Small nuclear RNAs from Saccharomyces cerevisiae: unexpected diversity in abundance, size, and molecular complexity.

Abstract

Previous work showed that the simple eukaryote Saccharomyces cerevisiae contains a group of RNAs with the general structural properties predicted for small nuclear RNAs (snRNAs), including possession of the characteristic trimethylguanosine 5'-terminal cap. It was also demonstrated that, unlike their metazoan counterparts, the yeast snRNAs are present in low abundance (200-500 molecules per haploid cell). We have now used antibody directed against the 5' cap to investigate the total set size of snRNAs in this organism. We present evidence that the number of distinct yeast snRNAs is on the order of several dozen, that the length of the capped RNAs can exceed 1000 nucleotides, and that the relative abundance of a subset of these RNAs is 1/5th to 1/20th that ofthe class ofsnRNAs described previously. These findings suggest that the six highly abundant species of snRNAs (Ul-U6) typically reported in metazoans may represent a serious underestimation of the total diversity of snRNAs in eukaryotes.Studies of small nuclear RNAs (snRNAs) have focused on the six so-called U-RNAs (U1-U6), first discovered some 15 years ago (for review, see ref. 1). In metazoans, these RNAs range in length from 90 to 216 nucleotides (nt), are extremely abundant (there are =106 U1 molecules in a HeLa cell, for example) and are encoded by multigene families (2, 3). Although functional studies have lagged behind structural characterization, these RNAs are widely believed to mediate a spectrum of RNA-processing reactions in eukaryotic cells. Indeed, recent information from in vitro analyses directly implicates the involvement of U1 (4-6), U2 (7,8), and probably U5 (9) in pre-mRNA splicing. Similarly, one or more U-RNAs appear to be required for 3' end formation/ polyadenylylation in vitro (10, 11), although the specific suggestion that these are U4/U6 (12) has not been verified. Finally, the involvement of U3 in rRNA maturation is deemed likely because U3 is restricted to the nucleolus and appears to be hydrogen-bonded to 14).Several years ago, we initiated a search for snRNAs in the simple eukaryote Saccharomyces cerevisiae, with the goal of exploiting the powerful genetic tools available in this organism. As described (15, 16), a group of RNAs were identified that conformed to several predicted criteria. These included small size, nuclear localization, metabolic stability, and possession of the characteristic trimethylguanosine 5' cap. The latter characteristic provides a particularly powerful diagnostic in that snRNAs are the only class of RNAs known to possess this terminus. However, it was also shown that the yeast snRNAs differ in several potentially important ways from U1-U6. A significant asset to our genetic strategy was the finding (15,17,18) that the six yeast RNAs tested (designated snR3, snR4, snR7, snR8, snR9, and snR10) are encoded by single-copy genes (designated SNR3, etc.). In keeping with this lack ofgenetic redundancy, the yeast RNAs are present in low abundance: 200-500 molecules per haploid cell (15).A further surp...

Sgt1p and Skp1p Modulate the Assembly and Turnover of CBF3 Complexes Required for Proper Kinetochore Function

“…Bound proteins were eluted as indicated in text. The tandem affinity purification (TAP)-Ctf13p fusion was purified from 10 mg of yeast extract as described previously (Riedel et al, 1986;Rigaut et al, 1999). Equivalent amounts of starting extract, flow-through, and 5 l of each fraction were resolved by SDS-PAGE and visualized by immunoblot or standard silver staining techniques (Morrissey, 1981).…”

Low abundance of telomerase in yeast: Implications for telomerase haploinsufficiency

“…So our ACT1 estimate is nearly threefold higher than the highest published estimate. We estimated 480 U2 snRNAs per haploid cell, which falls within the 200-500 moleculeper-cell range determined by Northern analysis (Riedel et al 1986). …”

A handful of intron-containing genes produces the lion's share of yeast mRNA

“…One possibility is that there are introndependent regulatory events that help control global expression of ribosomal proteins+ Another is that introns may improve expression of the genes that contain them in yeast, as apparently they do in mice (Choi et al+, 1991;Palmiter et al+, 1991)+ The proposal that reverse transcripts mediate intron loss in yeast (Fink, 1987) might predict that the most abundantly transcribed genes would be the first to lose their introns, but ribosomal protein genes have defied this expectation+ For evolutionary reasons we have yet to divine, the few remaining intron-containing genes in yeast produce nearly a third of all mRNAs, so that at the level of RNA, introns are still very much the business of the yeast cell+ Table 1 also illuminates the extreme degree to which the work of the yeast splicing apparatus is devoted to ribosome biogenesis+ Hartwell's original rna mutants were characterized as unable to make RNA at a restrictive temperature in a metabolic labeling experiment that measures primarily rRNA accumulation (Hartwell et al+, 1970)+ Several years (as well as the discovery of splicing) ensued before it was noted that the rna mutations (since renamed prp) affect removal of introns (Rosbash et al+, 1981), and that most inactivate components of the splicing apparatus as assayed in vitro (Lustig et al+, 1986)+ In addition, many unusual trends in gene organization have been noted, including such curiosities as small nucleolar RNAs (involved in rRNA maturation) encoded within introns of ribosomal protein, translation factor, and ribosome assembly factor genes, or introns within snoRNA U3 genes (for review, see Spingola et al+, 1999)+ Thus, although almost 30 years of traditional genetic and molecular studies have hinted that the spliceosome and the ribosome are in close regulatory communication, an expression-based splicing budget (Table 1) illustrates immediately why inhibition of splicing should lead to inhibition of rRNA accumulation+ As genome-wide expression studies are performed on strains carrying mutations in the splicing machinery, it will be interesting to note the degree to which expression of genes involved in translation is secondarily affected, even if those genes do not themselves contain introns+ In fact, the great absence of introns makes yeast possibly the only system in which to sort out primary from secondary effects of inhibiting splicing+ A final observation that can be made from this data concerns the activity of the splicing apparatus+ Estimates of snRNA content of yeast using Northern blots indicate that there are 200-500 copies of the major snRNAs per haploid cell (Riedel et al+, 1986)+ As seen in Table 1, at least 10,000 introns must be removed from pre-mRNA per yeast cell per hour using 500 molecules of U2 (an estimate based on internally controlled primer extensions; M+H+ Pauling & M+ Ares, unpubl+)+ With the assumption that every U2 molecule is part of the active pool of splicing factors, this means that each U2 snRNA molecule helps remove about 20 introns per hour, or 1 every 3 min+ Splicing factors in greater or lesser abundance than U2 would act more slowly or more rapidly, respectively+ If a pathway for snRNP reutilization exists in yeast as sug...…”