Abstract
In Drosophila, dosage compensation globally upregulates the expression of genes located on male single X-chromosome. Maleless (MLE) helicase plays an essential role to incorporate the roX lncRNA into the dosage compensation complex (MSL-DCC), and such function is essentially dependent on its dsRNA-binding domains (dsRBDs). Here, we report a 2.90Å crystal structure of tandem dsRBDs of MLE in complex with a 55mer stem-loop of roX2 (R2H1). MLE dsRBDs bind to R2H1 cooperatively and interact with two successive minor grooves and a major groove of R2H1, respectively. The recognition of R2H1 by MLE dsRBDs involves both shape- and sequence-specificity. Moreover, dsRBD2 displays a stronger RNA affinity than dsRBD1, and mutations of key residues in either MLE dsRBD remarkably reduce their affinities for roX2 both in vitro and in vivo. In Drosophila, the structure-based mle mutations generated using the CRISPR/Cas9 system, are partially male-lethal and indicate the inter-regulation among the components of the MSL-DCC at multiple levels. Hence, our research provides structural insights into the interactions between MLE dsRBDs and R2H1 and facilitates a deeper understanding of the mechanism by which MLE tandem dsRBDs play an indispensable role in specific recognition of roX and the assembly of the MSL-DCC in Drosophila dosage compensation.
Highlights
X-chromosomal dosage compensation processes exist in a wide range of eukaryotic organisms [1]
We identify the key residues of MLE dsRNAbinding domains (dsRBDs) that are responsible for the specific recognition of R2H1
Previous studies have revealed that MLE utilizes its Nterminal tandem dsRBDs to recognize the 5 hairpin cluster of roX2 (R2H1, R2H2 and R2H3) in an adenosine triphoshate (ATP)-independent manner––especially the helical region of R2H1 [19,21]
Summary
X-chromosomal dosage compensation processes exist in a wide range of eukaryotic organisms [1]. This biological process is essential for balancing the expression levels of Xlinked genes caused by the unequal number of X chromosomes between males and females [2]. One of the two X chromosomes is transcriptionally inactivated, through a process called X chromosome inactivation [3,4]. The compensation process in male Drosophila is mediated by the double upregulation of the transcription of genes located on the single X chromosome in males [5]. Dosage compensation in Drosophila is achieved by a ribonucleoprotein complex MSL-DCC [6]. The MSL-DCC selectively binds to the discontinuous high-affinity sites (HASs) of the male X chromosome and X chromosome-widely acetylates histone H4 lysine 16 (H4K16Ac) [9,10]. The acetylation mediated by the acetyltransferase activity of MOF loosens the chromatin fiber, promotes active gene transcription and up-
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