UC Santa Cruz

RNA helicases drive necessary rearrangements and ensure fidelity during the pre-mRNA splicing cycle. DEAD-box helicase DDX41 has been linked to human disease and has recently been shown to interact with DEAH-box helicase PRP22 in the spliceosomal C* complex, yet its function in splicing remains unknown. Previous transcriptomic studies of C. elegans depleted of the DDX41 homolog SACY-1 uncovered predominantly changes in alternative 3’ splice site usage. We did a transcriptomic analysis of a viable sacy-1(G533R) allele in staged L3 animals; this allele causes alternative 3’ splicing in introns with pairs of 3’ splice sites separated by ≤ 18 nucleotides. We find that both SACY-1 depletion and the G533R allele lead to a striking unidirectional increase in the usage of proximal (upstream) 3’ splice sites. We have previously discovered a similar alternative splicing pattern between germline tissue and somatic tissue, in which there is a unidirectional increase in proximal 3’ splice site usage in the germline for ~200 events; many of the somatic SACY-1 alternative 3’ splicing events overlap with these developmentally regulated events. We generated a targeted mutant allele of the C. elegans homolog of PRP22, mog-5, in the region of MOG-5 that is predicted to interact with SACY-1 based on the human C* structural model. This viable allele also promotes usage of the proximal alternative adjacent 3’ splice sites in somatic cells. We show that mog-5 and sacy-1 have overlapping proofreading phenotypes against proximal alternative adjacent 3’ splice sites. This work demonstrates that C. elegans is tractable for the genetic study of 3’ss choice after early spliceosomal assembly. These findings are used to inform a program for future genetic research on 3’ss choice.