Chemistry of Life

Molecular genetics, biochemistry

nonsense-mediated decay

Nonsense-mediated mRNA decay (NMD) or mRNA surveillance is an RNA decay pathway of eukaryotic cells that most probably evolved to protect cells from potentially deleterious proteins produced by inevitable errors in gene expression and to fine-tune the translational expression of thousands of mRNAs.

AREs : AU-rich elements : CBPs : co-translational export model : EJC, exon junction complex : miRNAs : NMD genes : nuclear scanning model : premature termination codon, PTC+ : unstable mRNAs : Upf : ~50-55 nt rule :

In addition to decay of mRNA, NMD participates in protecting organisms from stresses. Starvation inhibits NMD and causes increased levels of RNAs and proteins that directly contribute to survival through more efficient use of available nutrients. Cellular stresses that induce the 'unfolded protein response' also inhibit NMD, providing a mechanism to promote the selective expression of factors that contribute to survival. Complete loss of NMD is incompatible with embryonic viability.[s]

Mutations in any of seven genes, smg-1 through smg-7, in C. elegans eliminate nonsense-mediated mRNA decay, stabilizing nonsense mutant mRNAs. Corresponding genes are Upf1, Upf2/Nmd2, Upf3 and Hrp1/Nab4 in S. cerevisiae, and the human orthologs are hUpf1, hUpf2, hUpf3a/hUpf3b.

The NMD pathways (DcpS, hDcp2) degrade two types of mRNA:
1) mRNAs that contain premature termination codons (PTC+ mRNAs), which arise either by transcription of genes containing nonsense or frameshift mutations, or by errors in transcription or processing
2) wild-type mRNAs (PTC-)

Levels of mRNAs that contain premature termination codons (PTC+) are generally 100 fold lower than those of wild-type mRNAs (PTC-) because of selective degradation of PTC+ transcripts by nonsense-mediated mRNA decay. NMD is associated with the nucleus despite the fact that ribosomal machinery is cytoplasmic. It has been believed that only cytoplasmic ribosomes are capable of detecting and binding to open reading frames, so it has been proposed that NMD degrades PTC+ mRNAs outside of the nuclear envelope while still associated with the nuclear pore complex ("co-translational export model"). According to this model, the reduced PTC+ mRNA levels are detected in the nuclear fraction even though the process of NMD is actually cytoplasmic. However, NMD is not affected by inhibition of mRNA export. The alternative model ("nuclear scanning model") proposes that the premature termination codon is recognized by a nuclear frame reader (probably a nuclear ribosome) prior to mRNA export from the nucleus. This model is supported by evidence for translation inside the nucleus of mammalian cells (Iborra et al., J Cell Sci. 2004 PM). [] diagram [] diagram2 [] PTC-dependent accumulation of unspliced immunoglobulin m and TCR-b pre-mRNA at the transcription locus further suggests intranuclear recognition of PTCs (Muhlemann et al., Mol Cell. 2001 Jul;8(1):33-43. PM).

Errors of gene expression often generate mRNAs with reading frames upstream of the normal reading frame, or frameshift mutations that generate nonsense codons, or nonsense mutations. Approximately 30% of genetic diseases result from frameshift and nonsense mutations that cause premature termination of translation. NMD affects transcripts of the majority of mammalian genes that prematurely terminate translation more than ~50-55 nt upstream of the final exon-exon junction.

NMD also down-regulates a number of naturally occurring transcripts, including alternatively spliced RNAs and selenoprotein mRNAs. In mammalian cells, errors include inaccuracies in transcription initiation or in pre-mRNA splicing, and ineffective somatic DNA rearrangements of genes for immunoglobulins and T-cell receptor proteins.

Nuclear pre-mRNA splicing impacts cytoplasmic mRNA translation by influencing mRNP structure when a complex of proteins is deposited on mRNA immediately upstream of exon-exon junctions. The complex comprises Upf3 or Upf3X, which is a mostly nuclear shuttling protein involved in NMD. The Upf3- or Upf3X-bound complex next recruits Upf2, which is a perinuclear, cytoplasmic protein that ultimately recruits cytoplasmic Upf1. EJC-associated. The ~50-55 nt rule reflects the fact that Upf1 elicits NMD if translation terminates more than ~50-55 nt upstream of an exon-exon junction that is marked by the Upf proteins. However, the mRNA is immune to NMD and translating ribosomes remove all exon junction complexes whenever translation terminates less than ~50-55 nt upstream of or downstream of the 3’-most exon-exon junction. [] diagram ~50-55 nt rule [] model NMD []

The substrate for NMD is mRNA bound by the cap binding protein (CBP) heterodimer CBP80 and CBP20. CBP80/20-bound mRNA undergoes a “pioneer” round of translation, during which nonsense codon recognition can lead to NMD. Whenever CBP80/20 has been replaced by eukaryotic initiation factor (eIF) 4E, the mRNA is immune to NMD. Because NMD targets CBP80/20-bound mRNA but not eIF4E-bound mRNA, the Upf-containing exon junction complexes are detected only on CBP80/20-bound mRNA. CBP80 promotes NMD by interacting directly with Upf1 and promoting the interaction of Upf1 with Upf2. [] diagram []

Many of the most powerful biological regulators of cell growth and proliferation are encoded by unstable mRNAs, which are targeted for rapid degradation by the cell. Messenger RNAs such as c-myc, c-fos and GM-CSF encode protein products that influence proliferation and differentiation. Their mRNAs are relatively unstable, with half-lives of an hour or less, so small changes in their turnover rates affect their steady-state levels over a relatively short time period. Short-term regulation maintains the concentrations of these mRNAs within a limited range, and precise regulation is necessary because inappropriate expression of such genes interferes with proliferation and differentiation.

The loss of rapid degradation of these growth-promoting mRNAs can result in oncogenic transformation of the cell. Rapid mRNA decay is attributable to cis-acting elements present in labile mRNAs. Many mRNAs encoding cytokines, G protein-coupled receptors, and oncoproteins are labile by virtue of the presence of A+U-rich elements (AREs) in their 3'-UTRs. Targeted degradation of proto-oncogene and transcription factor mRNAs, and short-lived interleukins and cytokines is controlled both by an AU-rich element (ARE) located in the 3' noncoding region, and by several proteins that bind the ARE sequence.

These ARE-binding proteins, Dicer1 and Argonaute (Ago)-family proteins Ago1 and Ago2, are all involved in miRNA processing. Activation of the ARE for mRNA decay involves cotranslation of the mRNA by ribosomes, and employs the ubiquitin-proteasome pathway.[s] MicroRNAs (miRNAs) participate in ARE-mediated mRNA decay. Human miR-15a and miR16 are both deleted or down-regulated in more than 66% of cases of B cell chronic lymphocytic leukemias (CLL) miR16 contains eight bases that are complementary to ARE, and its destruction increases the stability of ARE-RNA. Tristetraproline (TTP) is an ARE-binding protein that promotes ARE-RNA destabilization, and both miR16 and TTP are required to mediate ARE-RNA decay. TTP contains two CCCH-type zinc finger domains that are both necessary and sufficient for ARE binding. BRF-1 and BRF-2 are two paralogs of TTP that are involved in ARE-mediated decay. Ago proteins and miR16 are both thought to form part of the RNA-induced silencing complex (RISC). TTP interacts with RISC to facilitate or stabilize miR16 targeting of ARE, leading to mRNA decay.[s]

Defusing Dangerous Mutations: Scientists Discover A New Way By Which Cells Control Genetic Errors: Adapted: "Nonsense-Mediated Decay (NMD), is a process by which cells destroy potentially harmful molecules. Both healthy and damaged proteins begin as instructions in genes. Cells transcribe and process this information to create a mRNA molecule, a template that will be used to create proteins.

pre-mRNAs usually contain extra bits of code that have to be cut out before they can be used. During this cut-and-paste operation, cells attach a group of molecules called the exon junction complex (EJC) to the pre-mRNA. A pre-mRNA made from a mutant gene usually has an EJC in the wrong position, which activates NMD and destroys the pre-mRNA before it can be used to make flawed proteins. There are at least two kinds of NMD: one requires UPF2 and the other does not.

The presence or absence of UPF2 changes the composition of the EJC, giving it different surfaces to which other molecules attach. This affects the way that another component, called UPF1, fits onto the machine. UPF1 is directly responsible for calling up the NMD machinery. The study shows that UPF1 can be mounted on both EJC types; the final effect is the same – to efficiently destroy faulty pre-mRNAs. "

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway that ensures the rapid degradation of mRNAs containing premature translation termination codons (PTCs), thereby preventing the synthesis of truncated and potentially harmful proteins. In addition, this pathway regulates the expression of approximately 10% of the transcriptome and is essential in mice. Although NMD is conserved in eukaryotes, recent studies in several organisms have revealed that different mechanisms have evolved to discriminate natural from premature stop codons and to degrade the targeted mRNAs. With the elucidation of the first crystal structures of components of the NMD machinery, the way is paved towards a molecular understanding of the protein interaction network underlying this process. Conti E, Izaurralde E. Nonsense-mediated mRNA decay: molecular insights and mechanistic variations across species. Curr Opin Cell Biol. 2005 Jun;17(3):316-25.

Messenger RNAs containing premature termination codons (PTCs) are selectively eliminated by nonsense-mediated mRNA decay (NMD). Paradoxically, although cytoplasmic ribosomes are the only known species capable of PTC recognition, in mammals many PTC-containing mRNAs are apparently eliminated prior to release from the nucleus. To determine whether PTCs can influence events within the nucleus proper, we studied the immunoglobulin (Ig)-mu and T cell receptor (TCR)-beta genes using fluorescent in situ hybridization (FISH). Alleles containing PTCs, but not those containing a missense mutation or a frameshift followed by frame-correcting mutations, exhibited elevated levels of pre-mRNA, which accumulated at or near the site of transcription. Our data indicate that mRNA reading frame can influence events at or near the site of gene transcription. Muhlemann O, Mock-Casagrande CS, Wang J, Li S, Custodio N,
Carmo-Fonseca M, Wilkinson MF, Moore MJ. Precursor RNAs harboring nonsense codons accumulate near the site of transcription. Mol Cell. 2001 Jul;8(1):33-43.

Although it is frequently assumed that translation does not occur in eukaryotic nuclei, recent evidence suggests that some translation can take place and that it is closely coupled to transcription. The first evidence concerns the destruction of nuclear mRNAs containing premature termination codons by nonsense-mediated decay (NMD). Only ribosomes can detect termination codons, and as some NMD occurs within the nuclear fraction, active nuclear ribosomes could perform the required detection. The second evidence is the demonstration that tagged amino acids are incorporated into nascent polypeptides in a nuclear process coupled to transcription. The third evidence is that components involved in translation, NMD and transcription colocalize, coimmunoprecipitate and co-purify. All these results are simply explained if nuclear ribosomes scan nascent transcripts for premature termination codons at the site of transcription. Alternatively, the scanning needed for NMD might take place at the nuclear membrane, and contaminating cytoplasmic ribosomes might give the appearance of some nuclear translation. We argue, however, that the balance of evidence favours bona fide nuclear translation. Iborra FJ, Jackson DA, Cook PR. The case for nuclear translation. (Free Full Text Article) J Cell Sci. 2004 Nov 15;117(Pt 24):5713-20.

AREs : CBPs : co-translational export model : EJC, exon junction complex : miRNAs : NMD genes : nuclear scanning model : premature termination codon, PTC+ : unstable mRNAs : Upf : ~50-55 nt rule : ~ capping ~ cellular stress response ~ cytokines ~ decay ~ gene regulation ~ immunoglobulins ~ nonstop decay ~ polyadenylation ~

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