RESUMEN
Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using a CRISPR-dCas13 system. Using specific guide RNAs, we target dCas13 to the region downstream of canonical stop codons of mammalian AGO1 and VEGFA mRNAs, known to exhibit natural SCR. Readthrough assays reveal enhanced SCR of these mRNAs (both exogenous and endogenous) caused by the dCas13-gRNA complexes. This effect is associated with ribosomal pausing, which has been reported for several SCR events. Our data show that CRISPR-dCas13 can also induce SCR across premature termination codons (PTCs) in the mRNAs of green fluorescent protein and TP53. We demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC in HBB mRNA and hereditary spherocytosis-causing PTC in SPTA1 mRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ARN Guía de Sistemas CRISPR-Cas , Animales , Codón de Terminación/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Codón sin Sentido/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Biosíntesis de Proteínas , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
During translation, a stop codon on the mRNA signals the ribosomes to terminate the process. In certain mRNAs, the termination fails due to the recoding of the canonical stop codon, and ribosomes continue translation to generate C-terminally extended protein. This process, termed stop codon readthrough (SCR), regulates several cellular functions. SCR is driven by elements/factors that act immediately downstream of the stop codon. Here, we have analysed the process of SCR using a simple mathematical model to investigate how the kinetics of translating ribosomes influences the efficiency of SCR. Surprisingly, the analysis revealed that the rate of translation inversely regulates the efficiency of SCR. We tested this prediction experimentally in mammalian AGO1 and MTCH2 mRNAs. Reduction in translation either globally by harringtonine or locally by rare codons caused an increase in the efficiency of SCR. Thus, our study has revealed a hitherto unknown mode of regulation of SCR.
Asunto(s)
Codón de Terminación , Biosíntesis de Proteínas , ARN Mensajero , Ribosomas , Codón de Terminación/genética , Codón de Terminación/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Humanos , Células HEK293 , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismoRESUMEN
It is generally believed that human mature erythrocytes do not possess functional ribosomes and therefore cannot synthesize proteins. However, the absence of translation is not consistent with the long lifespan of mature erythrocytes. They stay viable and functional for about 115 d in the circulatory system. Here, using a highly pure preparation of human mature erythrocytes, we demonstrate the presence of translation by polysome profiling, [35S]methionine labeling, and RiboPuromycylation. [35S]methionine labeling revealed that the translation in mature erythrocytes is about 10% of that observed in reticulocytes. We could observe polysomes by transmission electron microscopy in these cells. RNA-seq and quantitative real-time PCR performed on polysome fractions of these cells revealed that HBA (α-globin) and HBB (ß-globin) transcripts are translated. Using a luciferase-based reporter assay and mutational studies, we show that the sequence of the 5' untranslated region is crucial for the translation of these transcripts. Furthermore, mature erythrocytes showed reduced expression of globin proteins (α- and ß-) when treated with translation inhibitors. Overall, we provide multiple lines of evidence for translation of globin mRNAs in human mature erythrocytes.
Asunto(s)
Eritrocitos , Globinas beta , Regiones no Traducidas 5' , Eritrocitos/metabolismo , Humanos , Metionina/metabolismo , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Globinas alfa/metabolismo , Globinas beta/genética , Globinas beta/metabolismoRESUMEN
Nonsense mutations that result in premature stop codons in the HBB gene cause ß-thalassemia. This disease is characterized by a reduced hemoglobin level due to the lack of ß-globin. Compounds that induce translational readthrough across the thalassemia-causing premature stop codon will have therapeutic benefits. Currently available molecules that induce translational readthrough lack specificity, and some of them show toxicity after prolonged use. In this study, we have developed an oligonucleotide-based approach to induce translational readthrough across the thalassemia-causing premature stop codon. Oligonucleotides that target HBB mRNA downstream of the premature stop codon could induce translational readthrough, generating a full-length ß-globin protein. We show this effect using fluorescence- and luminescence-based readthrough assays and by Western blot. Remarkably, the amount of oligonucleotide-induced translational readthrough product is comparable to that of the protein generated by normal translation when there was no premature stop codon. Thus, these oligonucleotides, with certain modifications, have the potential to be used as drugs for the treatment of ß-thalassemia. Also, this strategy can be extended to treat other genetic diseases caused by premature stop codons.
