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Mechanistic studies using a humanized yeast strain sensitive to splicing inhibitors


Gene expression is a critical process for cell viability and growth. One step in gene expression is RNA splicing, which removes non-coding introns from mRNA during gene expression. This work aims to answer the following outstanding questions in the field: 1) What are the consequences of blocking splicing for individual introns and 2) how does global gene expression change when splicing is blocked? Proper recognition of branchpoint sequence (BPS) and splice sites in the intron are required for splicing. U2 small nuclear RNA (snRNA) base pairing to the BPS is an early step in this recognition. The U2 snRNA-BPS helix contains a bulged branchpoint adenosine (BPA) that binds a pocket formed by HEAT repeats 15 and 16 of the budding yeast HSH155 protein. In human SF3B1 (homolog of HSH155), this pocket also binds splicing inhibitors such as Pladienolide-B (Plad-B). HSH155 differs from SF3B1 in the amino acids lining the pocket, rendering yeast resistant to Plad-B. To study the consequences of splicing inhibition on individual introns and gene expression, we created a yeast strain sensitive to inhibitors. We replaced the HSH155 BPA binding pocket with that of SF3B1, essentially “humanizing” splicing in budding yeast. We called this allele hsh155- ds for drug-sensitive hsh155 mutant. In vivo, splicing inhibition occurs minutes after addition of Plad-B. In vitro splicing extracts from hsh155-ds yeast treated with inhibitors show that spliceosomes are blockedat prespliceosomes (A-complex). RNA-sequencing of cells carrying WT HSH155 treated with Plad-B produces no significant splicing changes compared to untreated cells carrying either HSH155 or hsh155-ds, showing that (1) wild type cells are completely resistant to Plad-B and (2) the humanizing mutation does not affect splicing. In contrast, treatment of the hsh155-ds strain with Plad-B results in a dramatic accumulation of pre-mRNA for intron-containing genes. The effect of a block to splicing reverberates across the transcriptome, leading to gene expression changes transcriptome-wide. Intronless ribosomal protein genes are downregulated, presumably in response to reduced expression of intron-containing ribosomal protein genes. The ability to chemically block splicing in a cell with few introns may reveal conserved regulatory connections between splicing and other steps of gene expression.

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