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

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

doi:10.1093/nar/gkz125

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

Nucleic Acids Res. 2019 May 7;47(8):4319-4333. doi: 10.1093/nar/gkz125.

Affiliations

¹ Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL) Heidelberg, 69117 Heidelberg, Germany.
² Biomedical Center and Center for Integrated Protein Science, Ludwig-Maximilians-University, 82152 Martinsried, Germany.
³ Collaboration for joint PhD degree between EMBL and Heidelberg University, Faculty of Biosciences.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Animals
Binding Sites
Chromosomal Proteins, Non-Histone / chemistry*
Chromosomal Proteins, Non-Histone / genetics
Chromosomal Proteins, Non-Histone / metabolism
Cloning, Molecular
DNA Helicases / chemistry*
DNA Helicases / genetics
DNA Helicases / metabolism
Dosage Compensation, Genetic
Double-Stranded RNA Binding Motif
Drosophila Proteins / chemistry*
Drosophila Proteins / genetics
Drosophila Proteins / metabolism
Drosophila melanogaster / chemistry*
Drosophila melanogaster / genetics
Drosophila melanogaster / metabolism
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Gene Expression Regulation
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Kinetics
Male
Models, Molecular
Nucleic Acid Conformation
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
RNA, Long Noncoding / chemistry*
RNA, Long Noncoding / genetics
RNA, Long Noncoding / metabolism
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Sequence Alignment
Sequence Homology, Amino Acid
Transcription Factors / chemistry*
Transcription Factors / genetics
Transcription Factors / metabolism

Substances

Chromosomal Proteins, Non-Histone
Drosophila Proteins
RNA, Long Noncoding
Recombinant Proteins
Transcription Factors
mle protein, Drosophila
roX1 protein, Drosophila
DNA Helicases