Nonsense mutations in close proximity to the initiation codon fail to trigger full nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs containing premature translation termination codons. In mammalian cells, a termination codon is ordinarily recognized as "premature" if it is located greater than 50-54 nucleotides 5' to the final exon-exon junction. We have described a set of naturally occurring human beta-globin gene mutations that apparently contradict this rule. The corresponding beta-thalassemia genes contain nonsense mutations within exon 1, and yet their encoded mRNAs accumulate to levels approaching wild-type beta-globin (beta(WT)) mRNA. In the present report we demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are intermediate between beta(WT) mRNA and beta-globin mRNA carrying a prototype NMD-sensitive mutation in exon 2 (codon 39 nonsense; beta 39). Functional analyses of these mRNAs with 5'-proximal nonsense mutations demonstrate that their relative resistance to NMD does not reflect abnormal RNA splicing or translation re-initiation and is independent of promoter identity and erythroid specificity. Instead, the proximity of the nonsense codon to the translation initiation AUG constitutes a major determinant of NMD. Positioning a termination mutation at the 5' terminus of the coding region blunts mRNA destabilization, and this effect is dominant to the "50-54 nt boundary rule." These observations impact on current models of NMD.     

An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3'-->5' degradation

Eukaryotic mRNAs containing premature termination codons are subjected to accelerated turnover, known as nonsense-mediated decay (NMD). Recognition of translation termination events as premature requires a surveillance complex, which includes the RNA helicase Upf1p. In Saccharomyces cerevisiae, NMD provokes rapid decapping followed by 5'-->3' exonucleolytic decay. Here we report an alternative, decapping-independent NMD pathway involving deadenylation and subsequent 3'-->5' exonucleolytic decay. Accelerated turnover via this pathway required Upf1p and was blocked by the translation inhibitor cycloheximide. Degradation of the deadenylated mRNA required the Rrp4p and Ski7p components of the cytoplasmic exosome complex, as well as the putative RNA helicase Ski2p. We conclude that recognition of NMD substrates by the Upf surveillance complex can target mRNAs to rapid deadenylation and exosome-mediated degradation.     

Context analysis of termination codons in mRNA that are recognized by plant NMD

The nonsense-mediated mRNA decay (NMD) system is an RNA surveillance system that degrades mRNAs possessing premature translation termination codons (PTCs). Although NMD factors are well conserved in eukaryotes, it is speculated that the contexts of those termination codons that are subject to NMD are different depending on the organism. Context analysis of termination codons that are recognized by the plant NMD system would clarify NMD target mRNAs in plants, and contribute to our understanding of its biological relevance in plants. In the present study we analyzed the positions of termination codons that were recognized as PTCs using an Agrobacterium transient expression assay, i.e. the accumulation of a series of plant mRNAs with nonsense mutations in different contexts was tested in plants. The results indicated that termination codons that are located distant from the mRNA 3' termini or >50 nucleotides upstream of the 3'-most exon-exon junction are recognized as substrates for NMD.     

Suppression of the acuH13 and acuH31 nonsense mutations in the carnitine/acylcarnitine translocase (acuH) gene of Aspergillus nidulans by the G265S substitution in the domain 2 of the release factor eRF1

A search for suppressors of the carnitine/acylcarnitine translocase (CACT) deficiency in Aspergillus nidulans permitted the identification of the suaE7 mutation, mapping at a new translational suppressor (suaE) gene. The suaE gene is essential in A. nidulans and encodes the eukaryotic release factor 1 (eRF1). The suaE7 mutation suppresses two acuH alleles (acuH13 and acuH31), both carrying nonsense mutations in the CACT encoding gene that involve the replacement of a CAG (Gln) codon with a premature TAG stop codon. In contrast, the suaE7 gene does not suppress the acuH20 amber nonsense mutation involving a TGG-->TAG change. The phenotype associated to the suaE7 mutation strictly resembles that of mutants at the suaA and suaC genes, two translational suppressor genes previously identified, suggesting that their gene products might functionally interact in translation termination. Sequencing of the suaE7 gene allowed the identification of a mutation in the domain 2 of the omnipotent class-1 eukaryotic release factor involving the Gly265Ser substitution in the A. nidulans eRF1. This mutation creates a structural context unfavourable for normal eRF binding that allows the misreading of stop codons by natural suppressor tRNAs, such as the tRNAs(Gln). Structural analysis using molecular modelling of A. nidulans eRF1 domain 2 bearing the G265S substitution and computer simulation results suggest that this mutation might impair the necessary conformational changes in the eRF1 to optimally recognize the stop codon and simultaneously interact with the peptidyl transferase centre of the 60S ribosomal subunit.     

蓝氏贾第虫无义mRNA的降解

无义介导的mRNA降解途径（nonsense-mediated mRNA decay,NMD）作为细胞内的一种重要的mRNA质量监控机制,可以降解含有提前终止密码子（premature termination codon,PTC）的异常转录本,从而避免截短蛋白质对细胞的毒害,但其详细的分子机制有待进一步阐释。蓝氏贾第虫（Giardia lamblia)作为一种寄生性单细胞原生动物,进化地位特殊,对其NMD途径的研究有利于阐明基因表达调控的分子和进化机制。本研究通过酵母双杂交及体外pull-down实验分析了贾第虫NMD途径因子上游移码蛋白1(Giardia lamblia up-frameshift 1,GlUPF1)、贾第虫RNA结合蛋白（Giardia lamblia HRP1, GlHRP1）、贾第虫核糖核酸外切酶（Giardia lamblia Ski7p,GlSki7p、Giardia lamblia XRN1,GlXRN1）之间的相互作用关系。结果表明,GlUPF1全长与GlHRP1、GlXRN1(1~500 aa)、GlSki7p间均可发生相互作用。而且GlUPF1的CH结构域和C端结构域分别与GlHRP1、GlXRN1（1~500 aa）、GlSki7p相互作用。说明GlUPF1在贾第虫NMD途径中作为招募平台,在无义mRNA识别和降解过程中发挥重要作用。为此,结合本实验室之前的研究结果,我们提出原生动物贾第虫的NMD途径：在提前终止密码子处SURF(SMG1-UPF1-eRF1-eRF3)复合物形成后,GlUPF1被磷脂酰肌醇3-激酶(suppressor with morphogenetic effect on genitalia 1,SMG1)磷酸化修饰, NMD途径激活,随后GlUPF1与HRP1相互作用,将转录本标记为NMD底物;GlUPF1进而招募下游贾第虫5′-3′核糖核酸降解酶GlXRN1、贾第虫3′-5′ 核糖核酸降解因子GlSki7p,最终降解靶标mRNA。