Biology subjects

Muhlemann, O.

Publications and source records attributed to Muhlemann, O..

3 recordsLinked to original sources

bioRxiv2019

Human NMD ensues independently of stable ribosome stalling

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty and the regulation of normal mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system revealed similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling revealed a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.

molecular biology↗

bioRxiv2019

Ribosome recycling factor ABCE1 depletion inhibits nonsense-mediated mRNA decay by promoting stop codon readthrough

Nonsense-mediated mRNA decay (NMD) is an essential post-transcriptional surveillance pathway in vertebrates that appears to be mechanistically linked with translation termination. To gain more insight into this connection, we interfered with translation termination by depleting human cells of the ribosome recycling factor ABCE1, which resulted in an upregulation of many but not all endogenous NMD-sensitive mRNAs. Notably, the suppression of NMD on these mRNAs occurs at a step prior to their SMG6-mediated endonucleolytic cleavage. Ribosome profiling revealed that ABCE1 depletion results in ribosome stalling at stop codons and increased ribosome occupancy in 3 UTRs, indicative of enhanced stop codon readthrough or re-initiation. Using reporter genes, we further demonstrate that the absence of ABCE1 indeed increases the rate of readthrough, which would explain the observed NMD inhibition, since enhanced readthrough has been previously shown to render NMD-sensitive transcripts resistant to NMD by displacing NMD triggering factors like UPF1 and exon junction complexes (EJCs) from the 3 UTR. Collectively, our results show that improper ribosome disassembly interferes with proper NMD activation. HighlightsO_LIABCE1 knockdown suppresses NMD of many NMD-sensitive mRNAs C_LIO_LIThe observed NMD inhibition occurs at a stage prior to SMG6-mediated cleavage of the mRNA C_LIO_LIABCE1 depletion enhances ribosome occupancy at stop codons and in the 3 UTR C_LIO_LIABCE1 depletion enhances readthrough of the stop codon C_LIO_LIEnhanced readthrough inhibits NMD, presumably by clearing the 3 UTR of NMD factors C_LI

molecular biology↗

bioRxiv2019

FUS-dependent phase separation initiates double-strand break repair

RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, 53BP1, and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient {gamma}H2AX foci formation. Finally, using super-resolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of {gamma}H2AX nano-foci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSBs repair complexes.

molecular biology↗