Applying nonsense-mediated mRNA decay research to the clinic: progress and challenges

doi:10.1016/j.molmed.2006.05.005

Trends in Molecular Medicine

Volume 12, Issue 7, July 2006, Pages 306-316

https://doi.org/10.1016/j.molmed.2006.05.005 Get rights and content

Premature termination codons (PTCs) are equivalent to nonsense sequences. They encode no amino acid, and their presence precludes the synthesis of full-length proteins. Furthermore, the resulting truncated proteins, if synthesized and stable, are likely to be non-functional or might even be deleterious to cellular metabolism. Approximately one third of genetic and acquired diseases are due to PTCs. In fact, PTCs are apt to cause at least some cases of all diseases that involve protein insufficiency. Cells have evolved a way to eliminate mRNAs that contain PTCs using a mechanism called nonsense-mediated mRNA decay (NMD). Here, we will review how to determine which PTCs elicit NMD, what is currently known about the mechanism of NMD, and additional information that is pertinent to establishing therapies for PTC-associated diseases.

Section snippets

Germline and somatic-cell origins of premature termination codons

Most human genes consist of exons – expressed sequences – that are interrupted by introns – intervening sequences (Figure 1). RNA polymerase transcribes genes into pre-messenger (pre-m)RNA in a co-linear fashion. Pre-mRNA is processed both co-transcriptionally and post-transcriptionally to form mRNA, which functions during translation as the template for protein synthesis. In 88% of pre-mRNAs that derive from intron-containing genes [1], the introns are removed by a complex series of reactions

Why NMD?

NMD is ubiquitous among the eukaryotes that have been examined 9, 10, 11, 12, 13, 14, 15. This, in itself, suggests that NMD has a crucial role in regulating gene expression. In fact, certain normal transcripts are targeted for NMD, indicating that NMD is required for a proper level of protein production from some genes. Results of microarray analyses have revealed that transcripts from ∼9% of human HeLa-cell genes are upregulated upon downregulation of UPF1, which is a protein required for NMD

NMD can prevent autosomal recessive disease: how PTCs trigger NMD

As a rule, only those PTCs that are located more than 50–55 nucleotides upstream of a splicing-generated exon–exon junction within mRNA elicit NMD [25] (Figure 2). However, because there are exceptions to this rule 12, 21, 26, and exceptions are not usually predictable from mRNA sequence alone, it is advisable to confirm that an mRNA is an NMD target by quantitating the level of nonsense-containing mRNA relative to nonsense-free mRNA whenever possible. The importance of the exon–exon junction

NMD occurs as a consequence of a pioneer round of translation

It has long been known that NMD in mammalian cells does not target steady-state mRNAs. Instead, NMD is restricted to newly synthesized mRNAs that, in the case of most transcripts that have been examined, have yet to be released from an association with nuclei into the cytoplasm 2, 12. Although a conundrum initially because there was no compelling evidence that nonsense-codon recognition occurs within nuclei [34], restriction of NMD to newly synthesized, nucleus-associated mRNA now makes sense.

Therapies of nonsense-associated diseases

Most nonsense-containing alleles produce mRNA that is targeted for NMD. The majority of nonsense-associated diseases result from insufficient levels of full-length protein. Therefore, nonsense-associated diseases are generally due to two defects in gene expression: degradation of the newly synthesized PTC-containing mRNA by NMD and failure of the abnormally low level of PTC-containing mRNA that escaped NMD to generate full-length and, thus, functional protein. As noted above, NMD is rarely 100%

Suppression of in-frame nonsense codons

Of the three nonsense codons, UAA codons are generally the most effective and UGA codons are the least effective at directing translation termination 39, 40. Furthermore, although nucleotide context both upstream and downstream of a nonsense codon influences termination efficiency, the first nucleotide downstream seems to be the most important [41]. The efficiency of termination is also dictated by cellular constituents of the translational machinery [42], some of which will be more amenable to

Suppression of out-of-frame nonsense codons

As noted earlier, out-of-frame nonsense codons are due to frameshift mutations. The function of genes carrying out-of-frame nonsense codons can be restored either by direct repair of the frameshift mutation or by introducing a compensating frameshift mutation that restores translation termination to the normal site, provided that the frameshifts do not preclude the production of functional protein.

Errors in pre-mRNA splicing that generate frameshifts or other types of deleterious insertions or

Upregulating a functional substitute for the protein encoded by a PTC-containing allele

Finding a cellular protein that can functionally substitute for the protein encoded by a nonsense-containing allele offers another possible therapeutic opportunity (Figure 3e) (Table 1). Such is the hope for DMD, where upregulation of the cellular level of utrophin might compensate for the deficiency in dystrophin because the two proteins seem to be functionally redundant [57]. Advantages of this approach are that it would apply to all DMD patients, regardless of their specific genetic defect,

Suppressing steps during the pioneer round of translation before nonsense codon recognition

As described earlier, NMD is a consequence of nonsense codon recognition during a pioneer round of translation. Therefore, any process that inhibits NMD and enables a nonsense-containing mRNA to be remodeled to become immune to NMD will result in higher levels of that mRNA that can then be targeted for drug-mediated nonsense codon suppression during subsequent rounds of steady-state translation.

Global cellular NMD has been inhibited by inactivating specific NMD factors. For example, wortmannin

Concluding remarks

NMD is a mechanism evolved by cells that functions to eliminate mRNAs containing PTCs and, thus, minimizes production of potentially deleterious truncated proteins. Nonsense-associated diseases are caused by insufficient levels of full-length protein. This is because newly synthesized, PTC-containing mRNAs are degraded, and those that escape decay (because NMD is not 100% efficient) are not translated into full-length proteins. Here, we have provided an overview of possible therapies for

Acknowledgements

The authors were supported by NIH R01 grants GM 05961 and GM 074593 to L.E.M.

Glossary

Antisense oligonucleotides (AOs):: oligomers that anneal to the cellular transcript to be therapeutically modified. These oligomers can target either pre-mRNA to suppress or redirect splicing or, at least in theory, mRNA to disrupt the connection between translation termination and a downstream EJC.
Cap binding protein (CBP) heterodimer CBP80–CBP20:: a heterodimer of 80-kDa and 20-kDa subunits that binds to the 5′-cap structures of pre-mRNAs during gene transcription and remains bound during the

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Trends in Molecular Medicine

Applying nonsense-mediated mRNA decay research to the clinic: progress and challenges

Section snippets

Germline and somatic-cell origins of premature termination codons

Why NMD?

NMD can prevent autosomal recessive disease: how PTCs trigger NMD

NMD occurs as a consequence of a pioneer round of translation

Therapies of nonsense-associated diseases

Suppression of in-frame nonsense codons

Suppression of out-of-frame nonsense codons

Upregulating a functional substitute for the protein encoded by a PTC-containing allele

Suppressing steps during the pioneer round of translation before nonsense codon recognition

Concluding remarks

Acknowledgements

Glossary

Trends Genet.

Curr. Opin. Struct. Biol.

Curr. Opin. Cell Biol.

Cell

Curr. Opin. Cell Biol.

Trends Biochem. Sci.

Biochim. Biophys. Acta

Mol. Cell

Cell

Biochimie

DNA Repair (Amst.)

DNA Repair (Amst.)

Trends Biochem. Sci.

Trends Mol. Med.

Neuromuscul. Disord.

Biochem. Biophys. Res. Commun.

Mol. Cell

Curr. Biol.

Biochim. Biophys. Acta

Computational prediction of SEG (single exon gene) function in humans

Front. Biosci.

From birth to death: the complex lives of eukaryotic mRNAs

Science

Nonsense-mediated mRNA decay in health and disease

Hum. Mol. Genet.

Defects in pre-mRNA processing as causes of and predisposition to diseases

DNA Cell Biol.

Quantitative microarray profiling provides evidence against widespread coupling of alternative splicing with nonsense-mediated mRNA decay to control gene expression

Genes Dev.

Regulation of gene expression by coupling of alternative splicing and NMD

Features of nonsense-mediated mRNA decay in Saccharomyces cerevisiae

Nonsense-mediated mRNA decay: a comparative analysis of different species

Curr. Genomics

NMD in mammalian cells: a history

NMD in Caenorhabditis elegans

NMD in Drosophila: a snapshot into the evolution of a conserved mRNA surveillance pathway

NMD in plants

Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise

Nat. Genet.

Regulated degradation of replication-dependent histone mRNAs requires both ATR and Upf1

Nat. Struct. Mol. Biol.