M6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress.

Ryoma Yoneda,Riki Kurokawa,Naomi Ueda

doi:10.3390/ijms222011014

Ryoma Yoneda, Riki Kurokawa + Show 1 more

Open Access

https://doi.org/10.3390/ijms222011014

Copy DOI

Abstract

Translocated in LipoSarcoma/Fused in Sarcoma (TLS/FUS) is a nuclear RNA binding protein whose mutations cause amyotrophic lateral sclerosis. TLS/FUS undergoes LLPS and forms membraneless particles with other proteins and nucleic acids. Interaction with RNA alters conformation of TLS/FUS, which affects binding with proteins, but the effect of m6A RNA modification on the TLS/FUS–RNA interaction remains elusive. Here, we investigated the binding specificity of TLS/FUS to m6A RNA fragments by RNA pull down assay, and elucidated that both wild type and ALS-related TLS/FUS mutants strongly bound to m6A modified RNAs. TLS/FUS formed cytoplasmic foci by treating hyperosmotic stress, but the cells transfected with m6A-modified RNAs had a smaller number of foci. Moreover, m6A-modified RNA transfection resulted in the cells obtaining higher resistance to the stress. In summary, we propose TLS/FUS as a novel candidate of m6A recognition protein, and m6A-modified RNA fragments diffuse cytoplasmic TLS/FUS foci and thereby enhance cell viability.

Highlights

Translocated in LipoSarcoma/Fused in Sarcoma (TLS/FUS) is a nuclear RNA/DNA binding protein whose mutations lead to amyotrophic lateral sclerosis (ALS)
We generated two RNA fragments derived from pncRNA-D, an long noncoding RNA (lncRNA) expressed from cyclin D1 promoter (Figure 1A, black box, [29])
We report that TLS/FUS preferentially bound to the m6 A-modified RNA fragments, and mutations in nuclear localization signal (NLS) reduced the RNA binding specificity of TLS/FUS

Summary

Introduction

Translocated in LipoSarcoma/Fused in Sarcoma (TLS/FUS) is a nuclear RNA/DNA binding protein whose mutations lead to amyotrophic lateral sclerosis (ALS). IDRs interact by themselves, forming membraneless particles, which could isolate contents from external environments. Subcellular organelles, such as nucleoli, stress granules, P bodies, and paraspeckles, are formed as a result of LLPS [9]. LLPS of TLS/FUS are regulated by various factors including post-translational modification, external environment, or interaction with proteins and RNAs. The arginine methylation of TLS/FUS alters cation–pie interaction [19], and phosphorylation of TLS/FUS IDR disrupts aggregation [20]. The arginine methylation of TLS/FUS alters cation–pie interaction [19], and phosphorylation of TLS/FUS IDR disrupts aggregation [20] The external environment, such as salt concentration, pH, temperature, or shear stress, could affect TLS/FUS LLPS [19,21]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations

Methods

Results

Conclusion