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

ABSTRACT: Branchpoint (bp) recognition is an early event that is required for spliceosome recruitment to a pre-mRNA substrate. The bp sequence can range from nearly invariant in yeast (UACUAAC) to quite degenerate in higher eukaryotes (YUNAY in humans), which correlates with the frequency of alternative splicing observed in the latter. The closely related KH-type RNA binding proteins (RBPs) SF1 and Quaking (QKI) can bind to a similar bp-like sequence, but the extent to which competition for the bp sequence between these two proteins might influence gene expression regulatory mechanisms is unknown. Here we find genomewide evidence for competition between these two RBPs for a subset of alternatively spliced exons that have ACUAAY-like bp sequences. Using a combination of molecular genetic and biochemical techniques with a model intron containing two bp-like sequences, we find that either can be used as a bp but high affinity binding and strong splicing repression by QKI requires both sites. This directly prevents SF1 binding and the subsequent recruitment of spliceosome-associated factors. Finally the ectopic expression of QKI in S. cerevisiae (which lacks QKI) is lethal, due at least in part to widespread splicing repression. In conclusion, QKI can function as a splicing repressor by directly competing with SF1/BBP for a subset of bp sequences that closely mirror its high affinity binding site. This suggests that QKI and degenerate bp sequences may have co-evolved which allowed for expansion of the alternative splicing repertoire in metazoans, plants, and animals.

ORGANISM(S): Saccharomyces cerevisiae BY4741

PROVIDER: GSE273838 | GEO | 2024/11/22

REPOSITORIES: GEO

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Project description:The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability among muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy, and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu. Interestingly, knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of Kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a powerful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.

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

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