Production of human cell lines with mutations and deletions in endogenous NMD factors using CRISPR/Cas genome editing

Nasif, Sofia; Mühlemann, Oliver (22 January 2016). Production of human cell lines with mutations and deletions in endogenous NMD factors using CRISPR/Cas genome editing (Unpublished). In: Swiss RNA Workshop 2016. Bern, Switzerland. 22.01.16.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA degradation pathway that modulates the abundance of 3-10% of all cellular mRNAs in mammalian cells. NMD constitutes a cellular quality control system that prevents the production of potentially harmful truncated proteins by targeting for degradation mRNAs with premature termination codons. Additionally, NMD targets many physiological mRNAs that encode full-length functional proteins and has therefore been implicated in the regulation of many different biological processes like stress response and cellular differentiation. Mammalian NMD relies on, at least, two different pathways for degrading its target mRNAs: the endonucleolytic pathway that depends on SMG6 endonuclease activity and the deadenylation-coupled exonucleolytic pathway for which the SMG5-SMG7 heterodimer is required. Available data suggests that these two degradation pathways act redundantly in human cells, but the accurate quantitative assessment of the relative contribution of each pathway to overall NMD has been elusive until now, mainly because of the use of knockdown strategies that never result in complete inactivation of the respective pathway. We therefore plan to investigate this by generating genome-edited human cell lines in which we mutate or delete SMG6 and SMG7 to inactivate, respectively, the endo- or exo- nucleolytic degradation pathways. The experimental approaches to generate the mutant cell lines by CRISPR/Cas genome editing will be presented. Characterization of these mutant cell lines will enable us to quantitatively assess the contribution of each decay pathway to NMD and to determine if different transcripts are preferentially targeted by one or the other pathway, providing further insight into the different roles of NMD as a general post-transcriptional gene regulatory mechanism.

Interest & Impact

Downloads

0 since deposited on 26 Jan 2017
0 in the past 12 months

Citations

5 Citations in Web of Science ®
6 Citations in Scopus

Search

in Google Scholar™

Services

Actions (login required)

Edit item

Actions (login required)

Edit item