Correction: Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome.

[This corrects the article DOI: 10.1371/journal.pgen.1008249.].

Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome.

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these "hungry spliceosome" conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of "intronization", whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function.

Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation.

Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA ("STAR" motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation.

A high-throughput splicing assay identifies new classes of inhibitors of human and yeast spliceosomes.

The spliceosome is the macromolecular machine responsible for pre-mRNA splicing, an essential step in eukaryotic gene expression. During splicing, myriad subunits join and leave the spliceosome as it works on the pre-mRNA substrate. Strikingly, there are very few small molecules known to interact with the spliceosome. Splicing inhibitors are needed to capture transient spliceosome conformations and probe important functional components. Such compounds may also have chemotherapeutic applications, as links between splicing and cancer are increasingly uncovered. To identify new splicing inhibitors, we developed a high-throughput assay for in vitro splicing using a reverse transcription followed by quantitative PCR readout. In a pilot screen of 3080 compounds, we identified three small molecules that inhibit splicing in HeLa extract by interfering with different stages of human spliceosome assembly. Two of the compounds similarly affect spliceosomes in yeast extracts, suggesting selective targeting of conserved components. By examining related molecules, we identified chemical features required for the activity of two of the splicing inhibitors. In addition to verifying our assay procedure and paving the way to larger screens, these studies establish new compounds as chemical probes for investigating the splicing machinery.

Rearrangement of competing U2 RNA helices within the spliceosome promotes multiple steps in splicing.

Nuclear pre-messenger RNA (pre-mRNA) splicing requires multiple spliceosomal small nuclear RNA (snRNA) and pre-mRNA rearrangements. Here we reveal a new snRNA conformational switch in which successive roles for two competing U2 helices, stem IIa and stem IIc, promote distinct splicing steps. When stem IIa is stabilized by loss of stem IIc, rapid ATP-independent and Cus2p-insensitive prespliceosome formation occurs. In contrast, hyperstabilized stem IIc improves the first splicing step on aberrant branchpoint pre-mRNAs and rescues temperature-sensitive U6-U57C, a U6 mutation that also suppresses first-step splicing defects of branchpoint mutations. A second, later role for stem IIa is revealed by its suppression of a cold-sensitive allele of the second-step splicing factor PRP16. Our data expose a spliceosomal progression cycle of U2 stem IIa formation, disruption by stem IIc, and then reformation of stem IIa before the second catalytic step. We propose that the competing stem IIa and stem IIc helices are key spliceosomal RNA elements that optimize juxtaposition of the proper reactive sites during splicing.