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

Abstract

The X chromosome provides an ideal model system to study the contribution of RNA-protein interactions in epigenetic regulation. In male flies, roX long noncoding RNAs (lncRNAs) harbor several redundant domains to interact with the ubiquitin ligase male-specific lethal 2 (MSL2) and the RNA helicase Maleless (MLE) for X-chromosomal regulation. However, how these interactions provide the mechanics of spreading remains unknown. By using the uvCLAP (UV cross-linking and affinity purification) methodology, which provides unprecedented information about RNA secondary structures in vivo, we identified the minimal functional unit of roX2 RNA. By using wild-type and various MLE mutant derivatives, including a catalytically inactive MLE derivative, MLE GET , we show that the minimal roX RNA contains two mutually exclusive stem-loops that exist in a peculiar structural arrangement: When one stem-loop is unwound by MLE, an alternate structure can form, likely trapping MLE in this perpetually structured region. We show that this functional unit is necessary for dosage compensation, as mutations that disrupt this formation lead to male lethality. Thus, we propose that roX2 lncRNA contains an MLE-dependent affinity switch to enable reversible interactions of the MSL complex to allow dosage compensation of the X chromosome.[Keywords: dosage compensation; H4K16ac; MLE; X chromosome; acetylation; roX RNA] Supplemental material is available for this article. RNA helicases are a large family of proteins that have important roles in many aspects of RNA biology. Although the name "helicase" suggests that the main function of these proteins is to destabilize and remove RNA secondary structures, there are several RNA helicases, especially of the DEAD-box subfamily, that use their helicase domains to simply interact with RNA (such as eIF4A3) and not for remodeling. On the other hand, a closely related RNA helicase, eIF4A1, can unwind RNA structures but only when they are relatively weak and short (Rogers et al. 2001). At the other extreme, several helicases are proposed to act as annealers that facilitate the formation of structured RNA rather than the other way around (Jankowsky 2011).Thus, although the helicase family is quite large, specific functions assigned to helicases that involve the actual removal of secondary structures in vivo are rather sparse. Maleless (MLE), known primarily for its role in Drosophila dosage compensation, is an RNA helicase of the DExH family, which is composed of relatively large multidomain helicases that (unlike the DEAD-box family) are thought to work as processive RNA helicases.

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

“…Furthermore, MLE mutated in K54E alone (which was expressed to similar levels as K225E, (see Figure S8C), or in combination with K53E immunoprecipitated roX2 much better. We conclude that the main determinants of roX2 binding in cells are located in dsRBD2, in agreement with earlier work (17,18). (6) A functional MSL complex localizes to the X chromosome territory and can be easily identified by MSL2 immunostaining (41).…”

“…Previous structural and biochemical studies had only identified the role of dsRBD2 and G-patch for dsRNA binding (17)(18)(19)(20). The role of dsRBD1 in dsRNA binding was not clear due to no or just minor binding observed in electro mobility shift assays (EMSA) (9,19).…”

“…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). MLE's domain architecture consists of two N-terminal double-stranded RNA binding domains (dsRBDs), followed by the helicase core (RecA1, RecA2, HA2 and OB-fold domains) and a C-terminal glycine-rich region (Figure 1B).…”

The simultaneous interaction of MSL2 with CLAMP and DNA provides redundancy in the initiation of dosage compensation in Drosophila males

“…The interaction between MSL1 and MSL2 proteins forms the structural core of the DCC, and the C-terminal PEHE domain of MSL1 is responsible for interactions with MSL3 and MOF (Li et al, 2005;Kadlec et al, 2011;Hallacli et al, 2012). The MLE protein, an ATP-dependent RNA/DNA helicase of the DEAD subfamily, interacts with two noncoding roX RNAs, roX1 and roX2, to induce their unwinding (Maenner et al, 2013;Ilik et al, 2017), which allows them to bind to MSL2. Thus, roX RNAs link MLE and MSL2 proteins into a single complex.…”

CLAMP directly interacts with MSL2 to facilitateDrosophiladosage compensation

“…The MLE protein, an ATP-dependent RNA/DNA helicase of the DEAD subfamily, interacts with two noncoding roX RNAs, roX1 and roX2, and induces their unwinding (Ilik et al, 2017;Maenner et al, 2013), as a result, making both roX RNAs capable of binding MSL2. Thus, roX RNAs link the MLE and MSL2 proteins into a single complex.…”