Systematic identification of NF90 target RNAs by iCLIP analysis

Valeria Lodde,Catello Mario Panu Napodano,Matteo Floris,Ji Heon Noh,M Laura Idda,Sergio Uzzau,Myriam Gorospe,Rachel Munk,Jennifer L Martindale,Francesco Cucca,Kotb Abdelmohsen,Davide Piredda

doi:10.1038/s41598-021-04101-1

Abstract

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNAs directing numerous essential roles in cellular physiology. Nuclear Factor 90 (NF90) is an RBP encoded by the interleukin enhancer-binding factor 3 (ILF3) gene that has been found to influence RNA metabolism at several levels, including pre-RNA splicing, mRNA turnover, and translation. To systematically identify the RNAs that interact with NF90, we carried out iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) analysis in the human embryonic fibroblast cell line HEK-293. Interestingly, many of the identified RNAs encoded proteins involved in the response to viral infection and RNA metabolism. We validated a subset of targets and investigated the impact of NF90 on their expression levels. Two of the top targets, IRF3 and IRF9 mRNAs, encode the proteins IRF3 and IRF9, crucial regulators of the interferon pathway involved in the SARS-CoV-2 immune response. Our results support a role for NF90 in modulating key genes implicated in the immune response and offer insight into the immunological response to the SARS-CoV-2 infection.

Highlights

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNAs directing numerous essential roles in cellular physiology
After circularization, followed by linearization and PCR amplification, the RNA fragments were identified by RNA-sequencing. (C) Most abundant Nuclear Factor 90 (NF90) RNA targets identified by iCLIP analysis; p-value of the association, chromosome (Chr) from where the RNA is transcribed and type of RNA are indicated. (D) Percentage of all NF90 iCLIP reads mapping to RNA subdivided by transcript type/region. (E) Percentage of RNA type identified by our analysis. (F) Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis of the NF90-target mRNAs identified by iCLIP
In order to systematically identify the collection of NF90 target mRNAs and further determine the sites of interaction with target mRNAs, we carried out iCLIP analysis in Human Embryonic Kidney 293 (HEK-293) cells

Summary

Introduction

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNAs directing numerous essential roles in cellular physiology. Nuclear Factor 90 (NF90) is an RBP encoded by the interleukin enhancer-binding factor 3 (ILF3) gene that has been found to influence RNA metabolism at several levels, including pre-RNA splicing, mRNA turnover, and translation. We found several RNAs that encoded proteins implicated in RNA metabolism, the response to viral infection, and signaling through RAP1 We validated these interactions by ribonucleoprotein (RNP) immunoprecipitation (RIP) analysis and investigated the impact of NF90 on the stability and translation of these target mRNAs. Among the most interesting NF90 targets, we identified several mRNAs encoding transcription factors in the interferon regulatory factor (IRF) family[20] and YWHAH, a protein implicated in susceptibility for schizophrenia and Parkinson’s d isease[21,22], as well as the lncRNA XIST, a known NF90 target[23]. Our results support a role of NF90 as a major regulator of RNA metabolism in a range of cellular responses, viral infection

Methods

Results

Conclusion