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 ce..., 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, ..., , # 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 replicat...

RNA结合蛋白（RNA-binding proteins, RBPs）的利用是病毒RNA转录后调控的重要环节。诸如甲型流感病毒（influenza A viruses, IAV）这类病毒会与RBPs相互作用，调控剪接、核输出与转运等过程，同时还会在其基因组中编码NP、NS1等RBPs。但人类基因组编码了近千种RBPs，其中多数蛋白在病毒复制过程中所发挥的作用仍未明确。

本研究借助RNA交互组捕获（RNA interactome capture, RIC）技术，从甲型流感病毒感染的细胞中分离RBPs，以解析受感染人类细胞中病毒mRNA结合的RBP组。研究中鉴定出了特定的RBPs MKRN2，该蛋白可与IAV mRNA结合并对其产生正向调控。经进一步验证，我们确定MKRN2参与了IAV mRNA的核质转运过程，其机制可能与RNA输出介导因子GLE1的结合有关。在MKRN2缺失的情况下，IAV mRNA会在感染细胞的细胞核中积累…… 免疫荧光实验

约1.5×10^5个A549-MKRN2细胞接种于1.5号厚度的盖玻片上，培养24小时后以感染复数（multiplicity of infection, MOI）3接种A/WSN/33病毒。感染初期通过4℃孵育1小时实现同步化感染。在指定时间点用4%多聚甲醛（PFA）固定细胞10分钟，随后进行间接免疫荧光染色。具体操作如下：先用0.2% Triton-X-100透化细胞20分钟，再用3%牛血清白蛋白（BSA）封闭30分钟。随后将样本与含3% BSA的一抗溶液孵育3小时，一抗包括鼠抗-DYKDDDDK标签抗体（Proteintech, 66008-3-Ig, 1:500）、兔抗-NP抗体（ThermoFisher, 1:500, PA5-32242）；之后用含3% BSA的二抗溶液孵育1小时，二抗包括AlexaFluor 647标记羊抗鼠抗体（Invitrogen, A32728, 1:500）、AlexaFluor 488标记羊抗兔抗体（Invitrogen, A-11008, 1:500）以及AlexaFluor 555标记鬼笔环肽（ThermoFisher, A30106, 1:400）。为便于核质分区分析，使用Hoechst染料对细胞核进行染色（Abcam, ……）。

# 鉴定感染过程中的细胞RNA结合蛋白揭示MKRN2在流感病毒mRNA转运中的功能

https://doi.org/10.5061/dryad.k98sf7mf5

## 数据与文件结构说明

`raw_data.xls` 包含了生成参考文献手稿中所有图表所需的全部数据。每个Excel工作表对应一幅图的不同子图，并以该子图的名称命名。所有重复实验数据均收录于对应工作表中，并为每个实验条件进行了恰当标注。

`MKRN2.zip` 包含了用于定量甲型流感病毒感染时间进程中MKRN2定位的全部图像文件，其中核组分与胞质组分的定量结果用于生成参考文献论文中的图5B。该压缩包的根目录包含4个子文件夹：3个文件夹对应实验的3次生物学重复，第4个名为“For figure”的文件夹包含用于生成图5A的代表性图像。每个重复……