Tandem Stem-Loops in roX RNAs Act Together to Mediate X Chromosome Dosage Compensation in Drosophila

Abstract

Summary Dosage compensation in Drosophila is an epigenetic phenomenon utilizing proteins and long noncoding RNAs (lncRNAs) for transcriptional upregulation of the male X chromosome. Here, by using UV crosslinking followed by deep sequencing, we show that two enzymes in the Male-Specific Lethal complex, MLE RNA helicase and MSL2 ubiquitin ligase, bind evolutionarily conserved domains containing tandem stem loops in roX1 and roX2 RNAs in vivo. These domains constitute the minimal RNA unit present in multiple copies in diverse arrangements for nucleation of the MSL complex. MLE binds to these domains with distinct ATP-independent and ATP-dependent behavior. Importantly, we show that different roX RNA domains have overlapping function, since only combinatorial mutations in the tandem stem loops result in severe loss of dosage compensation and consequently male-specific lethality. We propose that repetitive structural motifs in lncRNAs could provide plasticity during multiprotein complex assemblies to ensure efficient targeting in cis or in trans along chromosomes.

A mutually exclusive stem–loop arrangement in roX2 RNA is essential for X-chromosome regulation in Drosophila

“…1E; Supplemental Fig. S1A,B), which is consistent with our previous report and shows that the new method is indeed comparable with iCLIP (Ilik et al 2013). Second, in the relative absence of roX1 RNA in S2 cells (Johansson et al 2011), we also observed several mRNAs that were bound by MLE at their introns and some that were bound at their 3 ′ untranslated regions (UTRs; 145 peaks are located on introns of 58 genes, and 72 peaks are located on 3 ′ UTRs of 13 genes).…”

“…We showed previously that MLE has an unusually strong preference for two lncRNAs in Drosophila cells: roX1 and roX2 (Ilik et al 2013). Binding events on these two RNAs, as determined by iCLIP (individual nucleotide-resolution cross-linking and immunoprecipitation) (König et al 2010), comprise ∼80% of all binding events.…”

“…DHX9 was also shown to be able to unwind long stretches of dsRNA without apparent dissociation from its substrate in vitro (Koh et al 2014). In Drosophila, genetic and biochemical evidence suggests that MLE incorporates roX long noncoding RNAs (lncRNAs) into the malespecific lethal (MSL) complex that is composed also of MSL1, MSL2, MSL3, and MOF (Ilik and Akhtar 2009;Ilik et al 2013;Maenner et al 2013). However, how MLE carries out this function and why an RNA helicase is even required to incorporate these lncRNAs into the MSL complex are not known.lncRNAs have been proposed to have many functions, ranging from the classical assembly platforms or scaffolds that bring together various enzymatic activities for regulation of transcription to decoys that work by sequestering protein factors in a regulated way, while a few possess enzymatic activities themselves, such as RNaseP and the ribosome (Steitz and Moore 2003).…”

Dosage Compensation inDrosophila

“…The minimal protein core complex lacking roX RNAs can still specifically acetylate histone H4 on lysine 16 within nucleosomes in vitro (Morales et al 2004), and overexpression of MSL proteins can partially overcome the lack of roX RNAs, suggesting that the proteins possess all of the essential functions of dosage compensation but require the RNAs to stimulate assembly and spreading (Oh et al 2003). Assembly of the complex is initiated when the secondary structure of the roX RNAs is modified, allowing the binding of MSL2 and providing the core for the full recruitment of the other MSL subunits (Ilik et al 2013;Maenner et al 2013). …”

UNR facilitates the interaction of MLE with the lncRNA roX2 during Drosophila dosage compensation

“…The RNA helicase MLE colocalizes with MSL2 at HAS and facilitates the incorporation of roX into the complex. The RNA is important for the distribution of the MSL-DCC along the X-chromosome, where it activates target genes 2,[5][6][7] .…”