Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless.

RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We explored the interplay between the RecA and auxiliary domains of the RNA helicase maleless (MLE) from Drosophila using structural and functional studies. We discovered that MLE exists in a dsRNA-bound open conformation and that the auxiliary dsRBD2 domain aligns the substrate RNA with the accessible helicase tunnel. In an ATP-dependent manner, dsRBD2 associates with the helicase module, leading to tunnel closure around ssRNA. Furthermore, our structures provide a rationale for blunt-ended dsRNA unwinding and 3'-5' translocation by MLE. Structure-based MLE mutations confirm the functional relevance of our model for RNA unwinding. Our findings contribute to our understanding of the fundamental mechanics of auxiliary domains in DExH helicase MLE, which serves as a model for its human ortholog and potential therapeutic target, DHX9/RHA.

The XRN1-regulated RNA helicase activity of YTHDC2 ensures mouse fertility independently of m6A recognition.

The functional consequence of N6-methyladenosine (m6A) RNA modification is mediated by "reader" proteins of the YTH family. YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but its molecular function is poorly understood. Here, we identify U-rich motifs as binding sites of YTHDC2 on 3' UTRs of mouse testicular RNA targets. Although its YTH domain is an m6A-binder in vitro, the YTH point mutant mice are fertile. Significantly, the loss of its 3'→5' RNA helicase activity causes mouse infertility, with the catalytic-dead mutation being dominant negative. Biochemical studies reveal that the weak helicase activity of YTHDC2 is enhanced by its interaction with the 5'→3' exoribonuclease XRN1. Single-cell transcriptomics indicate that Ythdc2 mutant mitotic germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity that fail to progress further into meiosis. Finally, our demonstration that ythdc2 mutant zebrafish are infertile highlights its conserved role in animal germ cell development.

Sexually dimorphic RNA helicases DDX3X and DDX3Y differentially regulate RNA metabolism through phase separation.

Sex differences are pervasive in human health and disease. One major key to sex-biased differences lies in the sex chromosomes. Although the functions of the X chromosome proteins are well appreciated, how they compare with their Y chromosome homologs remains elusive. Herein, using ensemble and single-molecule techniques, we report that the sex chromosome-encoded RNA helicases DDX3X and DDX3Y are distinct in their propensities for liquid-liquid phase separation (LLPS), dissolution, and translation repression. We demonstrate that the N-terminal intrinsically disordered region of DDX3Y more strongly promotes LLPS than the corresponding region of DDX3X and that the weaker ATPase activity of DDX3Y, compared with DDX3X, contributes to the slower disassembly dynamics of DDX3Y-positive condensates. Interestingly, DDX3Y-dependent LLPS represses mRNA translation and enhances aggregation of FUS more strongly than DDX3X-dependent LLPS. Our study provides a platform for future comparisons of sex chromosome-encoded protein homologs, providing insights into sex differences in RNA metabolism and human disease.

YTHDC2 control of gametogenesis requires helicase activity but not m6A binding.

Mechanisms regulating meiotic progression in mammals are poorly understood. The N6-methyladenosine (m6A) reader and 3' → 5' RNA helicase YTHDC2 switches cells from mitotic to meiotic gene expression programs and is essential for meiotic entry, but how this critical cell fate change is accomplished is unknown. Here, we provide insight into its mechanism and implicate YTHDC2 in having a broad role in gene regulation during multiple meiotic stages. Unexpectedly, mutation of the m6A-binding pocket of YTHDC2 had no detectable effect on gametogenesis and mouse fertility, suggesting that YTHDC2 function is m6A-independent. Supporting this conclusion, CLIP data defined YTHDC2-binding sites on mRNA as U-rich and UG-rich motif-containing regions within 3' UTRs and coding sequences, distinct from the sites that contain m6A during spermatogenesis. Complete loss of YTHDC2 during meiotic entry did not substantially alter translation of its mRNA binding targets in whole-testis ribosome profiling assays but did modestly affect their steady-state levels. Mutation of the ATPase motif in the helicase domain of YTHDC2 did not affect meiotic entry, but it blocked meiotic prophase I progression, causing sterility. Our findings inform a model in which YTHDC2 binds transcripts independent of m6A status and regulates gene expression during multiple stages of meiosis by distinct mechanisms.

Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven lymphomagenesis.

DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.

Making ends meet: a universal driver of large ribosomal subunit biogenesis.

A common aspect of ribosome assembly, conserved across all domains of life, is the establishment of connections between the 5' and 3' ends of the large subunit (LSU) ribosomal RNA (rRNA) to initiate rRNA domain compaction and subunit assembly. We discuss the diverse mechanisms employed in different organisms to accomplish this important event.

Unveiling the DHX15-G-patch interplay in retroviral RNA packaging.

We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.

Spliceosomal helicases DDX41/SACY-1 and PRP22/MOG-5 both contribute to proofreading against proximal 3' splice site usage.

RNA helicases drive necessary rearrangements and ensure fidelity during the pre-mRNA splicing cycle. DEAD-box helicase DDX41 has been linked to human disease and has recently been shown to interact with DEAH-box helicase PRP22 in the spliceosomal C* complex, yet its function in splicing remains unknown. Depletion of DDX41 homolog SACY-1 from somatic cells has been previously shown to lead to changes in alternative 3' splice site (3'ss) usage. Here, we show by transcriptomic analysis of published and novel data sets that SACY-1 perturbation causes a previously unreported pattern in alternative 3' splicing in introns with pairs of 3' splice sites ≤18 nt away from each other. We find that both SACY-1 depletion and the allele sacy-1(G533R) lead to a striking unidirectional increase in the usage of the proximal (upstream) 3'ss. We previously discovered a similar alternative splicing pattern between germline tissue and somatic tissue, in which there is a unidirectional increase in proximal 3'ss usage in the germline for ∼200 events; many of the somatic SACY-1 alternative 3' splicing events overlap with these developmentally regulated events. We generated targeted mutant alleles of the Caenorhabditis elegans homolog of PRP22, mog-5, in the region of MOG-5 that is predicted to interact with SACY-1 based on the human C* structure. These viable alleles, and a mimic of the myelodysplastic syndrome-associated allele DDX41(R525H), all promote the usage of proximal alternative adjacent 3' splice sites. We show that PRP22/MOG-5 and DDX41/SACY-1 have overlapping roles in proofreading the 3'ss.

RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination.

Class switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus.

Cryo-EM Structures of MDA5-dsRNA Filaments at Different Stages of ATP Hydrolysis.

Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Long cytosolic dsRNA is recognized by MDA5. The cooperative assembly of MDA5 into helical filaments on dsRNA nucleates the assembly of a multiprotein type I interferon signaling platform. Here, we determined cryoelectron microscopy (cryo-EM) structures of MDA5-dsRNA filaments with different helical twists and bound nucleotide analogs at resolutions sufficient to build and refine atomic models. The structures identify the filament-forming interfaces, which encode the dsRNA binding cooperativity and length specificity of MDA5. The predominantly hydrophobic interface contacts confer flexibility, reflected in the variable helical twist within filaments. Mutation of filament-forming residues can result in loss or gain of signaling activity. Each MDA5 molecule spans 14 or 15 RNA base pairs, depending on the twist. Variations in twist also correlate with variations in the occupancy and type of nucleotide in the active site, providing insights on how ATP hydrolysis contributes to MDA5-dsRNA recognition.

The putative RNA helicase DDX1 associates with the nuclear RNA exosome and modulates RNA/DNA hybrids (R-loops).

The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A), followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing. We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.

Contrasting functions of ATP hydrolysis by MDA5 and LGP2 in viral RNA sensing.

Cytosolic long dsRNA, among the most potent proinflammatory signals, is recognized by melanoma differentiation-associated protein 5 (MDA5). MDA5 binds dsRNA cooperatively forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-ß responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. Consecutive rounds of ATP hydrolysis amplify the filament footprint, displacing tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling identifies an internally bound MDA5-LGP2-RNA complex, with the LGP2 C-terminal tail forming the key contacts with MDA5. These contacts are specifically required for NTP-dependent internal RNA binding. We conclude that NTPase-dependent binding of LGP2 to internal dsRNA sites complements NTPase-independent binding to dsRNA ends, via distinct binding modes, to increase the number and signaling output of MDA5-dsRNA complexes.

Complex interplay between FMRP and DHX9 during DNA replication stress.

Mutations in, or deficiency of, fragile X messenger ribonucleoprotein (FMRP) is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently reported that FXS patient cells lacking FMRP sustain higher level of DNA double-strand breaks (DSBs) than normal cells, specifically at sequences prone to forming R-loops, a phenotype further exacerbated by DNA replication stress. Moreover, expression of FMRP, and not an FMRPI304N mutant known to cause FXS, reduced R-loop-associated DSBs. We subsequently reported that recombinant FMRP directly binds R-loops, primarily through the carboxyl terminal intrinsically disordered region. Here, we show that FMRP directly interacts with an RNA helicase, DHX9. This interaction, which is mediated by the amino terminal structured domain of FMRP, is reduced with FMRPI304N. We also show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids and the inhibition is also dependent on the amino terminus. Furthermore, the FMRPI304N mutation causes both FMRP and DHX9 to persist on the chromatin in replication stress. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where their proper interaction leads to dissociation of both proteins from the fully resolved R-loop. We propose that the absence or the loss of function of FMRP leads to persistent presence of DHX9 or both proteins, respectively, on the unresolved R-loop, ultimately leading to DSBs. Our study sheds new light on our understanding of the genome functions of FMRP.

The Arabidopsis tDR Ala forms G-quadruplex structures that can be unwound by the DExH1 DEA(D/H)-box RNA helicase.

As in many other organisms, tRNA-derived RNAs (tDRs) exist in plants and likely have multiple functions. We previously showed that tDRs are present in Arabidopsis under normal growth conditions, and that the ones originating from alanine tRNAs are the most abundant in leaves. We also showed that tDRs Ala of 20 nt produced from mature tRNAAla (AGC) can block in vitro protein translation. Here, we report that first, these tDRs Ala (AGC) can be found within peculiar foci in the cell that are neither P-bodies nor stress granules and, second, that they assemble into intermolecular RNA G-quadruplex (rG4) structures. Such tDR Ala rG4 structures can specifically interact with an Arabidopsis DEA(D/H) RNA helicase, the DExH1 protein, and unwind them. The rG4-DExH1 protein interaction relies on a glycine-arginine domain with RGG/RG/GR/GRR motifs present at the N-terminal extremity of the protein. Mutations on the four guanine residues located at the 5' extremity of the tDR Ala abolish its rG4 structure assembly, association with the DExH1 protein, and foci formation, but they do not prevent protein translation inhibition in vitro. Our data suggest that the sequestration of tDRs Ala into rG4 complexes might represent a way to modulate accessible and functional tDRs for translation inhibition within the plant cell via the activity of a specific RNA helicase, DExH1.

Modulation of RNA-binding properties of the RNA helicase UPF1 by its activator UPF2.

The NMD helicase UPF1 is a prototype of the superfamily 1 (SF1) of RNA helicases that bind RNA with high affinity and translocate on it in an ATP-dependent manner. Previous studies showed that UPF1 has a low basal catalytic activity that is greatly enhanced upon binding of its interaction partner, UPF2. Activation of UPF1 by UPF2 entails a large conformational change that switches the helicase from an RNA-clamping mode to an RNA-unwinding mode. The ability of UPF1 to bind RNA was expected to be unaffected by this activation mechanism. Here we show, using a combination of biochemical and biophysical methods, that binding of UPF2 to UPF1 drastically reduces the affinity of UPF1 for RNA, leading to a release of the bound RNA. Although UPF2 is capable of binding RNA in vitro, our results suggest that dissociation of the UPF1-RNA complex is not a consequence of direct competition in RNA binding but rather an allosteric effect that is likely mediated by the conformational change in UPF1 that is induced upon binding its activator. We discuss these results in light of transient interactions forged during mRNP assembly, particularly in the UPF1-dependent mRNA decay pathways.

The DDX6/KIFC1 signaling axis, as regulated by YY1, contributes to the malignant behavior of pancreatic cancer.

Human DEAD/H box RNA helicase DDX6 acts as an oncogene in several different types of cancer, where it participates in RNA processing. Nevertheless, the role of DDX6 in pancreatic cancer (PC), together with the underlying mechanism, has yet to be fully elucidated. In the present study, compared with adjacent tissues, the level of DDX6 was abnormally increased in human PC tissues, and this increased level of expression was associated with poor prognosis. Furthermore, the role of DDX6 in PC was investigated by overexpressing or silencing the DDX6 in the PC cell lines, SW1990 and PaTu-8988t. A xenograft model was established by injecting nude mice with either DDX6-overexpressing or DDX6-silenced SW1990 cells. DDX6 overexpression promoted the proliferation and cell cycle transition, inhibited the cell apoptosis of PC cells, and accelerated tumor formation, whereas DDX6 knockdown elicited the opposite effects. DDX6 exerted positive effects on PC. RNA immunoprecipitation assay showed that DDX6 bound to kinesin family member C1 (KIFC1) mRNA, which was further confirmed by RNA pull-down assay. These results suggested that DDX6 positively regulated the expression of KIFC1. KIFC1 overexpression enhanced the proliferative capability of PC cells with DDX6 knockdown and inhibited their apoptosis. By contrast, DDX6 overexpression reversed the inhibitory effect of KIFC1 silencing on tumor proliferation. Subsequently, the transcription factor Yin Yang 1 (YY1) was shown to negatively regulate DDX6 at both the mRNA and protein levels. Dual-luciferase reporter assay verified that YY1 targeted the promoter of DDX6 and inhibited its transcription. High expression levels of YY1 decreased the proliferation of PC cells and promoted cell apoptosis, although these effects were reversed by DDX6 overexpression. Taken together, YY1 may target the DDX6/KIFC1 axis, thereby negatively regulating its expression, leading to an inhibitory effect on pancreatic tumor.

Regulation of m6A Transcripts by the 3'→5' RNA Helicase YTHDC2 Is Essential for a Successful Meiotic Program in the Mammalian Germline.

N6-methyladenosine (m6A) is an essential internal RNA modification that is critical for gene expression control in most organisms. Proteins with a YTH domain recognize m6A marks and are mediators of molecular functions like RNA splicing, mRNA decay, and translation control. Here we demonstrate that YTH domain-containing 2 (YTHDC2) is an m6A reader that is essential for male and female fertility in mice. High-throughput mapping of the m6A transcriptome and expression analysis in the Yhtdc2 mutant testes reveal an upregulation of m6A-enriched transcripts. Our biochemical studies indicate that YTHDC2 is an RNA-induced ATPase with a 3'→5' RNA helicase activity. Furthermore, YTHDC2 recruits the 5'→3' exoribonuclease XRN1 via Ankyrin repeats that are inserted in between the RecA modules of the RNA helicase domain. Our studies reveal a role for YTHDC2 in modulating the levels of m6A-modified germline transcripts to maintain a gene expression program that is conducive for progression through meiosis.

HIV-1 hypermethylated guanosine cap licenses specialized translation unaffected by mTOR.

Appended to the 5' end of nascent RNA polymerase II transcripts is 7-methyl guanosine (m7G-cap) that engages nuclear cap-binding complex (CBC) to facilitate messenger RNA (mRNA) maturation. Mature mRNAs exchange CBC for eIF4E, the rate-limiting translation factor that is controlled through mTOR. Experiments in immune cells have now documented HIV-1 incompletely processed transcripts exhibited hypermethylated m7G-cap and that the down-regulation of the trimethylguanosine synthetase-1-reduced HIV-1 infectivity and virion protein synthesis by several orders of magnitude. HIV-1 cap hypermethylation required nuclear RNA helicase A (RHA)/DHX9 interaction with the shape of the 5' untranslated region (UTR) primer binding site (PBS) segment. Down-regulation of RHA or the anomalous shape of the PBS segment abrogated hypermethylated caps and derepressed eIF4E binding for virion protein translation during global down-regulation of host translation. mTOR inhibition was detrimental to HIV-1 proliferation and attenuated Tat, Rev, and Nef synthesis. This study identified mutually exclusive translation pathways and the calibration of virion structural/accessory protein synthesis with de novo synthesis of the viral regulatory proteins. The hypermethylation of select, viral mRNA resulted in CBC exchange to heterodimeric CBP80/NCBP3 that expanded the functional capacity of HIV-1 in immune cells.

The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage.

DEAD-box RNA helicases play important roles in a wide range of metabolic processes. Regulatory proteins can stimulate or block the activity of DEAD-box helicases. Here, we show that LOTUS (Limkain, Oskar, and Tudor containing proteins 5 and 7) domains present in the germline proteins Oskar, TDRD5 (Tudor domain-containing 5), and TDRD7 bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. Our crystal structure of the LOTUS domain of Oskar in complex with the C-terminal RecA-like domain of Vasa reveals that the LOTUS domain occupies a surface on a DEAD-box helicase not implicated previously in the regulation of the enzyme's activity. We show that, in vivo, the localization of Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins. The binding and stimulation of Vasa DEAD-box helicases by LOTUS domains are widely conserved.

The RES complex is required for efficient transformation of the precatalytic B spliceosome into an activated Bact complex.

The precise function of the trimeric retention and splicing (RES) complex in pre-mRNA splicing remains unclear. Here we dissected the role of RES during the assembly and activation of yeast spliceosomes. The efficiency of pre-mRNA splicing was significantly lower in the absence of the RES protein Snu17, and the recruitment of its binding partners, Pml1 (pre-mRNA leakage protein 1) and Bud13 (bud site selection protein 13), to the spliceosome was either abolished or substantially reduced. RES was not required for the assembly of spliceosomal B complexes, but its absence hindered efficient Bact complex formation. ΔRES spliceosomes were no longer strictly dependent on Prp2 activity for their catalytic activation, suggesting that they are structurally compromised. Addition of Prp2, Spp2, and UTP to affinity-purified ΔRES B or a mixture of B/Bact complexes formed on wild-type pre-mRNA led to their disassembly. However, no substantial disassembly was observed with ΔRES spliceosomes formed on a truncated pre-mRNA that allows Prp2 binding but blocks its activity. Thus, in the absence of RES, Prp2 appears to bind prematurely, leading to the disassembly of the ΔRES B complexes to which it binds. Our data suggest that Prp2 can dismantle B complexes with an aberrant protein composition, suggesting that it may proofread the spliceosome's RNP structure prior to activation.