DOI: 10.5061/dryad.k98sf7mf5
title: Identifying cellular RNA-binding proteins during infection uncovers a role for MKRN2 in influenza mRNA trafficking

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
 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.
description: <strong>Immunofluorescence</strong>
   Approximately 1.5*10^5 A549-MKRN2
 cells were cultured on glass coverslips (thickness #1.5) for 24h prior to
 infection at MOI 3 with A/WSN/33. The infection was synchronised by 1h
 incubation at 4°C at the beginning of the infection. Cells were fixed in
 4% PFA for 10’ at the appropriate time points and subjected to indirect
 immunofluorescence. Briefly, cells were permeabilised by 20’ treatment
 with 0.2% Triton-X-100 and blocked with 3% BSA for 30’. Then samples were
 incubated for 3h with the primary antibody solution containing 3% BSA, Ms
 anti-DYKDDDDK tag (Proteintech, 66008-3-Ig, 1:500) Rb anti-NP
 (ThermoFisher, 1:500, PA5-32242), followed by 1h incubation with the
 secondary antibody solution comprising 3% BSA, AlexaFluor 647 anti-Ms
 (Invitrogen, A32728, 1:500) AlexaFluor 488 anti-Rb (Invitrogen, A-11008,
 1:500), AlexaFluor 555 Phalloidin (ThermoFisher, A30106, 1:400). In order
 to facilitate nuclear/cytoplasmic segmentation, nuclei were stained with
 Hoechst (Abcam, ab228551, 1:4000), while the cytosol with Phalloidin.
 Finally, coverslips were mounted on slides using ProLong Diamond Antifade
 Mountant (Invitrogen, P36961).  
 The samples were imaged on a Leica Stellaris SP8 confocal
 system, using an HC PL APO CS2   100x/1.40 OIL objective resulting in a
 voxel size of 114x114x300 nm. Whole z-stacks of fields of view comprising
 multiple cells were taken, for a total of at least 150 cells per condition
 across three biological replicates.  
 <strong>smiFISH imaging</strong>
   A549 cells were transfected with
 MKRN2-targeting or NSC siRNA at a concentration of 10nM exactly as
 described above. These cells were seeded in 24-well plates onto 12 mm
 coverslips. 48h post transfection, cells were infected with influenza
 A/WSN/33 at MOI 10. Cells were fixed at 6 hpi with 4% PFA and
 permeabilised with 70% ethanol for 2 hours at 4°C. Cells were briefly
 washed with FISH wash buffer (10% SSC, 10% formamide, 80% ddH2O) and
 incubated O/N at 37°C with smiFISH probes against WSN NP mRNA
 (Supplementary Table 5) annealed with Cy3 imager strands. Cells were
 washed twice with FISH wash buffer at 37°C for 30 minutes and
 counterstained with Hoechst and mounted prior to imaging. Coverslips were
 imaged with a Leica STELLARIS ONE 660 tauSTED Nanoscope with a 100x
 objective with 1.4 NA. Z stacks were acquired and analysed using
 Fiji/ImageJ version 1.54f.  
 <strong>Raw data for figure
 generation</strong>  
 An excel file of all raw data that was used to generate the
 graphs in the referenced publication has also been provided.
description: # Identifying cellular RNA-binding proteins during infection uncovers a
 role for MKRN2 in influenza mRNA trafficking.
 [https://doi.org/10.5061/dryad.k98sf7mf5](https://doi.org/10.5061/dryad.k98sf7mf5) ## Description of the data and file structure The raw_data.xls contains all the data used to generate each graph within the referenced manuscript. Each excel tab corresponds to a different panel in a figure, and is named after the figure panel. All replicate data is contained within the tab and labelled appropriately for each condition. The MKRN2.zip file contains all the image files used for quantification of MKRN2 localisation during an influenza A virus time course, with nuclear and cytoplasmic fractions quantified to generate Figure 5B of the referenced publication. The parent folder contains 4 subfolders, 3 correspond to the 3 biological replicates of the experiment and a 4th folder named 'For figure' which are the representative images selected for Figure 5A. Within each replicate folder are .tif files containing series of z-collapsed images for conditions of Mock, 4 h and 8 h post infection. The MKRN2_smiFISH.zip file contains all the image files used for the quantification of NP mRNA localisation in Figure 5D of the referenced manuscript. The parent folder contains 4 subfolders, 3 correspond to the 3 biological replicates of the experiment and are named Replicate 1, Replicate 2 and Replicate 3. Within each of these folders are .lif files containing sets of images and can be opened using the free ImageJ software for image processing. The files are titled Mock.lif, NSC.lif and MKRN2.lif, corresponding to the Mock infection, cells treated with an Non-Specific Control siRNA and infected, or cell treated with a MKRN2-specific siRNA and infected, respectively. The 4th folder contains the representative images selected for Figure 5C within sub folder for each condition, labeled mock, NSC and MKRN2. ## Sharing/Access information This data is not available anywhere else. ## Code/Software This is a compilation of raw unprocessed images and raw data used to generate graphs for the referenced manuscript.