Upstream open reading frames (uORFs) are tissue-specific cis-regulators of protein translation. Isolated reports have shown that variants that create or disrupt uORFs can cause disease. Here, in a systematic genome-wide study using 15,708 whole genome sequences, we show that variants that create new upstream start codons, and variants disrupting stop sites of existing uORFs, are under strong negative selection. This selection signal is significantly stronger for variants arising upstream of genes intolerant to loss-of-function variants. Furthermore, variants creating uORFs that overlap the coding sequence show signals of selection equivalent to coding missense variants. Finally, we identify specific genes where modification of uORFs likely represents an important disease mechanism, and report a novel uORF frameshift variant upstream of NF2 in neurofibromatosis. Our results highlight uORFperturbing variants as an under-recognised functional class that contribute to penetrant human disease, and demonstrate the power of large-scale population sequencing data in studying non-coding variant classes.

Background: Titin (TTN), the largest protein in humans, forms the molecular spring that spans half of the sarcomere to provide passive elasticity to the cardiomyocyte. Mutations that disrupt the TTN transcript are the most frequent cause of hereditary heart failure. We showed before that TTN produces a class of circular RNAs (circRNAs) that depend on RBM20 to be formed. In this study we show that the backsplice junction formed by this class of circRNAs creates a unique motif, which binds SRSF10 to enable it to regulate splicing. Furthermore, we show that one of these circRNAs (cTTN1) distorts both localization of and splicing by RBM20. Methods: We calculated genetic constraint of the identified motif in 125.748 exomes collected from the gnomAD database. Furthermore, we focused on the highest expressed RBM20-dependent circRNA in the human heart, which we named cTTN1. We used shRNAs directed to the backsplice junction to induce selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Results: Human genetics suggests reduced genetic tolerance of the generated motif, indicating that mutations in this motif might lead to disease. RNA immunoprecipitation confirmed binding of circRNAs with this motif to SRSF10. Selective loss of cTTN1 in hiPSC-CM induced structural abnormalities, apoptosis and reduced contractile force in engineered heart tissue. In line with its SRSF10 binding, loss of cTTN1 caused abnormal splicing of important cardiomyocyte SRSF10 targets like MEF2A and CASQ2 . Strikingly, loss of cTTN1 also caused abnormal splicing of TTN itself. Mechanistically, we show that loss of cTTN1 distorts both localization of and splicing by RBM20. Conclusions: We demonstrate that circRNAs formed from the TTN transcript are essential for normal splicing of key muscle genes by enabling splice regulators RBM20 and SRSF10. This shows that the TTN transcript also has regulatory roles, besides its well-known signaling and structural function. In addition, we demonstrate that the specific sequence created by the backsplice junction of these circRNAs has important functions. This highlights the existence of functionally important sequences that cannot be recognized as such in the human genome, but provides a yet unrecognized source for functional sequence variation.