The Search for Novel Splicing Factors: Mapping Mutations in a Temperature‐Sensitive Schizosaccharomyces pombe Library

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Gene expression, starting with the transcription of DNA to messenger RNA (mRNA) and the subsequent translation of mRNA transcripts to form proteins is a vital process for all living cells. Newly transcribed RNAs must be processed before they can serve as templates for protein synthesis. RNA splicing is a key step in RNA processing. Defective splicing disrupts the production of functional proteins and is linked to debilitating human diseases such as cancer, frontotemporal dementia, and Duchenne muscular dystrophy. However, the full set of factors involved in the splicing pathway remain unknown. Hence, uncovering the complete set of splicing factors is critical for the development of new therapeutic interventions.A recent sequencing‐based genetic screen 1, 2 of a temperature‐sensitive (ts) Schizosaccharomyces pombe library 3 identified a subset of mutants that displayed global splicing defects, which may highlight essential genes with potentially novel implications in splicing. The identities of the specific mutations for each of the strains in this ts library (N = ~2300) have never been previously mapped. Therefore, we aim to map the mutations in this library and quantify their impacts on RNA splicing, allowing us to identify the genes detected in the recent screen and possibly reveal additional novel splicing factors and functional connections. Here we use Tn5 tagmentation for genome fragmentation and Gibson assembly for the high‐throughput synthesis of a wild‐type (WT) cloning library, which will then be transformed into each mutant strain to identify the mutant alleles via complementation testing. Once the alleles have been identified, we will characterize their global splicing deficiencies by sequencing methods to measure the ratio of unspliced to spliced RNA transcripts for all introns. Our efforts will annotate the ts mutant library for general use and contribute to the knowledge of the elements involved in RNA splicing.