Summary We used a quantitative, high-density genetic interaction map, or E-MAP (Epistatic MiniArray Profile), to interrogate the relationships within and between RNA processing pathways. Due to their complexity, and the essential roles of many of the components, these pathways have been difficult to functionally dissect. Here we report the results for 107,155 individual interactions involving 552 mutations, 166 of which are hypomorphic alleles of essential genes. Our data enabled the discovery of links between components of the mRNA export and splicing machineries and Sem1/Dss1, a component of the 19S proteasome. In particular, we demonstrate that Sem1 has a proteasome-independent role in mRNA export as a functional component of the Sac3-Thp1 complex. Sem1 also interacts with Csn12, a component of the COP9 signalosome. Finally, we show that Csn12 plays a role in pre-mRNA splicing, which is independent of other signalosome components. Thus, Sem1 is involved in three separate and functionally distinct complexes.

While the core splicing machinery is highly conserved between budding yeast and mammals, the absence of alternative splicing in Saccharomyces cerevisiae raises the fundamental question of why introns have been retained in approximately 5% of the 6000 genes. Because ribosomal protein-encoding genes (RPGs) are highly overrepresented in the set of intron-containing genes, we tested the hypothesis that splicing of these transcripts would be regulated under conditions in which translation is impaired. Using a microarray-based strategy, we find that, within minutes after the induction of amino acid starvation, the splicing of the majority of RPGs is specifically inhibited. In response to an unrelated stress, exposure to toxic levels of ethanol, splicing of a different group of transcripts is inhibited, while the splicing of a third set is actually improved. We propose that regulation of splicing, like transcription, can afford rapid and specific changes in gene expression in response to the environment.

Appropriate expression of most eukaryotic genes requires the removal of introns from their pre–messenger RNAs (pre-mRNAs), a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well.

Ribonuclease P (RNase P) is an essential endonuclease responsible for the 5-end maturation of precursor tRNAs. Bacterial RNase P also processes precursor 4.5S RNA, tmRNA, 30S preribosomal RNA, and several reported protein-coding RNAs. Eukaryotic nuclear RNase P is far more complex than in the bacterial form, employing multiple essential protein subunits in addition to the catalytic RNA subunit. RNomic studies have shown that RNase P binds other RNAs in addition to tRNAs, but no non-tRNA substrates have previously been identified. Additional substrates were identified by using a multipronged approach in the budding yeast Saccharomyces cerevisiae. First, RNase P-dependant changes in RNA abundance were examined on whole-genome microarrays by using strains containing temperature sensitive (TS) mutations in two of the essential RNase P subunits, Pop1p and Rpr1r. Second, RNase P was rapidly affinity-purified, and copurified RNAs were identified by using a genome-wide microarray. Third, to identify RNAs that do not change abundance when RNase P is depleted but accumulate as larger precursors, >80 potential small RNA substrates were probed directly by Northern blot analysis with RNA from the RNase P TS mutants. Numerous potential substrates were identified, of which we characterized the box C/D intron-encoded small nucleolar RNAs (snoRNAs), because these both copurify with RNase P and accumulate larger forms in the RNase P temperature-sensitive mutants. It was previously known that two pathways existed for excising these snoRNAs, one using the pre-mRNA splicing path and the other that was independent of splicing. RNase P appears to participate in the splicing-independent path for the box C/D intron-encoded snoRNAs.RNA ͉ biogenesis R ibonuclease P (RNase P) is a conserved endoribonuclease responsible for removing the 5Ј leader sequence from precursor transfer RNAs (pre-tRNAs) found in bacteria, archaea, eukarya (1, 2). In all cases, with the possible exception of some organelles, RNase P is composed of both RNA and protein subunits. Bacterial RNase P is the simplest form of the holoenzyme, with one large RNA subunit and a single small protein subunit (1). Although the RNA subunit of bacterial RNase P is sufficient for catalysis in vitro at high salt concentrations (3), both the RNA and protein subunits are required in vivo. The protein subunit appears to stabilize the catalytically active conformation of RNase P RNA and assist with substrate binding (4-7). In addition to pre-tRNAs, bacterial RNase P is known to process several substrates that are proposed to contain tRNA-like structures: 4.5S RNA, tmRNA, viral RNAs, mRNAs, riboswitches, ColE1 replication origin control RNAs, and C4 antisense RNA from phages P1 and P7 (8-16). The presence of the protein subunit in the RNase P holoenzyme increases the substrate versatility of the enzyme over the RNA enzyme alone (17).The nature of the eukaryotic nuclear RNase P is much more complex. First, there are two very similar enzymes that are related to bacterial RNase P, termed RNa...

Gene expression in eukaryotic cells is profoundly influenced by the post-transcriptional processing of mRNAs, including the splicing of introns in the nucleus and both nuclear and cytoplasmic degradation pathways. These processes have the potential to affect both the steady-state levels and the kinetics of changes to levels of intron-containing transcripts. Here we report the use of a splicing isoform-specific microarray platform to investigate the effects of diverse stress conditions on pre-mRNA processing. Interestingly, we find that diverse stresses cause distinct patterns of changes at this level. The responses we observed are most dramatic for the RPGs and can be categorized into three major classes. The first is characterized by accumulation of RPG premRNA and is seen in multiple types of amino acid starvation regimes; the magnitude of splicing inhibition correlates with the severity of the stress. The second class is characterized by a rapid decrease in both pre-and mature RPG mRNA and is seen in many stresses that inactivate the TORC1 kinase complex. These decreases depend on nuclear turnover of the intron-containing pre-RNAs. The third class is characterized by a decrease in RPG pre-mRNA, with only a modest reduction in the mature species; this response is observed in hyperosmotic and cation-toxic stresses. We show that casein kinase 2 (CK2) makes important contributions to the changes in pre-mRNA processing, particularly for the first two classes of stress responses. In total, our data suggest that complex post-transcriptional programs cooperate to fine-tune expression of intron-containing transcripts in budding yeast.