DHX15-independent roles for TFIP11 in U6 snRNA modification, U4/U6.U5 tri-snRNP assembly and pre-mRNA splicing fidelity

Amandine Duchemin,Ludivine Wacheul,Franck Dequiedt,Catherine Michaux,Marc Thiry,Eric Hervouet,Paul Peixoto,Yann Audic,Denis L J Lafontaine,Agnès Mereau,Tina O’Grady,Denis Mottet,Sarah Hanache,Eric Perpète,Felix G M Ernst

doi:10.1038/s41467-021-26932-2

Abstract

The U6 snRNA, the core catalytic component of the spliceosome, is extensively modified post-transcriptionally, with 2’-O-methylation being most common. However, how U6 2’-O-methylation is regulated remains largely unknown. Here we report that TFIP11, the human homolog of the yeast spliceosome disassembly factor Ntr1, localizes to nucleoli and Cajal Bodies and is essential for the 2’-O-methylation of U6. Mechanistically, we demonstrate that TFIP11 knockdown reduces the association of U6 snRNA with fibrillarin and associated snoRNAs, therefore altering U6 2′-O-methylation. We show U6 snRNA hypomethylation is associated with changes in assembly of the U4/U6.U5 tri-snRNP leading to defects in spliceosome assembly and alterations in splicing fidelity. Strikingly, this function of TFIP11 is independent of the RNA helicase DHX15, its known partner in yeast. In sum, our study demonstrates an unrecognized function for TFIP11 in U6 snRNP modification and U4/U6.U5 tri-snRNP assembly, identifying TFIP11 as a critical spliceosome assembly regulator.

Highlights

The U6 snRNA, the core catalytic component of the spliceosome, is extensively modified post-transcriptionally, with 2’-O-methylation being most common
Acting as a co-factor of the ATP-dependent DEAH-box RNA helicase DHX15, the G-patch protein TFIP11 was initially described in yeast as a splicing factor that catalyzes the disassembly of the lariat-spliceosome[12,13,14,15]
In contrast to these studies, we reveal DHX15-independent functions for TFIP11 in human cells and demonstrate that TFIP11 should be considered a key assembly factor, regulating the 2’-O-methylation of U6 snRNA, U4/U6.U5 tri-snRNP formation and spliceosome assembly

Summary

Introduction

The U6 snRNA, the core catalytic component of the spliceosome, is extensively modified post-transcriptionally, with 2’-O-methylation being most common. We show U6 snRNA hypomethylation is associated with changes in assembly of the U4/U6.U5 tri-snRNP leading to defects in spliceosome assembly and alterations in splicing fidelity. This function of TFIP11 is independent of the RNA helicase DHX15, its known partner in yeast. It is thought that modifications of U snRNAs may influence numerous aspects of pre-mRNA splicing including the interaction of U snRNAs with each other, interaction of U snRNAs with spliceosomal proteins, and U snRNP assembly, stability, and catalytic activity in the spliceosome. Box H/ACA snoRNAs associate with a different set of core proteins (NHP2, NOP10, GAR1 and DKC1) and control site-specific pseudouridylation of RNA7. The full set of snoRNP assembly factors has not yet been fully identified

Methods

Results

Conclusion