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Proximity labelling of FLAG-TurboID-tagged UPF3B wild type and interaction-deficient mutant proteins followed by proteomics analysis

ABSTRACT: UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B in NMD, we have generated FLAG-TurboID-UPF3B wild type or mutant expressing human Flp-In T-Rex 293 UPF3B knockout cells using the PiggyBac transposon system. We generated proteomic data from the proximity labeled interactome of these UPF3B variants.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Prerana Wagle

LAB HEAD: Niels H. Gehring

PROVIDER: PXD029898 | Pride | 2022-05-04

REPOSITORIES: Pride

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			Action	DRS
	Q2_ColID_327_2755_01.raw	Raw
	Q2_ColID_327_2755_02.raw	Raw
	Q2_ColID_327_2755_03.raw	Raw
	Q2_ColID_327_2755_04.raw	Raw
	Q2_ColID_327_2755_05.raw	Raw

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Deciphering the cellular roles and dissecting functional regions of UPF3A and UPF3B in human NMD

Project description:The paralogous proteins UPF3A and UPF3B are involved in recognizing defective mRNAs that are degraded by nonsense-mediated mRNA decay (NMD). While UPF3B has been demonstrated to support NMD, UPF3A was reported to act either an NMD activator or an NMD inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using overexpression, knockdowns, knockouts (KO) and rescue experiments. Overexpression or knockout of UPF3A did not result in detectable changes in global NMD activity or other specific transcriptome alterations . NMD activity was also virtually unchanged in UPF3B KO cells. In contrast, the co-depletion or co-knockout of UPF3A and UPF3B resulted in a marked NMD inhibition and a global upregulation of PTC-containing transcripts. In rescue experiments UPF3B was fully functional when either UPF2- or EJC binding was impaired . However, the deletion of both interaction sites or one interaction site and a central region of UPF3B impaired its NMD function. Altogether, our work identifies critical functional domains of UPF3B and establishes redundant roles of UPF3A and UPF3B during NMD.

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The exon junction complex component CASC3 is a major effector of nonsense-mediated mRNA decay in human cells

Project description:In metazoans, the exon junction complex (EJC) is a central component of spliced messenger ribonucleoprotein particles (mRNPs). CASC3 has been reported to be a core component of the EJC and to be crucial for assembly, the splicing regulating function of the EJC and nonsense-mediated mRNA decay (NMD). However, recent evidence suggests that CASC3 functions differently from other EJC core components. To elucidate the cellular role of CASC3, we have established human cell lines in which CASC3 was inactivated by means of CRISPR-Cas9 genome editing. We show that in these cells CASC3 is dispensable for the splicing regulatory role of the EJC. However, we find that CASC3 depletion results in the upregulation of many known and novel NMD substrates, suggesting that CASC3 is required for the efficient execution of EJC-dependent NMD. Taken together, our results challenge the model of CASC3 as an assembly factor and core component of the EJC. Our data rather show that CASC3 is involved in the degradation of NMD substrates and therefore uncover the primary molecular function of CASC3 in human cells.

2021-09-09 | PXD019161 | Pride

The exon junction complex component CASC3 is a major effector of nonsense-mediated mRNA decay in human cells

2021-02-25 | PXD015754 | Pride

Proximity labelling of FLAG-TurboID-tagged UPF3B wild type and interaction-deficient mutant proteins followed by proteomics analysis

Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B in NMD, we have generated FLAG-TurboID-UPF3B wild type or mutant expressing human Flp-In T-Rex 293 UPF3B knockout cells using the PiggyBac transposon system. We generated proteomic data from the proximity labeled interactome of these UPF3B variants.

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Nonsense-mediated mRNA decay relies on "two-factor authentication" by SMG5-SMG7

Project description:Eukaryotic gene expression is constantly regulated and controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional hierarchy of the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm complete NMD inhibition resulting in massive transcriptomic alterations. The NMD activity conferred by SMG5-SMG7 is determined to varying degrees by their interaction with the central NMD factor UPF1, heterodimer formation and the initiation of deadenylation. Surprisingly, we find that SMG5 functionally substitutes SMG7 and vice versa. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.

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2022-04-25 | E-MTAB-11184 | biostudies-arrayexpress

Nonsense-mediated mRNA decay in Tetrahymena is EJC independent and requires a protozoa-specific nuclease

Project description:Nonsense-mediated mRNA decay (NMD) is essential for removing premature termination codon (PTC)-containing transcripts from cells. Studying the NMD pathway in model organisms can help to elucidate the NMD mechanism of humans and improves our understanding of how this biologically important process has evolved. Protozoa are among the earliest branching eukaryotes. Their NMD mechanism is poorly understood and may be primordial. We demonstrate that highly conserved Upf proteins (Upf1a, Upf2, and Upf3) are involved in the NMD pathway of the ciliate, Tetrahymena thermophila. We further show that a novel protozoa-specific nuclease, Smg6L, is responsible for destroying many NMD-targeted transcripts. The transcriptome-wide identification and characterization of NMD-targeted transcripts in vegetative Tetrahymena cells showed that many have exon–exon junctions downstream of the termination codon. However, Tetrahymena homologs of exon junction complex (EJC) core components do not form a complex and are dispensable for NMD, suggesting that NMD is EJC independent in this early branching eukaryote.

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RNA-Seq of WT Flp-In T-REx 293 and UPF3B knockout cell lines with additional siRNA-mediated control or UPF3A knockdowns

Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3B knockout human Flp-In T-REx 293 cells using CRISPR-Cas9. We generated RNA-Sequencing data for wildtype and UPF3B KO cells with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3A.

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Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3B knockout (KO) and UPF3A-UPF3B double KO (dKO) human Flp-In T-REx 293 cells using CRISPR-Cas9. We generated RNA-Sequencing data for wildtype, UPF3B KO and UPF3A-UPF3B dKO cells with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3B.

2022-04-25 | E-MTAB-10716 | biostudies-arrayexpress

RNA-Seq of WT Flp-In T-REx 293, UPF3A knockout and UPF3A overexpression cell lines in part with additional siRNA-mediated control or UPF3B knockdowns

Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3A knockout (KO) human Flp-In T-REx 293 cells using CRISPR-Cas9, as well as FLAG-tagged UPF3A overexpressing cells using the PiggyBac transposon system. We generated RNA-Sequencing data for wildtype, UPF3A KO and UPF3A overexpressing (OE) cells, in part with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3B.

2022-08-31 | E-MTAB-10718 | biostudies-arrayexpress

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