Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7

Emiko Uchikawa,Elodie Zhang,Xiao Han,Pierre Roblin,Bruno P Klaholz,Alexandre Durand,Anne-Catherine Dock-Bregeon,Kundhavai S Natchiar,Florence Proux

doi:10.1093/nar/gkv173

Abstract

The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3′-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3′ hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5′-end with the foot of the 3′ hairpin and has thus functional implications.

Highlights

The La-related proteins (LARPs) are involved in various important functions in RNA metabolism and are found in most eukaryotes [1]
Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5 -end with the foot of the 3 hairpin and has functional implications
Defining the domains of LARP7 required for the study

Summary

Introduction

The La-related proteins (LARPs) are involved in various important functions in RNA metabolism and are found in most eukaryotes [1]. Besides the essential role of the paradigmatic La protein in tRNA processing, and its involvement in transcription termination by binding to nascent transcripts generated by polymerase III [2], members of the LARP family are involved in the regulation of translation or demonstrate chaperoning activities [3]. LARP7 is the family member showing the highest sequence similarity to La, with the characteristic La module in the N-terminal third of the protein [3,8]. While La binds all nascent transcripts synthesized by RNA polymerase III via their shared termination motif, UUUOH, LARP7 binds almost exclusively to the non-coding RNA 7SK [9,10,11]. Other potential LARP7 homologs are found in ciliates, such as P65 in Tetrahymena thermophila, which has been found to assist in the correct folding of the telomerase RNA and hierarchical assembly of the RNP [14]

Methods

Results

Conclusion