Abstract
Pre-mRNA splicing is a critical step of gene expression in eukaryotes. Transcriptome-wide splicing patterns are complex and primarily regulated by a diverse set of recognition elements and associated RNA-binding proteins. The retention and splicing (RES) complex is formed by three different proteins (Bud13p, Pml1p and Snu17p) and is involved in splicing in yeast. However, the importance of the RES complex for vertebrate splicing, the intronic features associated with its activity, and its role in development are unknown. In this study, we have generated loss-of-function mutants for the three components of the RES complex in zebrafish and showed that they are required during early development. The mutants showed a marked neural phenotype with increased cell death in the brain and a decrease in differentiated neurons. Transcriptomic analysis of bud13, snip1 (pml1) and rbmx2 (snu17) mutants revealed a global defect in intron splicing, with strong mis-splicing of a subset of introns. We found these RES-dependent introns were short, rich in GC and flanked by GC depleted exons, all of which are features associated with intron definition. Using these features, we developed and validated a predictive model that classifies RES dependent introns. Altogether, our study uncovers the essential role of the RES complex during vertebrate development and provides new insights into its function during splicing.
Highlights
Splicing is critical step in eukaryotic gene expression and is an important source of transcriptomic complexity [1]
retention and splicing (RES) complex is essential for embryogenesis in zebrafish
To determine the role of the RES complex during vertebrate development, we first analyzed its expression pattern during development. bud13, rbmx2 and snip1 (Fig 1A) are maternally expressed (Fig 1B, S1D and S1F Fig), and later in development their mRNAs are strongly expressed in the central nervous system (CNS) (26 hours post fertilization, hpf), (Fig 1B) suggesting that RES complex may be required for brain development
Summary
Splicing is critical step in eukaryotic gene expression and is an important source of transcriptomic complexity [1]. Splicing is carried out by the spliceosome, a large macromolecular complex that includes five small nuclear ribonucleoproteins (snRNPs; U1, U2, U4, U5 and U6) and hundreds of core and accessory proteins that ensure the accurate removal of introns from pre-mRNAs [2]. Spliced introns are removed through two transesterification reactions during a complex process involving the recruitment and release of multiple core splicing factors [3]. Long introns surrounding short exons are recognized and spliced through “exon definition” mechanisms, in which the initial pairing bridges across the exon [4]. While these mechanisms are widely accepted, little is known about the specific factors associated with each process
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