Biology subjects

Donohue, J. P.

Publications and source records attributed to Donohue, J. P..

5 recordsLinked to original sources

bioRxiv2024

An ancient competition for the conserved branchpoint sequence influences physiological and evolutionary outcomes in splicing

Recognition of the intron branch point during spliceosome assembly is a multistep process that can influence mRNA structure and levels. A branch point sequence motif UACUAAC is variably conserved in eukaryotic genomes, but in some organisms, more than one protein can recognize it. Here, we show that SF1 and Quaking (QKI) compete for a subset of intron branch sites with the sequence ACUAA. SF1 activates exon inclusion through this sequence, but QKI represses the inclusion of alternatively spliced exons with this intron branch point sequence. Using mutant reporters derived from a natural intron with two branch site-like sequences, we find that when either branch point sequence is mutated, the other is utilized; however, when both are present, neither is used due to high-affinity binding and strong splicing repression by QKI. QKI occupancy at the dual branch site directly prevents SF1 binding and subsequent recruitment of spliceosome-associated factors. Finally, the ectopic expression of QKI in budding yeast (which lacks QKI) is lethal, at least in part due to the widespread repression of splicing. In conclusion, QKI can function as a splicing repressor by directly competing with SF1/BBP for a subset of branch point sequences that closely mirror its high-affinity binding site.

molecular biology↗

bioRxiv2024

Intron-lariat spliceosomes convert lariats to true circles: implications for intron transposition

Rare, full length circular intron RNAs distinct from lariats have been reported in several species, but their biogenesis is not understood. We envision and test a hypothesis for their formation using Saccharomyces cerevisiae, documenting full length and novel processed circular RNAs from multiple introns. Evidence implicates a previously undescribed catalytic activity of the intron-lariat spliceosome (ILS) in which the 3-OH of the lariat tail (with optional trimming and adenylation by the nuclear 3 processing machinery) attacks the branch, joining the intron 3 end to the 5 splice site in a 3-5 linked circle. Human U2 and U12 spliceosomes produce analogous full length and processed circles. Post-splicing catalytic activity of the spliceosome may promote intron transposition during eukaryotic genome evolution.

molecular biology↗

bioRxiv2023

Broad variation in response of individual introns to splicing inhibitors in a humanized yeast strain

Intron branch point (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity. To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeast Saccharomyces cerevisiae by changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Splicing is inhibited within minutes after addition of Plad-B, and different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed during co-transcriptional splicing in Plad-B using single-molecule intron tracking (SMIT) to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. Introns with variant BP sequences seem particularly sensitive, echoing observations from mammalian cells, where monitoring individual introns is complicated by multi-intron gene architecture and alternative splicing. The compact yeast system may hasten characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.

molecular biology↗

bioRxiv2020

Definition of germ cell lineage alternative splicing programs reveals a critical role for Quaking in specifying cardiac cell fate

Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.

molecular biology↗

bioRxiv2019

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 selection. 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.\n\nAuthor SummaryThe protein coding information in eukaryotic genes is broken by intervening sequences called introns that are removed from RNA during transcription by a large protein-RNA complex called the spliceosome. Where introns come from and how the spliceosome contributes to genome evolution are open questions. In this study, we find more than 150 new places in the yeast genome that are recognized by the spliceosome and spliced out as introns. Since they appear to have arisen very recently in evolution by sequence drift and do not appear to contribute to gene expression or its regulation, we call these protointrons. Protointrons are found in both protein-coding and non-coding RNAs and are not efficiently removed by the splicing machinery. Although most protointrons are not conserved, a few are spliced more efficiently, and are located where they might begin to play functional roles in gene expression, as predicted by the proposed process of intronization. The challenge now is to understand how spontaneously appearing splicing events like protointrons might contribute to the creation of new genes, new genetic controls, and new protein isoforms as genomes evolve.

genomics↗