De novo and inherited dominant variants in U4 and U6 snRNAs cause retinitis pigmentosa

Abstract

The U4 small nuclear RNA (snRNA) forms a duplex with the U6 snRNA and, together with U5 and ~30 proteins, is part of the U4/U6.U5 tri-snRNP complex, located at the core of the major spliceosome. Recently, recurrent de novo variants in the U4 RNA, transcribed from the RNU4-2 gene, and in at least two other RNU genes were discovered to cause neurodevelopmental disorder. We detected inherited and de novo heterozygous variants in RNU4-2 (n.18_19insA and n.56T>C) and in four out of the five RNU6 paralogues (n.55_56insG and n.56_57insG) in 135 individuals from 62 families with non-syndromic retinitis pigmentosa (RP), a rare form of hereditary blindness. We show that these variants are recurrent among RP families and invariably cluster in close proximity within the three-way junction (between stem-I, the 5' stem-loop and stem-II) of the U4/U6 duplex, affecting its natural conformation. Interestingly, this region binds to numerous splicing factors of the tri-snRNP complex including PRPF3, PRPF8 and PRPF31, previously associated with RP as well. The U4 and U6 variants identified seem to affect snRNP biogenesis, namely the U4/U6 di-snRNP, which is an assembly intermediate of the tri-snRNP. Based on the number of positive cases observed, deleterious variants in RNU4-2 and in RNU6 paralogues could be a significant cause of isolated or dominant RP, accounting for up to 1.2% of all undiagnosed RP cases. This study highlights the role of non-coding genes in rare Mendelian disorders and uncovers pleiotropy in RNU4-2, where different variants underlie neurodevelopmental disorder and RP.

Keywords: hereditary disease; non-coding; retinitis pigmentosa; snRNA; spliceosome; splicing.

Figure 1:. U4-U6 structure and rare variants found in RP cases and controls (gnomAD).

(A) 2D structure of U4-U6 duplex with recurrent variants identified in RP cases (in red for U4 and in green for U6), which cluster in the same region of the complex, the three-way junction. Nucleotides affected by variants previously observed in neurodevelopmental disorder cases are underlined. (B) Rare variants affecting RNU4-1, defined as AF<0.1% in gnomAD v.4.1, identified in RP cases and in controls. (C) same as (B) for RNU4-2 with recurrent variants displayed in red. (D) same as (B) for all five RNU6 paralogues combined, with recurrent variants displayed in green.

Figure 2:. 2D modeling of the U4-U6 three-way junction.

Wild-type (WT) structure of the U4/U6 duplex surrounding the internal loop (A, D) and structures including pathogenic variants affecting U4 (B, C), U6 (E, F). The gray circle gauges the normal size of the three-way junction, while the dashed lines show the normal orientations of the stems originating from the junction.

Figure 3:. 3D structure of the U4/U6 duplex and its interactions with the neighboring splicing factors PRPF3, PRPF6, PRPF8 and PRPF31.

(A) Naked U4/U6 pairing, showing the close proximity of the pathogenic variants identified (red and green). (B) Same as in (A), with interacting PRPF proteins. (C) Direct interactions of nucleotides of the U4/U6 duplex with PRPF31, by direct hydrogen bonds. (D) Same as (C) but for PRPF3.

Figure 4:. Expression and markers of transcriptional activity for RNU4 and RNU6 genes.

(A) Expression of RNU4-1 and RNU4-2 from RNA-seq of human donor choroid (n=13), neurosensory retina (NSR, n=4) and retinal pigmented epithelium (RPE, n=16). Data are represented in boxplots and the median value is written in the box. (B) Same for RNU4 genes (RNU4-1, RNU4-2 and their pseudogenes (in black)) and for all RNU6 genes (RNU6-1, RNU6-2, RNU6-7, RNU6-8, RNU6-9 and their pseudogenes (in black)). (C) ATAC-seq and H3K27ac signals for three RNU4 genes and 105 pseudogenes (D) ATAC-seq and H3K27ac signals for six RNU6 genes and 1,312 pseudogenes. ATAC-seq data is from Wang et al. and H3K27ac data is from Cherry et al.

Figure 5:. RP variants in RNU4-2 and RNU6 inhibit snRNP maturation.

(A) Immunoprecipitation of U4-MS2 (WT and variants) and (B) U6-MS2 (WT and variants). snRNPs were immunoprecipitated via MS2-YFP by anti-GFP antibodies and co-precipitated proteins were detected by Western blotting. The position of the MS2 loop (green) in snRNAs is indicated. Three independent experiments were quantified. Immunoprecipitated proteins are normalized to input and U4 or U6 wild-type controls. Statistical significance was analyzed by the two-tailed unpaired t-test. * indicates P-value ≤0.05, ** ≤0.01, and *** ≤0.001