ABSTRACT
De novo mutations cause a variety of neurodevelopmental disorders including autism. Recent whole genome sequencing from individuals with autism has shown that many de novo mutations also occur in untranslated regions (UTRs) of genes, but it is difficult to predict from sequence alone which mutations are functional, let alone causal. Therefore, we developed a high throughput assay to screen the transcriptional and translational effects of 997 variants from 5'UTR patient mutations. This assay successfully enriched for elements that alter reporter translation, identifying over 100 potentially functional mutations from probands. Studies in patient-derived cell lines further confirmed that these mutations can alter protein production in individuals with autism, and some variants fall in genes known to cause syndromic forms of autism, suggesting a diagnosis for these individual patients. Since UTR function varies by cell type, we further optimized this high throughput assay to enable assessment of mutations in neurons in vivo. First, comparing in cellulo to in vivo results, we demonstrate neurons have different principles of regulation by 5'UTRs, consistent with a more robust mechanism for reducing the impact of RNA secondary structure. Finally, we discovered patient mutations specifically altering the translational activity of additional known syndromic genes LRRC4 and ZNF644 in neurons of the brain. Overall our results highlight a new approach for assessing the impact of 5'UTR mutations across cell types and suggest that some cases of neurodevelopmental disorder may be caused by such variants.
ABSTRACT
Gene expression is regulated at multiple levels in eukaryotic cells. Regulation at the post-transcriptional level is modulated by various trans-acting factors that bind to specific sequences in the messenger RNA (mRNA). The binding of different trans factors influences various aspects of the mRNA such as degradation rate, translation efficiency, splicing, localization, etc. MicroRNAs (miRNAs) are short endogenous ncRNAs that combine with the Argonaute to form the microRNA-induced silencing complex (miRISC), which uses base-pair complementation to silence the target transcript. RNA-binding proteins (RBPs) contribute to post-transcriptional control by influencing the mRNA stability and translation upon binding to cis-elements within the mRNA transcript. RBPs have been shown to impact gene expression through influencing the miRISC biogenesis, composition, or miRISC-mRNA target interaction. While there is clear evidence that those interactions between RBPs, miRNAs, miRISC and target mRNAs influence the efficiency of miRISC-mediated gene silencing, the exact mechanism for most of them remains unclear. This review summarizes our current knowledge on gene expression regulation through interactions of miRNAs and RBPs.
ABSTRACT
Eukaryotic translation initiation factor 6 (eIF6) is essential for the synthesis of 60S ribosomal subunits and for regulating the association of 60S and 40S subunits. A mechanistic understanding of how eIF6 modulates translation in response to stress, specifically starvation-induced stress, is lacking. We here show a novel mode of eIF6 regulation by glycogen synthase kinase 3 (GSK3) that is predominantly active in response to serum starvation. Both GSK3α and GSK3ß phosphorylate human eIF6. Multiple residues in the C terminus of eIF6 are phosphorylated by GSK3 in a sequential manner. In response to serum starvation, eIF6 accumulates in the cytoplasm, and this altered localization depends on phosphorylation by GSK3. Disruption of eIF6 phosphorylation exacerbates the translation inhibitory response to serum starvation and stalls cell growth. These results suggest that eIF6 regulation by GSK3 contributes to the attenuation of global protein synthesis that is critical for adaptation to starvation-induced stress.
