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The RBPome of influenza A virus mRNA reveals a role for TDP-43 in viral replication

ABSTRACT: Recent technical advances have resulted in a significant increase in our understanding of the RNA-binding protein (RBP) repertoire present within eukaryotic cells. A particular focus has been on the RBPs that interact with cellular polyadenylated mRNAs. However, recent studies utilising the same technologies have begun to tease apart the RBP interactome of viral mRNAs, most notably SARS-CoV-2, demonstrating clear similarities but also differences between the RBP profiles of viral and cellular mRNAs. Herein, for the first time we comprehensively mapped the RBPs that associate with the NP mRNA of an influenza A virus. Moreover, we provide evidence that the viral polymerase is essential for the recruitment of RPBs to viral mRNAs through direct polymerase-RBP interactions during transcription. We show that loss of TDP-43, that associates with the viral mRNA, results in a decrease in viral mRNA availability within infected cells, with an impact on the accumulation of viral genomic RNA and the yield of infectious viral particles. Overall, we uncover an important role for TDP-43 in the influenza A virus replication cycle via a direct interaction with viral mRNAs and we demonstrate an essential role of the viral polymerase in orchestrating the assembly of viral mRNPs.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Line Cell

SUBMITTER: Thibaut Douché

LAB HEAD: Nadia Naffakh

PROVIDER: PXD049316 | Pride | 2024-04-16

REPOSITORIES: Pride

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			Action	DRS
	20191019-Homo_sapiens.fasta	Fasta
	20200107-Influenza_A.fasta	Fasta
	20200107-RNaseA	Other
	20230627_Q1_TD_ColL7_Courtney_M1.raw	Raw
	20230627_Q1_TD_ColL7_Courtney_M2.raw	Raw

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The RBPome of influenza A virus mRNA reveals a role for TDP-43 in viral replication

Project description:Recent technical advances have resulted in a significant increase in our understanding of the RNA-binding protein (RBP) repertoire present within eukaryotic cells. A particular focus has been on the RBPs that interact with cellular polyadenylated mRNAs. However, recent studies utilising the same technologies have begun to tease apart the RBP interactome of viral mRNAs, most notably SARS-CoV-2, demonstrating clear similarities but also differences between the RBP profiles of viral and cellular mRNAs. Herein, for the first time we comprehensively mapped the RBPs that associate with the NP mRNA of an influenza A virus. Moreover, we provide evidence that the viral polymerase is essential for the recruitment of RPBs to viral mRNAs through direct polymerase-RBP interactions during transcription. We show that loss of TDP-43, that associates with the viral mRNA, results in a decrease in viral mRNA availability within infected cells, with an impact on the accumulation of viral genomic RNA and the yield of infectious viral particles. Overall, we uncover an important role for TDP-43 in the influenza A virus replication cycle via a direct interaction with viral mRNAs and we demonstrate an essential role of the viral polymerase in orchestrating the assembly of viral mRNPs.

2024-04-16 | PXD040783 | Pride

Nucleocytoplasmic mRNA redistribution accompanies RNA binding protein mislocalisation in ALS motor neurons and is restored by VCP inhibition

Project description:Although the pathological hallmark of amyotrophic lateral sclerosis (ALS) is the nucleocytoplasmic mislocalisation of RNA binding proteins (RBPs), such as TDP-43 and FUS, the nucleocytoplasmic distribution of mRNA remains uncharacterised. Here, we used subcellular fractionation with RNA sequencing and proteomics to assess nucleocytoplasmic mRNA and protein localisation in human induced pluripotent stem cell-derived motor neurons (iPSNs) from ALS patients with TARDBP mutations, VCP mutations, and controls with VCP mutations knocked-in with CRISPR/Cas9. In each mutant group, we found substantial nucleocytoplasmic mRNA redistribution, particularly in transcripts involved in protein binding. Redistributed transcripts in ALS iPSNs were enriched in protein-coding biotypes, exhibited longer lengths, and had enhanced interactions with RBPs, including TDP-43 and FUS. Using mass spectrometry in TARDBP mutant, VCP mutant, and VCP mutant knock-in iPSNs, we reveal widespread protein mislocalisation, especially in RBPs. We find that mislocalised proteins exhibit substantially greater binding to redistributed transcripts than non-redistributed mRNAs, suggesting that RBP mislocalisation may be directly coupled to mRNA redistribution. Remarkably, treatment with the VCP D2 ATPase domain inhibitor ML240 restored both nucleocytoplasmic mRNA redistribution and protein mislocalisation not only in VCP mutants but also in TARDBP mutant iPSNs. Functional assays in VCP mutant iPSNs further confirmed that ML240 restores lysosomal localisation from the perinuclear region towards the neurites and reduces oxidative stress. Our findings demonstrate that ALS motor neurons exhibit concomitant nucleocytoplasmic mRNA redistribution and RBP

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TDP-43 prevents endogenous RNAs from triggering a lethal RIG-I-dependent interferon response

Project description:RIG-I-like receptors (RLRs) are involved in the discrimination of self vs non-self via the recognition of dsRNA. Emerging evidence suggests that immunostimulatory dsRNAs are ubiquitously expressed yet they are disrupted or sequestered by cellular RNA binding proteins (RBPs). TDP-43 is an RBP associated with multiple neurological disorders and is essential for cell viability. Here, we demonstrate that TDP-43 regulates the accumulation of immunostimulatory dsRNA. The immunostimulatory RNA was identified as RNA Polymerase III transcripts, including 7SL and Alu retrotransposons, and we demonstrate that the RNA binding activity of TDP-43 is required to prevent immune stimulation. The dsRNAs activate a RIG-I-dependent interferon response which promotes necroptosis. Genetic inactivation of the RLR-pathway rescues the interferon-mediated cell-death associated with loss of TDP-43. Collectively, our study describes a role for TDP-43 in preventing the accumulation of endogenous immunostimulatory dsRNAs and uncovers an intricate relationship between the control of cellular gene expression and IFN-mediated cell-death.

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Project description:We performed RNA immunoprecipitation (IP) and microarray (RIP-chip) analyses to identify and compare the biological mRNA targets of two RNA-binding protein (RBP), TDP-43 and FUS, associated to cytoplasmic ribonucleoprotein (RNP) complexes of motoneuronal NSC34 cells with the final aim to unravel their role in mRNA transport, stability, and translation in neuronal cells. To identify the transcripts contained and bound in the isolated RNP complexes, a chip analysis was performed using the recovered mRNAs from triplicate experimantal RIP samples (TDP-43-IP samples, the FUS-IP samples and the control IgG-IP sample).

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Identifying cellular RNA-binding proteins during infection uncovers a role for MKRN2 in influenza mRNA trafficking.

Project description:Utilisation of RNA-binding proteins (RBPs) is an important aspect of post-transcriptional regulation of viral RNA. Viruses such as influenza A viruses (IAV) interact with RBPs to regulate processes including splicing, nuclear export and trafficking, while also encoding RBPs within their genomes, such as NP and NS1. But with almost 1000 RBPs encoded within the human genome it is still unclear what role, if any, many of these proteins play during viral replication. Using the RNA interactome capture (RIC) technique, we isolated RBPs from IAV infected cells to unravel the RBPome of mRNAs from IAV infected human cells. This led to the identification of one particular RBP, MKRN2, that associates with and positively regulates IAV mRNA. Through further validation, we determined that MKRN2 is involved in the nuclear-cytoplasmic trafficking of IAV mRNA likely through an association with the RNA export mediator GLE1. In the absence of MKRN2, IAV mRNAs accumulate in the nucleus of infected cells, which we suspect leads to their degradation by the nuclear RNA exosome complex. MKRN2, therefore, appears to be required for the efficient nuclear export of IAV mRNAs in human cells.

2024-04-16 | PXD048870 | Pride

Splicing events and transcriptome changes upon TDP-43 and FUS knock-down in human neuroblastoma cells

Project description:We performed exon array analysis to define and compare the transcriptomic changes, in terms of splicing and gene expression profilings, occurring in human neuroblastoma cells upon knocking down TDP-43 and FUS RNA-binding proteins (RBP), with the final aim to unravel the biological role of these RBPs in neuronal cells.

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Functional TDP-43 amyloids bind sarcomeric mRNA to direct skeletal muscle formation

Project description:Cytoplasmic aggregates of TDP-43 are found in neuronal and skeletal muscle diseases yet it is not understood how these protein aggregates relate to the biological function of TDP-43. By examining normal skeletal muscle formation, we discovered TDP-43 redistributes from the nucleus into the cytoplasm and forms higher-ordered aggregates. These skeletal muscle TDP-43 aggregates adopt an amyloid-like structure capable of binding RNA. In skeletal muscle, TDP-43 binds UG-rich, sarcomeric mRNAs and oligomerizes on these long mRNA transcripts facilitating amyloid formation. Tissue specific genetic deletion of TDP-43 in skeletal muscle disrupts the formation and maintenance of the sarcomere and mislocalizes sarcomeric mRNAs. Thus, cytoplasmic, amyloid-like TDP-43 aggregates that traffic mRNAs could serve as a precursor to pathological TDP-43 aggregates common in degenerative neuromuscular disease.

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Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs

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2012-09-30 | E-GEOD-40653 | biostudies-arrayexpress

Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs (CLIP-Seq)

Project description:FUS/TLS and TDP-43 are RNA/DNA-binding proteins integrally involved in amyotrophic lateral sclerosis (ALS) and frontal temporal dementia. FUS/TLS is shown to bind RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU binding motif. A characteristic sawtooth-like binding pattern is identified, supporting co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system is shown to alter levels or splicing of >970 mRNAs, most of which are distinct from the RNAs whose maturation is dependent on TDP-43. Nonetheless, only 55 RNAs are reduced upon depletion of either TDP-43 or FUS/TLS from mouse brain and human neurons differentiated from pluripotent stem cells, including mRNAs transcribed from genes with exceptionally long introns and that encode proteins essential for neuronal integrity. A subset of these is significantly lowered in FUS/TLSR521G and TDP-43G298S mutant fibroblasts and in TDP-43 aggregate-containing motor neurons in sporadic ALS, evidence pointing to a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS. CLIP of Fus/Tls in 8 week mouse brain and adult human brain

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