Identifying cellular RNA-binding proteins during infection uncovers a role for MKRN2 in influenza mRNA trafficking
Stefano Bonazza, Hannah Leigh Coutts, Swathi Sukumar, Hannah Louise Turkington, David Gary Courtney
Abstract
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 potentially through an association with the RNA export mediator GLE1. In the absence of MKRN2, IAV mRNAs accumulate in the nucleus of infected cells, which may lead 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.
Introduction
In recent years, our understanding of the RNA-binding protein (RBP) interactome has become significantly more informed. The development of methods such as RNA-interactome capture (RIC), enhanced RIC (eRIC) and RNA affinity pulldown mass spectrometry (RAP-MS), has allowed researchers to further elucidate the true RBPome of total mRNAs, as well as individual RNAs, in a given cell population [1–4]. These techniques, in addition to viral cross-linking and solid-phase purification (VIR-CLASP) [5], have now been used to good effect to identify the RBPome of viral RNAs from Sindbis virus [6,7], Chikungunya virus (CHIKV) [5], and, in multiple studies, SARS-CoV-2 [8–10]. It has been reported previously, in cell culture, that influenza A virus (IAV) mRNAs can comprise up to 50% of the total mRNA population of an infected cell [11]. Therefore, we reasoned that use of the RIC method, resulting in the purification of total mRNAs and their interacting proteins, would be ideal for the identification of pro-viral host RBPs potentially hijacked during IAV replication. Unravelling the IAV RBPome is essential to better understand the post-transcriptional regulation of IAV mRNAs.
Methods
Cells
For this work immortalised A549 (ATCC; CCL-185), HEK 293T (ATCC; CRL-3216) and MDCK (CCL-34) cells were used. All cells were cultured in DMEM supplemented with 1% Pen-Strep (Thermo Fisher Scientific; 15140122) and 5% FBS (Thermo Fisher Scientific; 10270106) at 37°C and 5% CO2.
Dryad DOI https://doi.org/10.5061/dryad.k98sf7mf5.
Virus stocks and infections
WSN (A/WSN/33) viral stocks were generated from a reverse genetics system that has been described previously [36]. WSN-PA_mNeon virus was generated from the same reverse genetics system, where the pPolI-PA plasmid was substituted for the pPolI-PA_mNeon plasmid encoding for a C-terminal mNeon followed by a duplicated packaging signal (S2 Table). Cal09 (A/California/7/2009) and Norway14 (A/Norway/466/2014) viruses were grown from isolates acquired from the National Institute for Biological Standards and Control, UK. All stocks were grown on MDCK cells in IAV growth media consisting of DMEM, Pen-Strep, 0.2% BSA (Merck; A8412), 25mM HEPES (Merck; H0887) and TPCK-trypsin (Merck; T8802), and titred on MDCK cells by plaque assay.
Results
RIC methodology effectively identifies RBPs in influenza A virus-infected cells
To initially determine the RBPome of mRNAs from influenza A virus-infected cells, we employed the RIC approach referred to previously. This technique allows for the purification of all proteins bound to poly(A)+ RNA through conventional UV crosslinking, followed by mass spectrometry (Fig 1A). Minimally, influenza A virus encodes for 10 distinct poly(A)+ mRNA (Fig 1B). Following pulldown of all poly(A)+ mRNA and covalently crosslinked proteins, from mock, A/WSN/33 infected or A/California/7/2009 infected A549 cells, a silver stain was performed to confirm the presence of a diverse protein population suitable for mass spectrometric analysis (Fig 1C). Indeed, we suspected that viral NP protein was visible as a band at approximately 55kDa on the A/WSN/33 pulldown sample, and to a lesser extent in the A/California/7/2009 pulldown sample. Additionally, Western blot analysis confirmed the viral RBP NP was present only in infected samples, as well as in pulldown samples (Fig 1D).
Discussion
Influenza A virus represents a highly infectious human pathogen ripe with potential for zoonotic spillover. IAV utilises a plethora of host proteins and pathways to successfully transcribe, translate and replicate its genome. A particularly fascinating aspect of IAV biology, in stark contrast to most other RNA viruses, is that RNA synthesis occurs in the nucleus. IAV mRNA synthesis is further complicated by the fact that, minimally, 8 out of the 10 predominant IAV mRNAs are not spliced [13]. Splicing in eukaryotic cells generally leads to the direct recruitment of nuclear export factors such as NXF1 followed by the rapid trafficking of spliced mRNA to the cytoplasm [25]. However, this is not the case for the majority of IAV transcripts, and successful replication necessitates other means to effectively export IAV mRNAs.
Acknowledgments
This research received infrastructure support from the Wellcome-Wolfson Institute for Experimental Medicine at Queen’s University Belfast.
Citation: Bonazza S, Coutts HL, Sukumar S, Turkington HL, Courtney DG (2024) Identifying cellular RNA-binding proteins during infection uncovers a role for MKRN2 in influenza mRNA trafficking. PLoS Pathog 20(5): e1012231. https://doi.org/10.1371/journal.ppat.1012231
Editor: Martin Schwemmle, Freiburg University, GERMANY
Received: September 27, 2023; Accepted: April 29, 2024; Published: May 16, 2024
Copyright: © 2024 Bonazza et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The raw MS/MS data files are deposited on PRIDE partner repository and available via the unique identifier PXD048870. All raw data used to generate graphs within this manuscript and all imaging files used for the analysis of MKRN2 localisation or NP mRNA localisation are deposited on DRYAD under the following DOI 10.5061/dryad.k98sf7mf5. All other relevant data are within the manuscript and its Supporting information files.
Funding: This research was funded in part by an ERC-StG grant (PTFLU 949506 awarded to DGC). This ERC-StG grant funded the salaries of authors SB, HC, SS, HT and DGC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1012231#abstract0
