Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency.Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step ofsplicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA we observed that a second step splicing defect (the primary defect) was rapidly followed by a first step of splicing defect. Our results show that knocking down asplicing factor can quickly lead to a recycling defect with splicing factors sequestered installed complexes, thereby limiting new rounds of splicing. We demonstrate that this “feedback”effect can be minimized by depleting the target protein more gradually or only partially,allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.
- pre-mRNA splicing
- protein depletion