Abstract
The spliceosome consists of accessory proteins and small nuclear ribonucleoproteins (snRNPs) that remove introns from RNA. As splicing defects are associated with degenerative conditions, a better understanding of spliceosome formation and function is essential. We provide insight into the role of a spliceosome protein U4/U6.U5 tri-snRNP-associated protein 1, or Squamous cell carcinoma antigen recognized by T-cells (Sart1). Sart1 recruits the U4.U6/U5 tri-snRNP complex to nuclear RNA. The complex then associates with U1 and U2 snRNPs to form the spliceosome. A forward genetic screen identifying defects in choroid plexus development and whole-exome sequencing (WES) identified a point mutation in exon 12 of sart1 in Danio rerio (zebrafish). This mutation caused an up-regulation of sart1. Using RNA-Seq analysis, we identified additional upregulated genes, including those involved in apoptosis. We also observed increased activated caspase 3 in the brain and eye and down-regulation of vision-related genes. Although splicing occurs in numerous cells types, sart1 expression in zebrafish was restricted to the brain. By identifying sart1 expression in the brain and cell death within the central nervous system (CNS), we provide additional insights into the role of sart1 in specific tissues. We also characterized sart1’s involvement in cell death and vision-related pathways.
Highlights
The splicing process was discovered more than 30 years ago [1], the mechanisms of spliceosome assembly and splicing regulation are not well understood
While previous studies have identified the major role of Sart1 as recruiting the U4/U5/U6 tri-sRNP to the spliceosome, we provide evidence that it may directly affect specific classes of genes such as those involved in apoptosis along with eye formation and maintenance
Sart1 has been implicated in a number of different processes such as cell cycle arrest and apoptosis and has been suggested as a target for gene therapy due to its role as an antigen recognized by cytotoxic T-lymphocytes (CTLs) in certain types of cancer [17]
Summary
The splicing process was discovered more than 30 years ago [1], the mechanisms of spliceosome assembly and splicing regulation are not well understood. Over 100 proteins that regulate spliceosome assembly and function have been identified [2]. We have characterized one of these genes, sart, which was isolated from an N-ethyl-N-nitrosourea (ENU) mutagenesis forward genetic screen in the enhancer trap zebrafish line Et(cp:EGFP)sj to identify mutants in choroid plexus (CP) development [6]. In this genetic screen, zebrafish larvae were observed at 4 days postfertilization (dpf) to look for phenotypes in the CP. The green fluorescent protein (GFP), was expressed in CP epithelia so mutants in CP development could be observed under a fluorescent microscope. Referred to as cp27.5, Cells 2020, 9, 2340; doi:10.3390/cells9112340 www.mdpi.com/journal/cells
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