Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes¹. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome². We identify an 18 base pair region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals in whom it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologues. Using RNA sequencing, we show how 5′ splice-site use is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 base pair region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide.

Despite the low level expression of some long noncoding RNAs (lncRNAs), the differential expression of specific lncRNAs plays important roles during the development of many organisms. Schistosomes, parasitic flatworms that are responsible for schistosomiasis, infects over 200 million people resulting in chronic disease and hundreds of thousands of deaths. Schistosomes have a complex life cycle that transitions between molluscan and mammalian hosts. In a molluscan snail host, the sporocyst stage develops over 5 weeks undergoing asexual reproduction to give rise to free-swimming and infectious cercariae that penetrate human skin and eventually mature into egg producing worms in mammals. The tight integration of the sporocyst to the snail host hepatopancreas hinders the -omics study in the molluscan stage, so the sporocyst transcriptome has only been examined for lncRNAs in immature in vitro samples. Here we analyzed the in vivo mature sporocyst transcriptome to identify 4,930 total lncRNAs between the molluscan and mammalian stages of the parasite. We further demonstrate that the lncRNAs are differentially expressed in a development-dependent manner. In addition, we constructed a co-expression correlation network between lncRNAs and protein-coding (PC) genes that was used to identify clusters of lncRNA transcripts with potential functional relevance. We also describe lncRNA–lncRNA and lncRNA–kinome correlations that identify lncRNAs with prospective roles in gene regulation. Finally, our results show clear differential expression patterns of lncRNAs in host-dependent development stages of S. mansoni and ascribe potential functional roles in development based on predicted intracellular interaction.

Recently, de novo variants in an 18 nucleotide region in the centre of RNU4-2 were shown to cause ReNU syndrome, a syndromic neurodevelopmental disorder (NDD) that is predicted to affect tens of thousands of individuals worldwide ^1,2 . RNU4-2 is a non-protein-coding gene that is transcribed into the U4 small nuclear RNA (snRNA) component of the major spliceosome ³ . ReNU syndrome variants disrupt spliceosome function and alter 5’ splice site selection ^1,4 . Here, we performed saturation genome editing (SGE) of RNU4-2 to identify the functional and clinical impact of variants across the entire gene. The resulting SGE function scores, derived from variants’ effects on cell fitness, discriminate ReNU syndrome variants from those observed in the population and dramatically outperform in silico variant effect prediction. Using these data, we redefine the ReNU syndrome critical region at single nucleotide resolution, resolve variant pathogenicity for variants of uncertain significance, and show that SGE function scores delineate variants by phenotypic severity. Further, we identify variants impacting function in regions of RNU4-2 that are critical for interactions with other spliceosome components. We show that these variants cause a novel recessive NDD that is clinically distinct from ReNU syndrome. Together, this work defines the landscape of variant function across RNU4-2 , providing critical insights for both diagnosis and therapeutic development.

Genetic variants in RNU4-2 , which is transcribed into the U4 small nuclear RNA component of the major spliceosome, were recently shown to cause ReNU syndrome, a prevalent dominant neurodevelopmental disorder (NDD). These variants almost exclusively arise de novo and cluster within 18 nucleotides of RNU4-2 . Here we describe a new recessive NDD associated with homozygous and compound heterozygous variants in RNU4-2 . We identify 38 individuals with biallelic variants outside the 18-nucleotide ReNU syndrome region that cluster within other functionally important elements of U4: Stem II, the k-turn and the Sm protein binding site. We characterize the clinical phenotype in 31 individuals, demonstrating that the recessive disorder is clinically distinct from ReNU syndrome and is associated with distinctive white matter abnormalities, including enlarged perivascular spaces. Finally, we find reduced RNU4-2 transcript levels in individuals with the recessive disorder, suggesting a loss-of-function disease mechanism that is distinct from the mechanism underlying ReNU syndrome. Together, these findings expand the genotypic and phenotypic spectrum of RNU4-2 -associated NDDs.

Recently, de novo variants in an 18-nucleotide region in the centre of RNU4-2 were shown to cause ReNU syndrome, a syndromic neurodevelopmental disorder that is predicted to affect tens of thousands of individuals worldwide ^1,2 . RNU4-2 is a non-protein-coding gene that is transcribed into the U4 small nuclear RNA component of the major spliceosome ³ . ReNU syndrome variants disrupt spliceosome function and alter 5′ splice site selection ^1,4 . Here we performed saturation genome editing (SGE) of RNU4-2 to identify the functional and clinical impact of variants across the entire gene. The resulting SGE function scores, derived from variants’ effects on cell fitness, discriminate ReNU syndrome variants from those observed in the population and markedly outperform in silico variant effect prediction. Using these data, we redefine the ReNU syndrome critical region at single-nucleotide resolution, resolve variant pathogenicity for variants of uncertain significance and show that SGE function scores delineate variants by phenotypic severity and the extent of observed splicing disruption. Furthermore, we identify variants affecting function in regions of RNU4-2 that are critical for interactions with other spliceosome components. We show that these variants cause a new recessive neurodevelopmental disorder that is distinct from ReNU syndrome. Together, this work defines the landscape of variant function across RNU4-2 , providing critical insights for both diagnosis and therapeutic development.

Trypanosoma brucei is a parasitic protist that causes African sleeping sickness. The establishment of T. brucei cell lines has provided a significant advantage for the majority of T. brucei research. However, these cell lines were isolated and maintained in culture for decades, occasionally accumulating changes in gene expression. Since trypanosome strains have been maintained in culture for decades, it is possible that difference may have accumulated in fast-evolving non-coding RNAs between trypanosomes from the wild and those maintained extensively in cultures. To address this, we compared the lncRNA expression profile of trypanosomes maintained as cultured cell lines (CL) to those extracted from human patients, wildtype (WT). We identified lncRNAs from CL and WT from available transcriptomic data and demonstrate that CL and WT have unique sets of lncRNAs expressed. We further demonstrate that the unique and shared lncRNAs are differentially expressed between CL and WT parasites, and that these lncRNAs are more evenly up-regulated and down-regulated than protein-coding genes. We validated the expression of these lncRNAs using qPCR. Taken together, this study demonstrates that lncRNAs are differentially expressed between cell lines and wildtype T. brucei and provides evidence for potential evolution of lncRNAs, specifically in T. brucei maintained in culture.