Retinitis pigmentosa-linked mutation in DHX38 modulates its splicing activity

Abstract

Retinitis pigmentosa (RP) is a hereditary disease affecting tens of thousands of people world-wide. Here we analyzed the effect of an amino acid substitution in the RNA helicase DHX38 (Prp16) causing RP. DHX38 has been proposed as the helicase important for the 2nd step of splicing. We showed that DHX38 associates with key splicing factors involved in both splicing steps but did not find any evidence that the RP mutations changes DHX38 interaction profile with the spliceosome. We further downregulated DHX38 and monitored changes in splicing. We observed only minor perturbations of general splicing but detected modulation of ~70 alternative splicing events. Next, we probed DHX38 function in splicing of retina specific genes and found that FSCN2 splicing is dependent on DHX38. In addition, RHO splicing was inhibited specifically by expression of DHX38 RP variant. Finally, we showed that overexpression of DHX38 promotes usage of canonical as well as cryptic 5' splice sites in HBB splicing reporter. Together, our data show that DHX38 is a splicing factor that promotes splicing of cryptic splice sites and regulate alternative splicing. We further provide evidence that the RP-linked substitution G332D modulates DHX38 splicing activity.

Fig 1. DHX38 associates with multiple spliceosomal components.

DHX38^WT-FLAG and DHX38^G332D-FLAG were transiently expressed in HEK293 cells, immunoprecipitated and copurification of selected spliceosomal proteins monitored. Immunoprecipitation was performed in normal NET2 buffer containing 150 mM NaCl (A) or in stringent NET2 buffer containing 300 mM NaCl (B).

Fig 2. DHX38 downregulation changes primarily gene expression.

Summary of three independent RNA-seq analysis of HEK293 cells treated either with anti-DHX38 siRNA or negative control siRNA. Analysis of (A) splicing efficiency, (B) alternative splicing and (C) gene expression. Expression of selected genes that were downregulated (D) or upregulated (E) after DHX38 downregulation was analyzed by RT-qPCR. RNA was isolated from control cells or cells treated with cycloheximide (CHX) to reveal potential targets of non-sense mediated decay. Average of three independent experiment with SEM is shown. Expression value determined by RNA-seq analysis is shown in the same graph for comparison (RNA seq).

Fig 3. DHX38 is important for splicing of retina gene-derived reporters.

Splicing of (A) FSCN2-derived reporter and (B) RHO-derived reporter was analyzed after DHX38 downregulation and after expression of siRNA-resistant DHX38^WT-FLAG and DHX38^G332D-FLAG proteins. Normalized ratio of pre-mRNA/mRNA from at least three independent experiments is shown in a graph together with the mean and SEM. Statistical significance was assessed by two-tail unpaired t-test (** indicates p≤0.01). Representative gels are shown. * next to RHO gel marks an unspecific product.

Fig 4. DHX38 promotes splicing of the HBB-derived reporter.

(A) A representative gel of HBB-derived reporter splicing after DHX38 knockdown and expression of siRNA-resistant DHX38^WT-FLAG and DHX38^G332D-FLAG. (B) A scheme of HBB-derived reporter and M1 and M2 mutants that block usage of normal splice site and promote usage of upstream 5’ cryptic splice site. (C) Quantification of three independent experiments with the mean and SEM. Statistical significance was assessed by two tail unpaired t-test (* indicates p≤0.05, ** p≤0.01).