A systematic screen identifies Saf5 as a link between splicing and transcription in fission yeast

TL;DR

This study identifies Saf5 as a key factor linking splicing and transcription in fission yeast, especially for highly transcribed genes.

Contribution

The novel finding is that Saf5 connects splicing efficiency with transcription rate, particularly under stress conditions.

Findings

Saf5 is essential for splicing highly transcribed genes but not for low-transcribed ones.

Cells lacking Saf5 show impaired growth and splicing defects under stress conditions.

Saf5's role is not determined by cis elements but by gene transcription rates.

Abstract

Splicing is an important step of gene expression regulation in eukaryotes, as there are many mRNA precursors that can be alternatively spliced in different tissues, at different cell cycle phases or under different external stimuli. We have developed several integrated fluorescence-based in vivo splicing reporter constructs that allow the quantification of fission yeast splicing in vivo on intact cells, and we have compared their splicing efficiency in a wild type strain and in a prp2-1 (U2AF65) genetic background, showing a clear dependency between Prp2 and a consensus signal at 5’ splicing site (5’SS). To isolate novel genes involved in regulated splicing, we have crossed the reporter showing more intron retention with the Schizosaccharomyces pombe knock out collection. Among the candidate genes involved in the regulation of splicing, we have detected strong splicing defects in two of the mutants -Δcwf12, a member of the NineTeen Complex (NTC) and Δsaf5, a methylosome subunit that acts together with the survival motor neuron (SMN) complex in small nuclear ribonucleoproteins (snRNP) biogenesis. We have identified that strains with mutations in cwf12 have inefficient splicing, mainly when the 5’SS differs from the consensus. However, although Δsaf5 cells also have some dependency on 5’SS sequence, we noticed that when one intron of a given pre-mRNA was affected, the rest of the introns of the same pre-mRNA had high probabilities of being also affected. This observation points Saf5 as a link between transcription rate and splicing.

Diversity in gene expression extends to various levels, and splicing is no exception. Unlike more straightforward organisms like budding yeast, which feature a fundamental set of splicing components and a limited count of constitutively-processed introns, fission yeast stands out. In fission yeast, there is a greater abundance of introns, and the splicing machinery is more intricate, encompassing non-essential splicing factors. In this study, we conducted a genetic screening to identify non-essential genes regulating basal splicing. Notably, Saf5 emerged as a standout candidate. Cells lacking Saf5 exhibited significantly impaired growth, with viability limited to 30°C, indicating heightened sensitivity to both cold and heat stress. A detailed analysis of splicing efficiency revealed that Saf5’s role in splicing is not dictated by cis elements but is intricately tied to the transcription rate of individual genes. Consequently, Saf5 proves indispensable for the proper splicing of highly transcribed genes, yet dispensable for those with lower transcription rates. This pivotal role positions Saf5 as a crucial link bridging splicing and transcription processes.

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