Studies of nonsense-mediated mRNA decay in mammalian cells have proffered unforeseen insights into changes in mRNA–protein interactions throughout the lifetime of an mRNA. Remarkably, mRNA acquires a complex of proteins at each exon–exon junction during pre-mRNA splicing that influences the subsequent steps of mRNA translation and nonsense-mediated mRNA decay. Complex-loaded mRNA is thought to undergo a pioneer round of translation when still bound by cap-binding proteins CBP80 and CBP20 and poly(A)-binding protein 2. The acquisition and loss of mRNA-associated proteins accompanies the transition from the pioneer round to subsequent rounds of translation, and from translational competence to substrate for nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics

Many metazoan gene transcripts exhibit neuron-specific splicing patterns, but the developmental control of these splicing events is poorly understood. We show that the splicing of a large group of exons is reprogrammed during neuronal development by a switch in expression between two highly similar polypyrimidine tract-binding proteins, PTB and nPTB (neural PTB). PTB is a well-studied regulator of alternative splicing, but nPTB is a closely related paralog whose functional relationship to PTB is unknown. In the brain, nPTB protein is specifically expressed in post-mitotic neurons, whereas PTB is restricted to neuronal precursor cells (NPC), glia, and other nonneuronal cells. Interestingly, nPTB mRNA transcripts are found in NPCs and other nonneuronal cells, but in these cells nPTB protein expression is repressed. This repression is due in part to PTB-induced alternative splicing of nPTB mRNA, leading to nonsense-mediated decay (NMD). However, we find that even properly spliced mRNA fails to express nPTB protein when PTB is present, indicating contributions from additional post-transcriptional mechanisms. The PTB-controlled repression of nPTB results in a mutually exclusive pattern of expression in the brain, where the loss of PTB in maturing neurons allows the synthesis of nPTB in these cells. To examine the consequences of this switch, we used splicing-sensitive microarrays to identify different sets of exons regulated by PTB, nPTB, or both proteins. During neuronal differentiation, the splicing of these exon sets is altered as predicted from the observed changes in PTB and nPTB expression. These data show that the post-transcriptional switch from PTB to nPTB controls a widespread alternative splicing program during neuronal development.

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A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons

It appears that no organism is immune to the effects of nonsense codons on mRNA abundance. The study of how nonsense codons alter RNA metabolism is still at an early stage, and our current understanding derives more from incidental vignettes than from experimental undertakings that address molecular mechanisms. Challenges for the future include identifying the gene products and RNA sequences that function in nonsense mediated RNA loss, resolving the cause and consequences of there apparently being more than one cellular site and mechanism for nonsense-mediated RNA loss, and understanding how these sites and mechanisms are related to both constitutive and specialized pathways of pre-mRNA processing and mRNA decay.

When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells.

Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.

https://www.embopress.org/doi/pdf/10.1093/emboj/17.12.3484

Binary specification of nonsense codons by splicing and cytoplasmic translation

A Regulatory Mechanism That Detects Premature Nonsense Codons in T-cell Receptor Transcripts in Vivo Is Reversed by Protein Synthesis Inhibitors in Vitro

Mutations in the gene encoding alpha-L-iduronidase (IDUA) are the cause of Hurler syndrome. Fibroblasts from patients homozygous for nonsense IDUA alleles have much reduced mRNA detectable by Northern analysis, as has been observed in many other instances of premature translation termination. Yet RT-PCR (reverse transcription followed by PCR amplification) showed a normal level of a segment covering exons 1 and 2 in Hurler cells homozygous for alleles bearing the nonsense mutations, Q70X or W402X. The 3' end of the segment was between exons 2 and 4. The results indicate that the nonsense RNA was degraded to fragment(s), independent of the position of the mutation (exon 2 or exon 9, respectively). Treatment of the cells with cycloheximide resulted in some increase of intact mRNA, suggesting that translation is required for mRNA degradation.

K P Menon

Papers

Evidence for degradation of mRNA encoding alpha-L-iduronidase in Hurler fibroblasts with premature termination alleles.