Understanding the molecular principles of UPF1-dependent mRNA substrate recognition and degradation
Cell Biology
Final Report Abstract
Gene expression is a highly regulated, multi-layered process that involves several steps, including transcription, translation, and the turnover of mRNAs and proteins. Among the pathways involved in regulating gene expression is nonsense-mediated mRNA decay (NMD), a quality control mechanism that targets and degrades faulty mRNAs to prevent the production of aberrant proteins. A critical component of the NMD machinery is UPF1, an RNA helicase, and ATPase that links translation termination with mRNA degradation. Despite extensive research, the exact molecular role of UPF1 in the different stages of NMD remains unclear. While it is known that UPF1’s ATPase and helicase activities are essential for NMD, the precise steps where these activities function have not yet been established. It is hypothesized that UPF1’s ATPase activity is primarily required for remodeling messenger ribonucleoprotein particles (mRNPs) after mRNA degradation has started, facilitating the dissociation of the surveillance complex from the substrate mRNA. However, there is growing evidence suggesting that ATP hydrolysis and helicase activity may have additional functions in NMD that have yet to be identified. Recent findings also show that UPF1 phosphorylation affects its activity, either directly or by promoting the recruitment of regulatory factors involved in mRNA decay. Despite its central role in NMD, UPF1 has proven difficult to study. Surprisingly, there is a limited amount of high-quality RNA sequencing (RNA-Seq) data from UPF1-depleted cells, and the data that does exist shows only moderate effects on NMD. In contrast, stronger effects are observed when other NMD factors, such as SMG6 and SMG7, are depleted, raising questions about whether the tools currently available for studying UPF1 are sufficient or whether UPF1’s role in NMD is inherently challenging to investigate. During the project challenges arose due to incomplete NMD inhibition by the UPF1 knockdown. To overcome this, we adopted the auxin-inducible degron (AID) system, which allowed for rapid and precise depletion of UPF1 within two hours. This method minimized the secondary effects seen with siRNA and enabled us to observe the immediate consequences of UPF1 loss on NMD, resulting in more reliable data and clearer insights into UPF1’s molecular role. Rapid UPF1 depletion lead to widespread upregulation of NMD targets: The depletion of UPF1 led to a significant upregulation of NMD-targeted genes, with the core of upregulated genes being more than ten times larger than the number of downregulated genes. This confirms UPF1’s role as an RNA helicase involved in mRNA degradation and highlights the extensive impact of NMD on the transcriptome. The study identified both canonical NMD targets, such as transcripts with premature termination codons, and a subset of transcripts regulated through alternative mechanisms. Interestingly, depletion of UPF1 also resulted in an increase in long non-coding RNA (lncRNA) transcripts, many of which were shown to be translated, based on ribosome profiling data. In conclusion, our findings provide, for the first time, a high-confidence set of annotated NMD- targeted transcripts. These results enhance our understanding of UPF1’s role in NMD and suggest that while UPF1 is a central factor in this pathway, its regulation of mRNA decay is highly dynamic and involves a complex interplay with other NMD components. We have gained valuable insights into how UPF1 contributes to mRNA surveillance and degradation, setting the stage for future research into the NMD pathway and its implications for cellular health and disease.
Publications
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Plasmid transfection influences the readout of nonsense-mediated mRNA decay reporter assays in human cells. Scientific Reports, 7(1).
Gerbracht, Jennifer V.; Boehm, Volker & Gehring, Niels H.
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Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates. RNA, 23(8), 1224-1236.
Ottens, Franziska; Boehm, Volker; Sibley, Christopher R.; Ule, Jernej & Gehring, Niels H.
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Rapid UPF1 depletion illuminates the temporal dynamics of the NMD-regulated transcriptome in human cells.
Boehm, Volker; Wallmeroth, Damaris; Wulf, Paul O.; Alves, Luiz Gustavo Teixeira; Popp, Oliver; Riedel, Maximilian; Wyler, Emanuel; Franitza, Marek; Gerbracht, Jennifer V.; Becker, Kerstin; Polkovnychenko, Karina; Del Giudice, Simone; Benlasfer, Nouhad; Mertins, Philipp; Landthaler, Markus & Gehring, Niels H.
