The Coupling of Alternative Splicing and Nonsense-Mediated mRNA Decay

Most human genes exhibit alternative splicing, but not all alternatively spliced transcripts produce functional proteins. Computational and experimental results indicate that a substantial fraction of alternative splicing events in humans result in mRNA isoforms that harbor a premature termination codon (PTC). These transcripts are predicted to be degraded by the nonsense-mediated mRNA decay (NMD) pathway. One explanation for the abundance of PTC-containing isoforms is that they represent splicing errors that are identified and degraded by the NMD pathway. Another potential explanation for this startling observation is that cells may link alternative splicing and NMD to regulate the abundance of mRNA transcripts. This mechanism, which we call "Regulated Unproductive Splicing and Translation" (RUST), has been experimentally shown to regulate expression of a wide variety of genes in many organisms from yeast to human. It is frequently employed for autoregulation of proteins that affect the splicing process itself. Thus, alternative splicing and NMD act together to play an important role in regulating gene expression

Intranuclear degradation of nonsense codon-containing mRNA

Most vertebrate mRNAs with premature termination codons (PTCs) are specifically recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD) while still associated with the nucleus. However, it is still a matter of debate whether PTCs can be identified by intranuclear scanning or only by ribosomes on the cytoplasmic side of the nuclear envelope. Here we show that inhibition of mRNA export by two independent approaches does not affect the downregulation of PTC-containing T-cell receptor β transcripts in the nuclear fraction of mammalian cells, providing strong evidence for intranuclear NMD. Our results are fully consistent with recently reported evidence for nuclear translation and suggest that an important biological role for nuclear ribosomes is the early elimination of nonsense mRNA during a pioneer round of translation

Boundary-independent polar nonsense-mediated decay

Nonsense-mediated decay (NMD) is an RNA surveillance mechanism that degrades mRNAs containing premature termination (nonsense) codons. The second signal for this pathway in mammalian cells is an intron that must be at least ∼55 nucleotides downstream of the nonsense codon. Although the functional significance of this ‘–55 boundary rule’ is not known, it is widely thought to reflect the important role of an exon junction protein complex deposited just upstream of exon–exon junctions after RNA splicing. Here we report that a T-cell receptor (TCR)-β gene did not conform to this rule. Rather than a definitive boundary position, nonsense codons had a polar effect, such that nonsense codons distant from the terminal downstream intron triggered robust NMD and proximal nonsense codons caused modest NMD. We identified a region of the TCR-β gene that conferred this boundary-independent polar expression pattern on a heterologous gene. Collectively, our results suggest that TCR-β transcripts contain one or more sequence elements that elicit an unusual NMD response triggered by a novel second signal that ultimately causes boundary-independent polar regulation. TCR genes may have evolved this unique NMD response because they frequently acquire nonsense codons during normal development

Human MRE11 is inactivated in mismatch repair-deficient cancers

Mutations of the ATM and NBS1 genes are responsible for the inherited Ataxia-Telangiectasia and Nijmegen Breakage Syndrome, both of which are associated with a predisposition to cancer. A related syndrome, the Ataxia-Telangiectasia-like disorder, is due to mutations of the MRE11 gene. However, the role of this gene in cancer development has not been established. Here we describe an often homozygous mutation of the poly(T)11 repeat within human MRE11 intron 4 that leads to aberrant splicing, impairment of wild-type MRE11 expression and generation of a truncated protein. This mutation is present in mismatch repair-deficient, but not proficient, colorectal cancer cell lines and primary tumours and is associated with reduced expression of the MRE11–NBS1–RAD50 complex, an impaired S-phase checkpoint and abrogation of MRE11 and NBS1 ionizing radiation-induced nuclear foci. Our findings identify MRE11 as a novel and major target for inactivation in mismatch repair-defective cells and suggest its impairment may contribute to the development of colorectal cancer

Turnover of primary transcripts is a major step in the regulation of mouse H19 gene expression

In the gene expression pathway, RNA biogenesis is a central multi-step process where both message fidelity and steady-state levels of the mature RNA have to be ascertained. An emerging question is whether RNA levels could be regulated at the precursor stage. Until recently, because it was technically very difficult to determine the level of a pre-mRNA, discrimination between changes in transcriptional activity and in pre-mRNA metabolism was extremely difficult. H19 RNA, the untranslated product of an imprinted gene, undergoes post-transcriptional regulation. Here, using a quantitative real-time RT–PCR approach, we accurately quantify its precursor RNA levels and compare these with the transcriptional activity of the gene, assessed by run-on assays. We find that the levels of H19 precursor RNA are regulated during physiological processes and this regulation appears to be related to RNA polymerase II transcription termination. Our results provide direct evidence that turnover of polymerase II primary transcripts can regulate gene expression in mammals