Small uORFs favor translation re-initiation but do not protect mRNAs from nonsense-mediated decay

It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation re-initiation. However, termination in the 5ʹ UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability. Using custom 5ʹ UTRs and uORF sequences, we show that re-initiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives and mining available mRNA half-life datasets for cumulative uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to evade NMD. Together, these data support a model where uORFs have evolved to balance coding capacity, translational control, and mRNA stability.

Premature Termination Codon in 5′ Region of Desmoplakin and Plakoglobin Genes May Escape Nonsense-Mediated Decay through the Reinitiation of Translation

Arrhythmogenic cardiomyopathy is a heritable heart disease associated with desmosomal mutations, especially premature termination codon (PTC) variants. It is known that PTC triggers the nonsense-mediated decay (NMD) mechanism. It is also accepted that PTC in the last exon escapes NMD; however, the mechanisms involving NMD escaping in 5′-PTC, such as reinitiation of translation, are less known. The main objective of the present study is to evaluate the likelihood that desmosomal genes carrying 5′-PTC will trigger reinitiation. HL1 cell lines were edited by CRISPR/Cas9 to generate isogenic clones carrying 5′-PTC for each of the five desmosomal genes. The genomic context of the ATG in-frame in the 5′ region of desmosomal genes was evaluated by in silico predictions. The expression levels of the edited genes were assessed by Western blot and real-time PCR. Our results indicate that the 5′-PTC in PKP2, DSG2 and DSC2 acts as a null allele with no expression, whereas in the DSP and JUP gene, N-truncated protein is expressed. In concordance with this, the genomic context of the 5′-region of DSP and JUP presents an ATG in-frame with an optimal context for the reinitiation of translation. Thus, 5′-PTC triggers NMD in the PKP2, DSG2* and DSC2 genes, whereas it may escape NMD through the reinitiation of the translation in DSP and JUP genes, with no major effects on ACM-related gene expression.

Intron Retention Coupled with Nonsense-Mediated Decay is Involved in Cellulase Biosynthesis in Cellulolytic Fungi

Background Knowledge on regulatory networks associated with cellulase biosynthesis is prerequisite for exploitation of such regulatory systems in ehancing cellulase production with low cost. The biological functions of intron retention (IR) and nonsense-mediated mRNA decay (NMD) in filamentous fungi is lack of study, let alone their roles in cellulase biosynthesis. Result We found that major cellulase genes (cel7a, cel7b, and cel3a) exhibited concomitant decrease in IR rates and increase in their gene expression in T. reesei under cellulase-producing condition (cellulose and lactose) that was accompanied with a more active NMD pathway, as compared to non cellulase-producing condition (glucose). In the presence of the NMD pathway inhibitor that successfully repressed the NMD pathway, the mRNA levels of cellulase genes were sharply down-regulated, but the rates of IR in these genes were significantly up-regulated. Consistently, the cellulase activities were severely inhibited. In addition, the NMD pathway inhibitor caused the downregulated mRNA levels of two important genes of the target of rapamycin (TOR) pathway, trfkbp12 and trTOR1. The absence of gene trfkbp12 made the cellulase production in T. reesei more sensitive to the NMD pathway inhibitor. Conclusion All these findings suggest that the IR of cellulase genes regulates their own gene expression by coupling with the NMD pathway, which might involve the TOR pathway. Our results provide better understanding on intron retention, the NMD pathway, and cellulase production mechanism in filamentous fungi.

Global SLAM-Seq for accurate mRNA decay determination and identification of NMD targets

Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-Linked Alkylation for the Metabolic Sequencing of RNA (SLAM-Seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5 % of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-Seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the Nonsense-mediated decay (NMD) pathway. There are currently no global reports of half-lives in both wild type and NMD defective yeast cells; instead steady-state RNA level changes are used as a proxy. With SLAM-Seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 230 novel targets, and observe a strong agreement with previous reports of NMD targets, 60 % of our candidates being identified in previous studies. This indicates that SLAM-Seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.

Genetic, parental and lifestyle factors influence telomere length

The average length of telomere repeats (TL) declines with age and is considered to be a marker of biological ageing. Here, we measured TL in six blood cell types from 1,046 individuals using the clinically validated Flow-FISH method. We identified remarkable cell-type-specific variations in TL. Host genetics, environmental, parental and intrinsic factors such as sex, parental age, and smoking are associated to variations in TL. By analysing the genome-wide methylation patterns, we identified that the association of maternal, but not paternal, age to TL is mediated by epigenetics. Coupling these measurements to single-cell RNA-sequencing data for 62 participants revealed differential gene expression in T-cells. Genes negatively associated with TL were enriched for pathways related to translation and nonsense-mediated decay. Altogether, this study addresses cell-type-specific differences in telomere biology and its relation to cell-type-specific gene expression and highlights how perinatal factors play a role in determining TL, on top of genetics and lifestyle.

Hypofibrinogenemia with preserved hemostasis and protection from thrombosis in mice with a Fga truncation mutation

Genetic variants within the fibrinogen Aa-chain encoding the aC-region commonly result in hypodysfibrinogenemia in patients. However, the (patho)physiological consequences and underlying mechanisms of such mutations remain undefined. Here, we generated Fga270 mice carrying a premature termination codon within the Fga gene at residue 271. The Fga270 mutation was compatible with Mendelian inheritance for offspring of heterozygous crosses. Adult Fga270/270 mice were hypofibrinogenemic with ~10% plasma fibrinogen levels relative to FgaWT/WTmice, linked to 90% reduction in hepatic Fga mRNA due to nonsense-mediated decay of the mutant mRNA. Fga270/270 mice had preserved hemostatic potential in vitro and in vivo in models of tail bleeding and laser-induced saphenous vein injury, while Fga-/- mice had continuous bleeding. Platelets from FgaWT/WTandFga270/270 mice displayed comparable initial aggregation following ADP stimulation, but Fga270/270 platelets quickly disaggregated. Despite ~10% plasma fibrinogen, the fibrinogen level in Fga270/270 platelets was ~30% of FgaWT/WT platelets with a compensatory increase in fibronectin. Notably, Fga270/270 mice showed complete protection from thrombosis in the inferior vena cava stasis model. In a model of Staphylococcus aureus peritonitis, Fga270/270 mice supported local, fibrinogen-mediated bacterial clearance and host survival comparable to FgaWT/WT, unlike Fga-/- mice. Decreasing the normal fibrinogen levels to ~10% with siRNA in mice also provided significant protection from venous thrombosis without compromising hemostatic potential and antimicrobial function. These findings both reveal novel molecular mechanisms underpinning fibrinogen aC-region truncation mutations and highlight the concept that selective fibrinogen reduction may be efficacious for limiting thrombosis while preserving hemostatic and immune protective functions.

Identification and Functional Characterization of a Novel Nonsense Variant in ARR3 in a Southern Chinese Family With High Myopia

ARR3 has been associated with X-linked, female-limited, high myopia. However, using exome sequencing (ES), we identified the first high myopia case with hemizygous ARR3-related mutation in a male patient in a Southern Chinese family. This novel truncated mutation (ARR3: c.569C>G, p.S190*) co-segregated with the disease phenotype in affected family members and demonstrated that high myopia caused by ARR3 is not X-linked, female-limited, where a complicated X-linked inheritance pattern may exist. Thus, our case expanded the variant spectrum in ARR3 and provided additional information for genetic counseling, prenatal testing, and diagnosis. Moreover, we characterized the nonsense-mediated decay of the ARR3 mutant mRNA and discussed the possible underlying pathogenic mechanisms.

Integrity and Stability of PTC Bearing CFTR mRNA and Relevance to Future Modulator Therapies in Cystic Fibrosis

Major advances have recently been made in the development and application of CFTR (cystic fibrosis transmembrane conductance regulator) mutation class-specific modulator therapies, but to date, there are no approved modulators for Class I mutations, i.e., those introducing a premature termination codon (PTC) into the CFTR mRNA. Such mutations induce nonsense-mediated decay (NMD), a cellular quality control mechanism that reduces the quantity of PTC bearing mRNAs, presumably to avoid translation of potentially deleterious truncated CFTR proteins. The NMD-mediated reduction of PTC-CFTR mRNA molecules reduces the efficacy of one of the most promising approaches to treatment of such mutations, namely, PTC readthrough therapy, using molecules that induce the incorporation of near-cognate amino acids at the PTC codon, thereby enabling translation of a full-length protein. In this study, we measure the effect of three different PTC mutations on the abundance, integrity, and stability of respective CFTR mRNAs, using CFTR specific RT-qPCR-based assays. Altogether, our data suggest that optimized rescue of PTC mutations has to take into account (1) the different steady-state levels of the CFTR mRNA associated with each specific PTC mutation; (2) differences in abundance between the 3′ and 5′ regions of CFTR mRNA, even following PTC readthrough or NMD inhibition; and (3) variable effects on CFTR mRNA stability for each specific PTC mutation.

Comprehensive Analysis of Combinatorial Pharmacological Treatments to Correct Nonsense Mutations in the CFTR Gene

Cystic fibrosis (CF) is caused by loss of function of the CFTR chloride channel. A substantial number of CF patients carry nonsense mutations in the CFTR gene. These patients cannot directly benefit from pharmacological correctors and potentiators that have been developed for other types of CFTR mutations. We evaluated the efficacy of combinations of drugs targeting at various levels the effects of nonsense mutations: SMG1i to protect CFTR mRNA from nonsense-mediated decay (NMD), G418 and ELX-02 for readthrough, VX-809 and VX-445 to promote protein maturation and function, PTI-428 to enhance CFTR protein synthesis. We found that the extent of rescue and sensitivity to the various agents is largely dependent on the type of mutation, with W1282X and R553X being the mutations most and least sensitive to pharmacological treatments, respectively. In particular, W1282X-CFTR was highly responsive to NMD suppression by SMG1i but also required treatment with VX-445 corrector to show function. In contrast, G542X-CFTR required treatment with readthrough agents and VX-809. Importantly, we never found cooperativity between the NMD inhibitor and readthrough compounds. Our results indicate that treatment of CF patients with nonsense mutations requires a precision medicine approach with the design of specific drug combinations for each mutation.