Fig 1. Mutations in KIN17/dxbp-1 and PRCC suppress cryptic splicing, promoting an unusual /UU 5’ splice site.

(A) Schematic diagram of the 16th intron of the C. elegans gene unc-73, showing genomic coordinates and relative loci of splice sites and PCR primer locations used to assess splice site usage. Below, aligned sequences of the unc-73 sequence and exon/intron boundary in wild type, unc-73(e936), and in the CRISPR engineered allele unc-73(az63). The cryptic splice sites activated in the competition assay are labeled -1 and +23 and define introns beginning with /GU that are both out of frame. Note that the wild-type splicing position is still denoted “wt ss” even though that intron now begins /UU. The slash mark (/) denotes the splice site. (B) Poly-acrylamide gel showing Cy-3 labeled unc-73 PCR products amplified from unc-73 cDNA. RNA was extracted from plates of the following 6 mixed-stage strains of C. elegans: wild-type N2, unc-73(e936), and four independent original suppressed strains identified in the genetic screen whose genotypes are indicated below, each bears both the unc-73(e936) allele and an extragenic suppressor of both the movement defect. The same PCR primers are used on all samples; band positions and intensities are indicative of relative use of the three available 5’ splice sites, labeled -1, wt, and +23. Strains are, in lane order, N2, SZ181, SZ162, SZ283, SZ280, and SZ281, see Methods for genetic details. (C) Putative suppressor identities were verified by de novo recreations of mutations using CRISPR/Cas9 and homology-directed repair into unc-73 reporter strains. Image is a scan of a denaturing polyacrylamide gel showing Cy-3 labeled unc-73 PCR products from unc-73 cDNA. RNA was extracted from mixed-stage strains with the indicated unc-73, dxbp-1, and prcc-1 alleles shown below. Strains are, in lane order, N2, SZ181, SZ219, SZ391, SZ222, SZ308, SZ348, see Methods for genetic details. Unless otherwise mentioned, CRISPR-engineered mimic alleles are used for all subsequent experiments and figures in this report. (D) Four new suppressors of cryptic splicing represent a new class of suppressors, with a distinct molecular phenotype compared to previously identified suppressors. Indicated are the suppressor class (I, II, or III) [16,17,20], genotype of unc-73, genotype of suppressor, average percent spliced in (PSI), n≥3, at the /GU splice site at position -1 relative to wild type, average PSI at /UU splice site in wild-type position, and average PSI at the /GU at position +23. Conditional grayscale shading highlights patterns in numerical data. All 4 Type III suppressors have a statistically significant difference in usage of the -1 splice site when compared to all of the Type II suppressors, p< 0.01 by Student’s T-Test. (S1 Table). Note that the values in 1D for Type II suppressors and control vary slightly from previous publications, but the trends are all consistent. This variation may be due to the use of the new Cy-3-labeled primer assay, see Methods.