimming of linear intron substrates (16)(17)(18). To address the detailed mechanism of snoRNA production from the host intron, an in vitro system where both splicing and snoRNA processing occurs has been developed from HeLa nuclear extracts.…”

“…A common feature of mammalian snoRNA host gene transcripts is a 5Ј terminal oligopyrimidine sequence, whose precise function with respect to snoRNA synthesis is unknown (14,15). In most cases, it appears that snoRNAs are released from excised, debranched introns by exonucleolytic trimming (16)(17)(18); a minor pathway involves endonucleolytic cleavage of flanking intron sequences (19,20). Many yeast snoRNAs are transcribed as monocistronic or polycistronic precursors from independent transcription units, and proteins involved in their processing have been characterized (21)(22)(23).…”

Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability

“…Comparison of different box CϩD snoRNAs revealed some variation in the residual levels on depletion of Nop58p+ For U3, a longer species accumulated, clearly showing an effect on synthesis of the snoRNA+ The 59 end of mature U3 corresponds to the tri-methyl guanosine cap of the primary transcript, and the extended form is therefore very likely to be 39 extended+ In vertebrates and yeast, spliceosomal snRNAs are processed from precursors with 39 extensions (Madore et al+, 1984a(Madore et al+, , 1984bYuo et al+, 1985;Chanfreau et al+, 1997;Abou Elela & Ares, 1998), and U3 may be similarly processed+ Longer forms of U24 were also accumulated, together with low levels of shorter forms+ The 59 end of U24 was unaffected by depletion of Nop58p whereas RNase protection experiments detected species 39 extended by 5-6 nt, showing these changes to be due to alterations in 39 processing+ Yeast U24 is encoded in the intron of the BEL1 gene (Qu et al+, 1995) and the snoRNA is obligatorily synthesized from the intron following debranching of the intron lariat; very low levels of mature U24 are synthesized in a dbr1-⌬ strain that lacks debranching activity (Ooi et al+, 1998;Petfalski et al+, 1998)+ This strongly indicates that both ends of U24 are synthesized by exonucleolytic activities and, indeed, the 59 r 39 exonucleases Rat1p and Xrn1p were identified as the activities responsible for the 59 processing (Petfalski et al+, 1998)+ In vertebrates, the 59 and 39 ends of all tested intronencoded snoRNAs are synthesized by exonucleases (Cecconi et al+, 1995;Kiss & Filipowicz, 1995;Caffarelli et al+, 1996;Xia et al+, 1997)+…”