The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast

Ribosomal protein genes rp 39(10-78), rp 39(11-40), rp 51, and rp 52 are not contiguous to other ribosomal protein genes in the Saccharomyces cerevisiae genome

“…This latter observation suggests that the effect of rna2 is specific for individual rp genes and does not affect adjacent (or nearby) genes. A similar observation has been made for the rp 51 gene (17). A single large transcript hybridized to X13-86(l) (Figure 4, lanes 1 and 2), consistent with the fact that a single large polypeptide (, 95,000 daltons) was detected by cell-free translation of 9*.-''l200bp * * %^-"600bp 12 34 56 7 8 Northern blotting experiments demonstrate that the levels of the mature mRNA coding for ribosomal protein 51 are depleted approximately ten-+ fold in poly(A) RNA from rna2 strains grown at 36°C, and that levels of higher molecular weight transcripts, precursors to the ribosomal protein 51 mRNA, are increased in the mutant (17).…”

“…Plasmid DNA was purified from Brij deoxycholate treated bacterial spheroplasts by CsCl ethidium bromide density gradient centrifugation (14), with the modifications of Petes et al (13). Gradient (17).…”

Structure and expression of the Saccharomyces cerevisiae CRY1 gene: a highly conserved ribosomal protein gene.

“…The sequence TACTAAC, present within the CRY) intron, has been found in similar locations in all known yeast intervening sequences (57,66). The 5' exon of CRY) consists of a short untranslated leader that is 21 to 38 nucleotides long plus 2¼3 codons, whereas the 3' exon contains the remaining 1342¼3 codons and a 3' untranslated sequence that is 123 or 126 nucleotides long.…”

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...…”