Structure of the RNA Helicase MLE Reveals the Molecular Mechanisms for Uridine Specificity and RNA-ATP Coupling

Prabu, J.R.; Müller, Marisa; Thomae, Andreas W.; Schüssler, Steffen; Bonneau, Fabien; Becker, Peter B.; Conti, Elena

doi:10.1016/j.molcel.2015.10.011

Cited by 68 publications

(207 citation statements)

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“…Our results show that, as expected from our previous knowledge of MLE-roX interactions, the top target of MLE in S2 cells is roX2. Through the analysis of point mutants, we verified that the N-terminal dsRBDs (and especially dsRBD2) are crucial for MLE's interactions with RNA, in agreement with a recent crystal structure of MLE (Rajan Prabu et al 2015). While analyzing the enzymatically inactive MLE mutant MLE GET , we serendipitously discovered that uvCLAP data, in addition to providing us with nucleotide-resolution binding sites, also contain crucial secondary structure information about MLE's in vivo RNA targets.…”

Section: Discussionsupporting

confidence: 82%

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

et al. 2017

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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.

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Section: Discussionsupporting

confidence: 82%

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

et al. 2017

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“…The basic features of this mechanism are the same as determined for other members of the DEAH-box family (31,33). These basic features, including a requirement for translocation, probably reflect the presence in DEAH-box proteins of C-terminal domains that surround nucleic acid substrates and are likely important for translocation (33,(43)(44)(45), as well as conserved sequence motifs within the helicase core. While we established this mechanism using principally DNA G4s, analogous RNA constructs were processed by DHX36 with similar rates, indicating that DHX36 likely uses the same mechanism to disrupt DNA and RNA G4s.…”

Section: Discussionmentioning

confidence: 60%

“…6D), indicating that they dissociate more slowly. Interesting, the MLE helicase has an analogous sequence preference, binding tightly to U-rich ssRNAs (43). The maximal rate constant for DHX36-mediated disruption of a G4 with a T 15 extension is also lower than that for a G4 with an A 15 extension, as shown in the model in Fig.…”

Section: Discussionmentioning

confidence: 89%

“…The requirement for the extension to be approximately 10 nt for optimal activity is consistent with a model in which its role is in loading of the helicase core. A recentlysolved crystal structure of the related helicase MLE suggests a binding region for single-stranded nucleotides of approximately this length (43).…”

Section: Efficient Dhx36 Activity Requires a Singlestranded 3′ Extensmentioning

confidence: 99%

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confidence: 99%

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The G-quadruplex (G4) resolvase DHX36 efficiently and specifically disrupts DNA G4s via a translocation-based helicase mechanism

Yangyuoru

Bradburn

et al. 2018

Journal of Biological Chemistry

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Single-stranded DNA (ssDNA) and RNA regions that include at least four closely spaced runs of three or more consecutive guanosines strongly tend to fold into stable G-quadruplexes (G4s). G4s play key roles as DNA regulatory sites and as kinetic traps that can inhibit biological processes, but how G4s are regulated in cells remains largely unknown. Here, we developed a kinetic framework for G4 disruption by DEAH-box helicase 36 (DHX36), the dominant G4 resolvase in human cells. Using tetramolecular DNA and RNA G4s with four to six G-quartets, we found that DHX36-mediated disruption is highly efficient, with rates that depend on G4 length under saturating conditions (k cat ) but not under subsaturating conditions (k cat /K M ). These results suggest that a step during G4 disruption limits the k cat value and that DHX36 binding limits k cat /K M . Similar results were obtained for unimolecular DNA G4s. DHX36 activity depended on a 3′ ssDNA extension and was blocked by a polyethylene glycol linker, indicating that DHX36 loads onto the extension and translocates 3′-5′ toward the G4. DHX36 unwound dsDNA poorly compared with G4s of comparable intrinsic lifetime. Interestingly, we observed that DHX36 has striking 3′-extension sequence preferences that differ for G4 disruption and dsDNA unwinding, most likely arising from differences in the rate-limiting step for the two activities. Our results indicate that DHX36 disrupts G4s with a conventional helicase mechanism that is tuned for great efficiency and specificity for G4s. The dependence of DHX36 on the 3′-extension sequence suggests that the extent of formation of genomic G4s may not track directly with G4 stability. ___________________________________Single-stranded DNA and RNA segments that include at least four closely-spaced runs of three or more consecutive guanosine nucleotides have a strong intrinsic propensity to fold into stable G-quadruplex structures (G4s) (1) (Fig. 1A, top left). The genomes of humans and many other eukaryotes are replete with putative G4-forming sequences, and pronounced, conserved patterns have been observed in their distribution, suggesting that Gquadruplex structures form at least transiently in cells and perform conserved functions (2-6). Supporting this hypothesis, G4s have been detected in cells for both .To function as regulatory elements, G4s must be folded and disrupted in a regulated way. In addition, processes that require single-stranded DNA or RNA, such as replication and translation, require that G4s be temporarily disrupted. The high stability and long intrinsic lifetime of G4s suggest Mechanism of G4 disruption by DHX362 that their efficient disruption requires the activity of ATP-dependent enzymes such as helicases.Supporting this view, incorporation of sequences predicted to form particularly stable G4s leads to genetic instability in yeast, suggesting that these G4 structures are not processed efficiently and pose blocks to replication (12). Indeed, several helicases have been shown to possess G4 disruption acti...

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Challenges in the analysis of long noncoding RNA functionality

Leone

Santoro

2016

FEBS Letters

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Long noncoding RNA (lncRNA) are emerging as important regulators of diverse biological functions. Although mechanistic models are starting to emerge, it is also clear that the lncRNA field needs appropriate model systems in order to better elucidate the functions of lncRNA and their roles in both physiological and pathological conditions. The field of lncRNA is new, and the biochemical and genetic methods used to address function and mechanisms of lncRNA have only recently been developed or adapted from techniques used to investigate protein-coding genes. In this review, we discuss the strengths and weaknesses of available techniques for the analysis of chromatin-associated lncRNA and emerging models for the recruitment to specific genomic sites such as triple-helix, RNA-protein-DNA recognition and proximity-guided search models.

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Structure of the RNA Helicase MLE Reveals the Molecular Mechanisms for Uridine Specificity and RNA-ATP Coupling

Cited by 68 publications

References 51 publications

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

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

The G-quadruplex (G4) resolvase DHX36 efficiently and specifically disrupts DNA G4s via a translocation-based helicase mechanism

Challenges in the analysis of long noncoding RNA functionality

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