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

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.

Methods
Immunofluorescence

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.

smiFISH imaging

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.

Raw data for figure generation

An excel file of all raw data that was used to generate the graphs in the referenced publication has also been provided.