UC Santa Cruz

Structural data suggest that human SNRP27 M141 and SNU-66 H734have a stacking interaction at the base of the U6 snRNA ACAGAGA box in the pre-B complex. Previously, we found that mutations in C. elegans at SNRP-27 M141 promote changes in cryptic 5’ splice site choice and alternative 5’ss usage at native alternative splicing events 1 . We wanted to better understand if the potential interaction between SNRP-27 M141 and SNU-66 H765 (the C. elegans equivalent position to human SNU66 H734) contributes to maintaining the 5’ splice site identity during spliceosome assembly. Mutations at SNU-66 H765 change alternative 5’ splice sites usage genome wide. SNU-66(H765G) is a suppressor of unc-73(e936) 5’ splice site cryptic splicing, similar to SNRP-27 (M141T). Many of the alternative 5’ splicing events affected by SNU-66 (H765G) and (H765L) overlapped with those affected SNRP-27 (M141T). Double mutants of snrp-27(M141T) with snu-66(H765G) are homozygous lethal. Our findings show a new role for SNU-66 in 5’ splice site selection in a similar way as SNRP-27 M141T. Mutations at SNRP-27 M141 and SNU-66 H765 allow the spliceosome to choose alternative 5’ splice sites with weaker consensus in the proximity of a stronger consensus 5’ss. While we observed alternative 5’ splicing events that could be promoted either upstream or downstream of the stronger 5’ss in these mutants, the mechanism by which SNRP-27 M141 and SNU-66 H765 DocuSign Envelope ID: 13C88DBB-B62D-4F32-A3B3-56B16F7842AC mutants affect 5’ splice sites is dependent on both the presence of a weaker consensus 5’ss in close proximity and other factors (perhaps splicing factor binding sites nearby) that are important for determining the directionality of alternative splicing.