Coupling of spliceosome complexity to intron diversity

Abstract

We determined that over 40 spliceosomal proteins are conserved between many fungal species and humans but were lost during the evolution of S. cerevisiae, an intron-poor yeast with unusually rigid splicing signals. We analyzed null mutations in a subset of these factors, most of which had not been investigated previously, in the intron-rich yeast Cryptococcus neoformans. We found they govern splicing efficiency of introns with divergent spacing between intron elements. Importantly, most of these factors also suppress usage of weak nearby cryptic/alternative splice sites. Among these, orthologs of GPATCH1 and the helicase DHX35 display correlated functional signatures and copurify with each other as well as components of catalytically active spliceosomes, identifying a conserved G-patch/helicase pair that promotes splicing fidelity. We propose that a significant fraction of spliceosomal proteins in humans and most eukaryotes are involved in limiting splicing errors, potentially through kinetic proofreading mechanisms, thereby enabling greater intron diversity.

Targeted high throughput mutagenesis of the human spliceosome reveals itsin vivooperating principles

“…We designed a single guide RNA (sgRNA) library targeting a hand-curated list of 153 human spliceosomal proteins which engage at various steps of the splicing cycle (Figure 1A), and are reproducibly detected through mass spectrometry (MS), interaction studies, and/or visualized in structural biology studies (see Figure 1B, Table S1)(Sales-Lee et al, 2021). Given that base editing outcomes are not fully predictable, to maximize mutagenesis we targeted every available NGG PAM sequence across all annotated exons plus 20 bp flanking intronic sequence (Figure 1C).…”

“…We designed a single guide RNA (sgRNA) library targeting a hand-curated list of 153 human spliceosomal proteins which engage at various steps of the splicing cycle (Figure 1A), and are reproducibly detected through mass spectrometry (MS), interaction studies, and/or visualized in structural biology studies (see Figure 1B, Table S1) (Sales-Lee et al, 2021) .…”

A dual role for the RNA helicase DHX34 in NMD and pre-mRNA splicing and its function in hematopoietic differentiation

“…1A). A recent study revealed that GPATCH1 copurifies with DHX35 and with components of catalytically active spliceosomes, strongly suggesting that the GPATCH1/DHX35 pair functions together to promote splicing fidelity (Sales- Lee et al 2021). Whether GPATCH1 also acts together with DHX34 in pre-mRNA splicing; or and/or whether DHX34 is regulated by a different GPATCH protein, remains to be determined.…”

Integrating phylogenetics with intron positions illuminates the origin of the complex spliceosome

“…These were included in the original list because these were also clearly described as spliceosomal in the literature. If an ortholog was not present in yeast, spliceosomal annotations for orthologs in S. pombe, A. thaliana (both in the UniProt database) or Cryptococcus neoformans (Sales-Lee et al 2021) were checked. If an ortholog was not present in human, the function of the A. thaliana ortholog was investigated.…”

“…Proteins that are involved in other processes (such as transcription and polyadenylation) and splice site selection and splicing regulation were removed. The list was supplemented with human spliceosomal proteins from recent literature (Bai et al 2021;Sales-Lee et al 2021;de Wolf et al 2021). Initial evolutionary scenarios of these proteins were inferred based on the approach of Van Hooff et al (2019.…”