The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts

Abstract

RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi) and microRNA (miRNA) pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss) RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.

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

“…NMR titrations for dsRBD1,2 were performed at 0.3 mM protein concentration. 15 N labelled dsRBD1,2 was titrated with various ratios of different RNA oligos described in the results section and monitored by recording 1 H, 15 N HSQC spectra. For SL7 14merLoop titrations, deuterated 15 N labelled dsRBD1,2 protein was used.…”

“…Therefore, we recorded 1 H, 15 N-HSQC spectra of the individual dsRBD domains and compared them with the spectrum of the tandem dsRBD1,2 construct (Figure 2A). The three spectra are very well superimposable and the chemical shifts of the individual dsRBD 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).…”

“…Therefore, we found it surprising that only dsRBD2 had been identified to bind RNA (19). To determine the contribution of each dsRBD domain for RNA binding, we performed 1 H, 15 N-HSQC NMR titrations with each individual dsRBD domain and dsRNA (Figure 4, S3). Uridine-rich stem-loop structures and single-stranded polyU sequences have been shown as preferred substrates for MLE (9)(10)(11) and remodeling of the uridine-rich roX-box regions in roX RNA has been shown to recruit MSL2 to the MLE-roX2 complex, suggesting a role of MLE during initial assembly of the MSL complex (10,17) ( Figure 1A).…”

“…To understand the binding of individual dsRBD domains in the context of dsRBD1,2 and to study if there is any cooperativity between the two domains for RNA binding, we performed filter binding assays and 1 H, 15 N-HSQC NMR titrations of dsRNA into tandem dsRBD1,2 domain ( Figure S5). As determined from filter binding experiments, the affinity of dsRBD1,2 for SL7 binding was a modest 2-fold higher than the dsRBD2 alone suggesting an additive effect between the two domains ( Figure S5D).…”

“…It shares 50% sequence identity with its human homologue DHX9, which has been shown to bind both DNA and RNA (13) and to be involved in diverse cellular functions ranging from DNA replication, microRNA processing, RNA processing and transport, transcription and translation regulation and maintenance of genomic stability (14). Besides its primarily known role in dosage compensation in Drosophila, MLE has been shown to be involved in RNA editing and siRNA processing (15,16). During Drosophila dosage compensation, MLE has been proposed to bind to SL3 and to a region around SL7 of roX2 to extensively remodel the RNA and to form an alternative stem loop (9,10,17) (Figure 1A).…”