Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

Pravin Kumar Ankush Jagtap,Pawel Masiewicz,Sören Von Bülow,Nele Merret Hollmann,Bernd Simon,Peter B Becker,Marisa Müller,Janosch Hennig,Po-Chia Chen,Andreas W Thomae

doi:10.1093/nar/gkz125

Pravin Kumar Ankush Jagtap, Pawel Masiewicz + Show 8 more

Open Access

https://doi.org/10.1093/nar/gkz125

Copy DOI

Abstract

Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR and SAXS data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAXK RNA binding motifs. NMR titrations combined with filter binding experiments and isothermal titration calorimetry (ITC) document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

Highlights

In sexually reproducing organisms, the number of X chromosomes differs between males and females
nuclear magnetic resonance spectroscopy (NMR) titrations combined with filter binding experiments and isothermal titration calorimetry (ITC) document the contribution of dsRBD1 to double-stranded RNA (dsRNA) binding in vitro
The three spectra are very well superimposable and the chemical shifts of the individual double-stranded RNA binding domains (dsRBDs) domains are very similar to the tandem dsRBD1,2 except for the expected changes for residues at respective termini caused by the changed chemical environment of the additional peptide bonds (Figure 2B)

Summary

Introduction

The number of X chromosomes differs between males and females. In Drosophila males, the male-specific-lethal (MSL) complex binds with remarkable X-chromosome specificity to PionX sites [4], spreads out along the X-chromosome and achieves two-fold hypertranscription [5]. In females, this would be lethal and the RNA binding proteins (RBPs) Sex-lethal (Sxl), Upstream-of-N-Ras (Unr) and Hrp repress translation of msl mRNA to prevent formation of the MSL complex [6,7,8,9]. The MSL complex consists of four core proteins, MSL1, MSL2, MSL3 and males-absent-onthe-first (MOF) and further accommodates, at least during certain stages of dosage compensation, the helicase Maleless (MLE) and two long non-coding RNAs (lncRNAs), roX1 and roX2 (for ‘RNA-on-the-X’) [2]. During assembly of the MSL complex a critical step is the remodeling of roX2 by MLE [11], which is further assisted by Unr [12]

Methods

Results

Conclusion