Transcript specificity in yeast pre-mRNA splicing revealed by mutations in core spliceosomal components - PubMed (original) (raw)

Transcript specificity in yeast pre-mRNA splicing revealed by mutations in core spliceosomal components

Jeffrey A Pleiss et al. PLoS Biol. 2007 Apr.

Abstract

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.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1. Properties of Yeast Introns

(A) Consensus sequences found in yeast introns at 5′ splice sites (5′SS), 3′ splice sites (3′SS), and branch points (BP) [37]. (B) Distribution of yeast intron lengths shown for all introns (black), introns only in ribosomal protein genes (green), or introns only in nonribosomal protein genes (red).

Figure 2. Monitoring Genome-Wide Changes in Pre-mRNA Splicing

(A) Oligos target either the pre-mRNA (P), the mature mRNA (M), or the total level of mRNA (T) for each intron-containing gene. (B) False color representation of the splicing response of 249 different intron-containing transcripts at a single timepoint after inactivation of Prp2 activity. Each horizontal line describes the behavior of a single transcript. (C) Time course from 0 to 30 min showing the kinetic response to inactivation of Prp2. The gene order is identical to that shown in (B).

Figure 3. Comparing the Cellular and Molecular Phenotypes of Three Different Spliceosomal Mutants

(A) Serial dilutions of a wild-type strain and strains containing the prp2-1, prp8-1, and prp5-1 mutations are grown at the temperatures indicated. (B) Time-resolved splicing profiles for each of the three mutant strains compared to a wild-type strain. (C) Time-resolved profiles resulting from microarrays directly comparing the indicated mutants. The order of genes is identical to that in (B). (D) Transcripts that encode ribosomal protein genes are indicated with a red line. The order of genes is identical to that in (B) and (C). (E) The identification and behavior of a subset of genes indicated with a green bar in (B) and (C). For these transcripts the comparison experiments reveal a different level of precursor accumulation for each of the mutants. (F) The identification and behavior of a subset of genes indicated with a red bar in (B) and (C). For these transcripts the comparison experiments show an identical splicing defect for all three mutants. (G) The identification and behaviors of a subset of genes whose splicing is affected by only one or two of the three mutants studied.

Figure 4. Two Different Alleles of Prp8 Produce Very Different Molecular Phenotypes

Time-resolved splicing profiles derived from comparisons of prp8-1 (A), prp8-101 (B), or prp16-302 (C), each compared to a wild-type strain. The order of the genes is the same for all three profiles.

Figure 5. A Global View of Defects in Pre-mRNA Splicing

Time-resolved splicing profiles derived from a panel of factors involved in pre-mRNA processing. The behavior of each individual transcript can be seen by following a vertical line. The particular mutations examined are shown on the left. For each mutant, the order is indicated at the top right corner of the figure.

Figure 6. A Transcript-Specific View of Defects in Pre-mRNA Splicing

Time-resolved splicing profiles for individual transcripts derived from the same panel of factors shown in Figure 5.

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Transcript specificity in yeast pre-mRNA splicing revealed by mutations in core spliceosomal components - PubMed (original) (raw)