Project

Discipline

RNA quality control; mRNA turnover; posttranscriptional gene regulation; Nonsense-mediated mRNA decay; mRNA surveillance; translation termination

Lead
Lay summary
Accurate expression of the genetic information is crucial for complex organisms and requires several different quality control (QC) mechanisms that recognize mistakes along the cascade of complex biochemical reactions from RNA synthesis to functional proteins. Among those, nonsense-mediated mRNA decay (NMD) has been initially characterized as a translation-dependent posttranscriptional QC system that selectively recognizes and degrades mRNAs with crippled protein-coding potential, thereby preventing the cell from production of truncated proteins with potentially toxic functions. Recent transcriptome profiling of NMD-deficient yeast, Drosophila, and human cells revealed that NMD affects the mRNA concentrations of 3-10% of all genes, indicating a role of NMD in gene regulation that extends beyond QC. NMD is essential in vertebrates and an important modulator of genetic disease phenotypes in humans, since about 30% of all known disease-causing mutations trigger NMD. While the phenomenon of NMD and its impact on gene expression and genetic diseases is well documented, the understanding of the underlying molecular mechanisms is still fragmented The specific projects supported by this grant are a continuation of more than 10 years of SNF-supported basic research aiming at unraveling the molecular mechanism and physiological role of NMD in human cells by using a combination of state-of-the-art biochemical, molecular biology, cell biology and reverse genetics methods. Specifically, we try to understand the features that identify an mRNA as a substrate for NMD and want to know which factors interact how, where and in which temporal order with the these mRNAs and with each other to achieve the rapid degradation of the target mRNAs. In this context, we also try to decipher the puzzling nuclear effects observed with NMD-targeted mRNAs. Given its tight coupling to translation, NMD is commonly assumed to occur in the cytoplasm, and the recurring effects of NMD-causing premature termination codons on nuclear processes like transcription and splicing are therefore enigmatic. Furthermore, we attempt to reveal the roles that NMD plays in regulating gene expression. To this end, we want to identify and characterize transcriptome-wide mRNAs differentially regulated by NMD during the differentiation of neuroblastoma into neuron-like cells and thereby discover specific biological pathways that are controlled by NMD. In particular, we will to test the hypothesis that spatial rearrangements of the 3’ UTR configuration represent a way by which the half-life of mRNAs can be differentially regulated by NMD. Concerning planned methodological progress, we will expand and optimize the use of lentiviral constructs to engineer cell lines that allow inducible expression of two or even three transgenes separately. We need such cell lines for the planned purification and characterization of specific mRNP populations that have been arrested at different stages along the NMD pathway. If successful, this method will have a wide range of interesting applications beyond NMD research.

Direct link to Lay Summary

Last update: 21.02.2013

Active participation

Self-organised

Communication	Title	Media	Place	Year

Number	Title	Start	Funding scheme

Accurate expression of the genetic information is achieved with the help of several quality control (QC) systems that recognize mistakes along the cascade of complex biochemical reactions from RNA synthesis to functional proteins and so prevent production of faulty gene products. One of these QC systems is “nonsense-mediated mRNA decay” (NMD), a translation-dependent process that degrades mRNAs with truncated open reading frames (ORFs). By recognizing and degrading mRNAs with premature termination codons (PTCs), many of which arise by alternative splicing, NMD protects the cell from accumulating C-terminally truncated proteins with potentially toxic functions. Transcriptome profiling of NMD-deficient yeast, Drosophila, and human cells revealed that NMD affects (directly or indirectly) the mRNA levels of 3 - 10% of all genes, indicating a role of NMD in gene regulation that extends beyond QC. NMD is essential in vertebrates and an important modulator of genetic disease phenotypes in humans, since 30% of all known disease-causing mutations are predicted to trigger NMD. While the phenomenon of NMD and its impact on gene expression and genetic diseases is well documented, the understanding of the underlying molecular mechanisms is still fragmented.Altogether, the projects proposed here aim at unraveling the molecular mechanism and physiological role of NMD in human cells. We use a combination of state-of-the-art biochemical, molecular biology, cell biology and reverse genetics methods to elucidate which features render an mRNA a NMD substrate, and which factors interact how, where and in which temporal order with these features and with each other to achieve the rapid degradation of target mRNAs. In this context, we also try to decipher the puzzling nuclear effects observed with NMD-targeted mRNAs. Given its tight coupling to translation, NMD is commonly assumed to occur in the cytoplasm, and the recurring NMD factor-dependent effects of PTCs on nuclear processes like transcription and splicing are enigmatic. Besides trying to get further insight into the mechanism of NMD, we also attempt to reveal the roles that NMD plays in regulating gene expression. To this end, we want to identify and characterize transcriptome-wide mRNAs regulated by NMD and thereby discover specific biological functions that are controlled by NMD. In particular, we also want to test the hypothesis that spatial rearrangements of the 3’ UTR configuration represent a way by which the half-life of mRNAs can be differentially regulated by NMD.In terms of methodological progress, we began to utilize lentiviral constructs to engineer cell lines that allow inducible expression of two or even three transgenes separately. We will need such cell lines to achieve our ambitious goal of purifying from cells and characterizing by mass spectrometry the composition of specific mRNP populations that have been arrested a different stages along the NMD pathway. If successful, this method will have a wide range of interesting applications in different areas of the molecular life sciences.