Project

Discipline

Nonsense-mediated mRNA decay; mRNA turnover; posttranscriptional gene regulation; translation termination; RNA quality control; mRNA surveillance; Nonsense-mediated mRNA decay (NMD); post-transcriptional gene regulation; RNA turnover; mRNA quality control

Lead
Lay summary
Different quality control mechanisms exist to recognize mistakes during the molecular processes involved in gene expression and to prevent production of faulty gene products. Among those, nonsense-mediated mRNA decay (NMD) represents a translation-dependent posttranscriptional process that selectively recognizes and degrades mRNAs whose open reading frame (ORF) is truncated by a premature translation-termination codon (PTC, also called "nonsense codon"). In doing so, NMD protects the cell from accumulating C-terminally truncated proteins with potentially deleterious functions. Because 30% of all known disease-causing mutations are predicted to trigger NMD, NMD represents an important modulator of genetic disease phenotypes in humans, and understanding the molecular mechanisms of NMD therefore is key for developing future therapies for many genetic diseases. Transcriptome profiling of NMD-deficient yeast, Drosophila, and human cells revealed that 3-10% of all mRNA levels are affected by NMD, indicating an important role of NMD in gene regulation that extends beyond quality control. While the phenomenon of NMD and its impact on gene expression is well documented, the understanding of the underlying molecular mechanisms is still fragmented.Our research attempts to solve two key questions about NMD:1.) Which features distinguish faulty mRNAs that will be subjected to NMD from "normal" mRNAs that will not be targeted by NMD? How does this substrate recognition work at the molecular level? Specifically, we will test a working model that we recently postulated based on our previous work, the so-called unified NMD model (see Mühlemann et al., Biochim Biophys Acta, 2008). This model posits that proper translation termination depends on stimulating signals from the 3' UTR and that in the absence of these signals, translation termination is slow, giving time for the assembly of a protein complex that mediates NMD. The model further predicts that spatial rearrangements of the 3' UTR structure of an mRNA may represent a novel mechanism for tissue-specific or developmentally regulated post-transcriptional control of gene expression.2.) Once identified as nonsense transcript, how are these mRNAs rapidly eliminated? We have recently discovered that one of the known NMD factors, SMG6, is an endonuclease that can cleave an mRNA internally at the site where it binds the RNA (Eberle et al., Nat Struct Mol Biol, 2009). On the other hand, other laboratories have reported that nonsense mRNAs are degraded exonucleolytically from both ends in human cells. We will scrutinze both decay pathways and and try to determine their relative contribution to NMD.

Direct link to Lay Summary

Last update: 21.02.2013

Active participation

Self-organised

Communication	Title	Media	Place	Year

Number	Title	Start	Funding scheme

Background:Many different quality control mechanisms have evolved to recognize mistakes during the molecular processes involved in gene expression and to prevent production of faulty gene products. Among those, nonsense-mediated mRNA decay (NMD) represents a translation-dependent posttranscriptional process that selectively recognizes and degrades mRNAs whose open reading frame (ORF) is truncated by a premature translation-termination codon (PTC, also called „nonsense codon“). In doing so, NMD protects the cell from accumulating C-terminally truncated proteins with potentially deleterious functions. Because 30% of all known disease-causing mutations are predicted to trigger NMD, NMD represents an important modulator of genetic disease phenotypes in humans, and understanding the molecular mechanisms of NMD therefore is key for developing future therapies for many genetic diseases. Transcriptome profiling of NMD-deficient yeast, Drosophila, and human cells revealed that 3-10% of all mRNA levels are regulated (directly or indirectly) by NMD, indicating an important role of NMD in gene regulation that extends beyond quality control. While the phenomenon of NMD and its impact on gene expression is well documented, the understanding of the underlying molecular mechanisms is still fragmented.Current and future research on nonsense-mediated mRNA decay (NMD):A central question of NMD research is how PTCs are recognized and discriminated from natural termination codons. Recent work from my lab and others led to a new model postulating an evolutionarily conserved mechanism for PTC recognition. This so-called “unified NMD model” and its implications for posttranscriptional gene regulation and certain genetic diseases need to be critically scrutinized in the coming years. To further investigate the molecular mechanism of PTC recognition, we concentrate on trying to understand the PABP-mediated promotion of translation termination and the exact role of UPF1 in NMD. For UPF1, we plan to systematically identify interaction partners during the different stages of NMD complex assembly with help of a new mass spectrometry-based technique that enables quantitative identification of interacting factors from affinity purified complexes (SILAC-IP). Another goal is to identify endogenous examples of the postulated posttranscriptional mechanism for gene regulation by spatial rearrangement of the 3’ UTR, a direct implication of the “unified NMD model”. Along these lines, we will also try to identify disease-related human mutations that change the 3’ UTR of the mRNA and experimentally validate those transcripts predicted by our model to have become NMD substrates. Substantial progress in understanding the degradation of nonsense mRNAs in human cells has been made by our recent demonstration that SMG6 is an endonuclease and cleaves the transcript near the PTC. In the future, we want to address if decay of all human NMD substrates is induced by SMG6-mediated endonucleolytic cleavage, or if additional parallel pathways exist. In the case of multiple pathways, we would like to identify the decisive parameters for one or another pathway.Nonsense-mediated transcriptional gene silencing (NMTGS) - quo vadis?Transcriptional silencing of PTC-containing Ig-mu minigenes stably integrated into the genome of HeLa cells was serendipitously discovered in our lab five years ago. Although the biological relevance of this observation still remains elusive, the mechanism itself is intriguing: a translation signal on an mRNA (the PTC) somehow feeds back to its cognate gene and silences its transcription. We are currently trying to establish a more robust experimental system that would enable us to continue our mechanistic dissection of NMTGS. Circumstantial evidence suggests the involvement of small RNAs and components of the RNAi machinery, but more direct evidence is needed. Various experiments are planned to gain more insight into this enigmatic process.