ABSTRACT
DEAD- and DExH-box ATPases (DDX/DHXs) are abundant and highly conserved cellular enzymes ubiquitously involved in RNA processing. By remodeling RNA-RNA and RNA-protein interactions, they often function as gatekeepers that control the progression of diverse RNA maturation steps. Intriguingly, most DDX/DHXs localize to membraneless organelles (MLOs) such as nucleoli, nuclear speckles, stress granules, or processing bodies. Recent findings suggest not only that localization to MLOs can promote interaction between DDX/DHXs and their targets but also that DDX/DHXs are key regulators of MLO formation and turnover through their condensation and ATPase activity.In this review, we describe the molecular function of DDX/DHXs in ribosome biogenesis, messenger RNA splicing, export, translation, and storage or decay as well as their association with prominent MLOs. We discuss how the enzymatic function of DDX/DHXs in RNA processing is linked to DDX/DHX condensation, the accumulation of ribonucleoprotein particles and MLO dynamics. Future research will reveal how these processes orchestrate the RNA life cycle in MLO space and DDX/DHX time.
Subject(s)
DEAD-box RNA Helicases , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/chemistry , Humans , Animals , RNA/metabolism , RNA/genetics , RNA/chemistry , RNA Splicing , Organelles/metabolism , Organelles/genetics , Ribosomes/metabolism , Ribosomes/genetics , RNA Folding , RNA Processing, Post-Transcriptional , Ribonucleoproteins/metabolism , Ribonucleoproteins/genetics , Cell Nucleolus/metabolism , Cell Nucleolus/genetics , RNA, Messenger/metabolism , RNA, Messenger/geneticsABSTRACT
DEAD-box ATPases constitute a very large protein family present in all cells, often in great abundance. From bacteria to humans, they play critical roles in many aspects of RNA metabolism, and due to their widespread importance in RNA biology, they have been characterized in great detail at both the structural and biochemical levels. DEAD-box proteins function as RNA-dependent ATPases that can unwind short duplexes of RNA, remodel ribonucleoprotein (RNP) complexes, or act as clamps to promote RNP assembly. Yet, it often remains enigmatic how individual DEAD-box proteins mechanistically contribute to specific RNA-processing steps. Here, we review the role of DEAD-box ATPases in the regulation of gene expression and propose that one common function of these enzymes is in the regulation of liquid-liquid phase separation of RNP condensates.
Subject(s)
DEAD-box RNA Helicases , RNA , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , DEAD-box RNA Helicases/chemistry , Gene Expression , Humans , RNA/metabolismABSTRACT
The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5' splice site (5'SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5'SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5'SS, producing a free 5' exon. Removal of the BP adenosine from the active site allows the 3'SS to bind, so that the 5' exon attacks the 3'SS to produce mature mRNA and an excised lariat intron.
Subject(s)
DEAD-box RNA Helicases/genetics , RNA Splicing Factors/genetics , RNA Splicing , RNA, Small Nuclear/genetics , Saccharomyces cerevisiae/genetics , Spliceosomes/metabolism , Catalytic Domain , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , Exons , Humans , Introns , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Structure, Secondary , RNA Helicases/chemistry , RNA Helicases/genetics , RNA Helicases/metabolism , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing Factors/chemistry , RNA Splicing Factors/metabolism , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spliceosomes/genetics , Spliceosomes/ultrastructureABSTRACT
Human Dicer (hDicer) is a multi-domain protein belonging to the RNase III family. It plays pivotal roles in small RNA biogenesis during the RNA interference (RNAi) pathway by processing a diverse range of double-stranded RNA (dsRNA) precursors to generate â¼22 nt microRNA (miRNA) or small interfering RNA (siRNA) products for sequence-directed gene silencing. In this work, we solved the cryoelectron microscopy (cryo-EM) structure of hDicer in complex with its cofactor protein TRBP and revealed the precise spatial arrangement of hDicer's multiple domains. We further solved structures of the hDicer-TRBP complex bound with pre-let-7 RNA in two distinct conformations. In combination with biochemical analysis, these structures reveal a property of the hDicer-TRBP complex to promote the stability of pre-miRNA's stem duplex in a pre-dicing state. These results provide insights into the mechanism of RNA processing by hDicer and illustrate the regulatory role of hDicer's N-terminal helicase domain.
Subject(s)
DEAD-box RNA Helicases/chemistry , MicroRNAs/chemistry , Ribonuclease III/chemistry , Cryoelectron Microscopy , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Electrophoretic Mobility Shift Assay , Humans , MicroRNAs/metabolism , Nuclear Receptor Coactivators/chemistry , Nuclear Receptor Coactivators/genetics , Nuclear Receptor Coactivators/metabolism , Nucleic Acid Conformation , Protein Binding , Protein Domains , Protein Structure, Quaternary , RNA Cleavage , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Ribonuclease III/genetics , Ribonuclease III/metabolismABSTRACT
The disassembly of the intron lariat spliceosome (ILS) marks the end of a splicing cycle. Here we report a cryoelectron microscopy structure of the ILS complex from Saccharomyces cerevisiae at an average resolution of 3.5 Å. The intron lariat remains bound in the spliceosome whereas the ligated exon is already dissociated. The step II splicing factors Prp17 and Prp18, along with Cwc21 and Cwc22 that stabilize the 5' exon binding to loop I of U5 small nuclear RNA (snRNA), have been released from the active site assembly. The DEAH family ATPase/helicase Prp43 binds Syf1 at the periphery of the spliceosome, with its RNA-binding site close to the 3' end of U6 snRNA. The C-terminal domain of Ntr1/Spp382 associates with the GTPase Snu114, and Ntr2 is anchored to Prp8 while interacting with the superhelical domain of Ntr1. These structural features suggest a plausible mechanism for the disassembly of the ILS complex.
Subject(s)
Introns , Spliceosomes/ultrastructure , Cryoelectron Microscopy , DEAD-box RNA Helicases/chemistry , Models, Molecular , RNA Precursors/chemistry , RNA Precursors/ultrastructure , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/ultrastructure , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Schizosaccharomyces/chemistry , Spliceosomes/chemistryABSTRACT
Dysregulated rRNA synthesis by RNA polymerase I (Pol I) is associated with uncontrolled cell proliferation. Here, we report a box H/ACA small nucleolar RNA (snoRNA)-ended long noncoding RNA (lncRNA) that enhances pre-rRNA transcription (SLERT). SLERT requires box H/ACA snoRNAs at both ends for its biogenesis and translocation to the nucleolus. Deletion of SLERT impairs pre-rRNA transcription and rRNA production, leading to decreased tumorigenesis. Mechanistically, SLERT interacts with DEAD-box RNA helicase DDX21 via a 143-nt non-snoRNA sequence. Super-resolution images reveal that DDX21 forms ring-shaped structures surrounding multiple Pol I complexes and suppresses pre-rRNA transcription. Binding by SLERT allosterically alters individual DDX21 molecules, loosens the DDX21 ring, and evicts DDX21 suppression on Pol I transcription. Together, our results reveal an important control of ribosome biogenesis by SLERT lncRNA and its regulatory role in DDX21 ring-shaped arrangements acting on Pol I complexes.
Subject(s)
DEAD-box RNA Helicases/metabolism , RNA Polymerase I/metabolism , RNA Precursors/genetics , RNA, Long Noncoding/metabolism , Allosteric Site , Animals , Carcinogenesis , Cell Line , Cell Line, Tumor , DEAD-box RNA Helicases/chemistry , Female , Gene Knockout Techniques , Humans , Mice , Mice, Nude , RNA Precursors/metabolism , Transcription, GeneticABSTRACT
MicroRNA maturation is initiated by RNase III DROSHA that cleaves the stem loop of primary microRNA. DROSHA functions together with its cofactor DGCR8 in a heterotrimeric complex known as Microprocessor. Here, we report the X-ray structure of DROSHA in complex with the C-terminal helix of DGCR8. We find that DROSHA contains two DGCR8-binding sites, one on each RNase III domain (RIIID), which mediate the assembly of Microprocessor. The overall structure of DROSHA is surprisingly similar to that of Dicer despite no sequence homology apart from the C-terminal part, suggesting that DROSHA may have evolved from a Dicer homolog. DROSHA exhibits unique features, including non-canonical zinc-finger motifs, a long insertion in the first RIIID, and the kinked link between Connector helix and RIIID, which explains the 11-bp-measuring "ruler" activity of DROSHA. Our study implicates the evolutionary origin of DROSHA and elucidates the molecular basis of Microprocessor assembly and primary microRNA processing.
Subject(s)
MicroRNAs/metabolism , RNA Processing, Post-Transcriptional , Ribonuclease III/chemistry , Amino Acid Sequence , Crystallography, X-Ray , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , Evolution, Molecular , Humans , MicroRNAs/chemistry , Models, Chemical , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Folding , Protein Structure, Tertiary , RNA-Binding Proteins/metabolism , Ribonuclease III/genetics , Ribonuclease III/metabolism , Sequence Alignment , Structural Homology, ProteinABSTRACT
DEAD-box ATPases are major regulators of biomolecular condensates and orchestrate diverse biochemical processes that are critical for the functioning of cells. How DEAD-box proteins are selectively recruited to their respective biomolecular condensates is unknown. We explored this in the context of the nucleolus and DEAD-box protein DDX21. We find that the pH of the nucleolus is intricately linked to the transcriptional activity of the organelle and facilitates the recruitment and condensation of DDX21. We identify an evolutionarily conserved feature of the C terminus of DDX21 responsible for nucleolar localization. This domain is essential for zebrafish development, and its intrinsically disordered and isoelectric properties are necessary and sufficient for the ability of DDX21 to respond to changes in pH and form condensates. Molecularly, the enzymatic activities of poly(ADP-ribose) polymerases contribute to maintaining the nucleolar pH and, consequently, DDX21 recruitment and nucleolar partitioning. These observations reveal an activity-dependent physicochemical mechanism for the selective recruitment of biochemical activities to biomolecular condensates.
Subject(s)
DEAD-box RNA Helicases , Zebrafish , Animals , Zebrafish/genetics , Zebrafish/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/chemistry , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Organelles/metabolism , Hydrogen-Ion ConcentrationABSTRACT
The exosome regulates the processing, degradation, and surveillance of a plethora of RNA species. However, little is known about how the exosome recognizes and is recruited to its diverse substrates. We report the identification of adaptor proteins that recruit the exosome-associated helicase, Mtr4, to unique RNA substrates. Nop53, the yeast homolog of the tumor suppressor PICT1, targets Mtr4 to pre-ribosomal particles for exosome-mediated processing, while a second adaptor Utp18 recruits Mtr4 to cleaved rRNA fragments destined for degradation by the exosome. Both Nop53 and Utp18 contain the same consensus motif, through which they dock to the "arch" domain of Mtr4 and target it to specific substrates. These findings show that the exosome employs a general mechanism of recruitment to defined substrates and that this process is regulated through adaptor proteins.
Subject(s)
DEAD-box RNA Helicases/metabolism , Exosomes/metabolism , Nuclear Proteins/metabolism , Ribosomal Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Animals , Ascomycota/chemistry , Ascomycota/classification , Ascomycota/genetics , DEAD-box RNA Helicases/chemistry , Humans , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/chemistry , Nucleic Acid Conformation , RNA, Fungal/chemistry , RNA, Fungal/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribosomal Proteins/chemistry , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Sequence AlignmentABSTRACT
Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites1-4. However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity in Arabidopsis, we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming.
Subject(s)
Codon, Initiator , Nucleic Acid Conformation , RNA, Double-Stranded , RNA, Messenger , Humans , Arabidopsis/genetics , Arabidopsis/immunology , Codon, Initiator/genetics , Innate Immunity Recognition , Open Reading Frames/genetics , Protein Biosynthesis/genetics , Protein Biosynthesis/immunology , Ribosomes/metabolism , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Messenger/genetics , Transcriptome , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Deep LearningABSTRACT
Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.
Subject(s)
Cryoelectron Microscopy , DEAD-box RNA Helicases , MicroRNAs , RNA Precursors , Ribonuclease III , Humans , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/ultrastructure , MicroRNAs/biosynthesis , MicroRNAs/chemistry , MicroRNAs/metabolism , MicroRNAs/ultrastructure , Mutation , Ribonuclease III/chemistry , Ribonuclease III/genetics , Ribonuclease III/metabolism , Ribonuclease III/ultrastructure , RNA Precursors/chemistry , RNA Precursors/metabolism , RNA Precursors/ultrastructure , RNA, Double-Stranded/metabolism , Substrate SpecificityABSTRACT
Ribosome assembly is catalyzed by numerous trans-acting factors and coupled with irreversible pre-rRNA processing, driving the pathway toward mature ribosomal subunits. One decisive step early in this progression is removal of the 5' external transcribed spacer (5'-ETS), an RNA extension at the 18S rRNA that is integrated into the huge 90S pre-ribosome structure. Upon endo-nucleolytic cleavage at an internal site, A1, the 5'-ETS is separated from the 18S rRNA and degraded. Here we present biochemical and cryo-electron microscopy analyses that depict the RNA exosome, a major 3'-5' exoribonuclease complex, in a super-complex with the 90S pre-ribosome. The exosome is docked to the 90S through its co-factor Mtr4 helicase, a processive RNA duplex-dismantling helicase, which strategically positions the exosome at the base of 5'-ETS helices H9-H9', which are dislodged in our 90S-exosome structures. These findings suggest a direct role of the exosome in structural remodeling of the 90S pre-ribosome to drive eukaryotic ribosome synthesis.
Subject(s)
DEAD-box RNA Helicases/chemistry , Endoribonucleases/chemistry , Exonucleases/chemistry , Exosome Multienzyme Ribonuclease Complex/ultrastructure , RNA, Ribosomal, 18S/chemistry , Ribosomes/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/genetics , Binding Sites , Cryoelectron Microscopy , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Endoribonucleases/genetics , Endoribonucleases/metabolism , Exonucleases/genetics , Exonucleases/metabolism , Exosome Multienzyme Ribonuclease Complex/genetics , Exosome Multienzyme Ribonuclease Complex/metabolism , Models, Molecular , Protein Binding , Protein Biosynthesis , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA Stability , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1 expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.
Subject(s)
DEAD-box RNA Helicases , Interferon Type I , NF-kappa B , RNA Virus Infections , Ribonuclease III , Animals , Humans , NF-kappa B/genetics , RNA Interference , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Ribonuclease III/chemistry , Ribonuclease III/genetics , Ribonuclease III/metabolism , RNA Virus Infections/enzymologyABSTRACT
MDA5, a viral double-stranded RNA (dsRNA) receptor, shares sequence similarity and signaling pathways with RIG-I yet plays essential functions in antiviral immunity through distinct specificity for viral RNA. Revealing the molecular basis for the functional divergence, we report here the crystal structure of MDA5 bound to dsRNA, which shows how, using the same domain architecture, MDA5 recognizes the internal duplex structure, whereas RIG-I recognizes the terminus of dsRNA. We further show that MDA5 uses direct protein-protein contacts to stack along dsRNA in a head-to-tail arrangement, and that the signaling domain (tandem CARD), which decorates the outside of the core MDA5 filament, also has an intrinsic propensity to oligomerize into an elongated structure that activates the signaling adaptor, MAVS. These data support a model in which MDA5 uses long dsRNA as a signaling platform to cooperatively assemble the core filament, which in turn promotes stochastic assembly of the tandem CARD oligomers for signaling.
Subject(s)
DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , RNA, Double-Stranded/metabolism , Amino Acid Sequence , Humans , Interferon-Induced Helicase, IFIH1 , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , RNA, Double-Stranded/chemistry , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/metabolism , Sequence Alignment , X-Ray DiffractionABSTRACT
In mammals, a single Dicer participates in biogenesis of small RNAs in microRNA (miRNA) and RNAi pathways. In mice, endogenous RNAi is highly active in oocytes, but not in somatic cells, which we ascribe here to an oocyte-specific Dicer isoform (Dicer(O)). Dicer(O) lacks the N-terminal DExD helicase domain and has higher cleavage activity than the full-length Dicer in somatic cells (Dicer(S)). Unlike Dicer(S), Dicer(O) efficiently produces small RNAs from long double-stranded (dsRNA) substrates. Expression of the Dicer(O) isoform is driven by an intronic MT-C retrotransposon promoter, deletion of which causes loss of Dicer(O) and female sterility. Oocytes from females lacking the MT-C element show meiotic spindle defects and increased levels of endogenous small interfering RNA (endo-siRNA) targets, phenocopying the maternal Dicer null phenotype. The alternative Dicer isoform, whose phylogenetic origin demonstrates evolutionary plasticity of RNA-silencing pathways, is the main determinant of endogenous RNAi activity in the mouse female germline.
Subject(s)
DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Oocytes/metabolism , RNA, Small Interfering/metabolism , Retroelements , Ribonuclease III/genetics , Ribonuclease III/metabolism , Animals , Base Sequence , DEAD-box RNA Helicases/chemistry , Female , Gene Expression , Infertility, Female , Mice , Molecular Sequence Data , Phylogeny , Promoter Regions, Genetic , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Small Interfering/chemistry , Ribonuclease III/chemistryABSTRACT
Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. In this study, we investigated the interaction of the RNA editing mechanism with the RNA interference (RNAi) machinery and found that ADAR1 forms a complex with Dicer through direct protein-protein interaction. Most importantly, ADAR1 increases the maximum rate (Vmax) of pre-microRNA (miRNA) cleavage by Dicer and facilitates loading of miRNA onto RNA-induced silencing complexes, identifying a new role of ADAR1 in miRNA processing and RNAi mechanisms. ADAR1 differentiates its functions in RNA editing and RNAi by the formation of either ADAR1/ADAR1 homodimer or Dicer/ADAR1 heterodimer complexes, respectively. As expected, the expression of miRNAs is globally inhibited in ADAR1(-/-) mouse embryos, which, in turn, alters the expression of their target genes and might contribute to their embryonic lethal phenotype.
Subject(s)
Adenosine Deaminase/metabolism , DEAD-box RNA Helicases/metabolism , RNA Interference , RNA Processing, Post-Transcriptional , Ribonuclease III/metabolism , Adenosine Deaminase/chemistry , Adenosine Deaminase/genetics , Animals , Base Sequence , DEAD-box RNA Helicases/chemistry , Dimerization , Embryo, Mammalian/metabolism , HEK293 Cells , HeLa Cells , Humans , Mice , MicroRNAs/metabolism , Molecular Sequence Data , Protein Interaction Domains and Motifs , RNA, Small Interfering/metabolism , RNA-Binding Proteins , Ribonuclease III/chemistry , Up-RegulationABSTRACT
RIG-I is a critical RNA virus sensor that serves to initiate antiviral innate immunity. However, posttranslational regulation of RIG-I signaling remains to be fully understood. We report here that RNA viruses, but not DNA viruses or bacteria, specifically upregulate lectin family member Siglecg expression in macrophages by RIG-I- or NF-κB-dependent mechanisms. Siglec-G-induced recruitment of SHP2 and the E3 ubiquitin ligase c-Cbl to RIG-I leads to RIG-I degradation via K48-linked ubiquitination at Lys813 by c-Cbl. By increasing type I interferon production, targeted inactivation of Siglecg protects mice against lethal RNA virus infection. Taken together, our data reveal a negative feedback loop of RIG-I signaling and identify a Siglec-G-mediated immune evasion pathway exploited by RNA viruses with implication in antiviral applications. These findings also provide insights into the functions and crosstalk of Siglec-G, a known adaptive response regulator, in innate immunity.
Subject(s)
DEAD-box RNA Helicases/metabolism , Gram-Negative Bacterial Infections/immunology , Immunity, Innate , Lectins/metabolism , RNA Virus Infections/immunology , Receptors, Antigen, B-Cell/metabolism , Animals , DEAD Box Protein 58 , DEAD-box RNA Helicases/chemistry , Dendritic Cells/immunology , Gram-Negative Bacteria/metabolism , Interferon Regulatory Factor-3/metabolism , Lectins/genetics , Lysine/metabolism , Macrophages/immunology , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Proto-Oncogene Proteins c-cbl/metabolism , RNA Viruses/metabolism , Receptors, Antigen, B-Cell/genetics , Sialic Acid Binding Immunoglobulin-like Lectins , UbiquitinationABSTRACT
In contrast to the bacterial translation machinery, mitoribosomes and mitochondrial translation factors are highly divergent in terms of composition and architecture. There is increasing evidence that the biogenesis of mitoribosomes is an intricate pathway, involving many assembly factors. To better understand this process, we investigated native assembly intermediates of the mitoribosomal large subunit from the human parasite Trypanosoma brucei using cryo-electron microscopy. We identify 28 assembly factors, 6 of which are homologous to bacterial and eukaryotic ribosome assembly factors. They interact with the partially folded rRNA by specifically recognizing functionally important regions such as the peptidyltransferase center. The architectural and compositional comparison of the assembly intermediates indicates a stepwise modular assembly process, during which the rRNA folds toward its mature state. During the process, several conserved GTPases and a helicase form highly intertwined interaction networks that stabilize distinct assembly intermediates. The presented structures provide general insights into mitoribosomal maturation.
Subject(s)
Mitochondrial Ribosomes/chemistry , RNA, Ribosomal/metabolism , Ribosome Subunits, Large/chemistry , Trypanosoma brucei brucei/metabolism , Cryoelectron Microscopy , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Mitochondrial Ribosomes/metabolism , Models, Molecular , Nucleic Acid Conformation , RNA, Ribosomal/chemistry , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosome Subunits, Large/metabolism , Trypanosoma brucei brucei/geneticsABSTRACT
During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1-4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2-BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2-BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing.
Subject(s)
DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Splicing , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae , Spliceosomes/enzymology , Actins/genetics , Adenosine/metabolism , Binding Sites , Cryoelectron Microscopy , DEAD-box RNA Helicases/ultrastructure , Models, Molecular , Mutation , Protein Domains , RNA Precursors/metabolism , RNA Precursors/ultrastructure , RNA Splicing/genetics , Ribonucleoprotein, U1 Small Nuclear/metabolism , Ribonucleoprotein, U2 Small Nuclear/chemistry , Ribonucleoprotein, U2 Small Nuclear/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/ultrastructure , Spliceosomes/chemistry , Spliceosomes/metabolismABSTRACT
During gene expression, RNA export factors are mainly known for driving nucleo-cytoplasmic transport. While early studies suggested that the exon junction complex (EJC) provides a binding platform for them, subsequent work proposed that they are only recruited by the cap binding complex to the 5' end of RNAs, as part of TREX. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are recruited to the whole mRNA co-transcriptionally via splicing but before 3' end processing. Consequently, Alyref alters splicing decisions and Chtop regulates alternative polyadenylation. Alyref is recruited to the 5' end of RNAs by CBC, and our data reveal subsequent binding to RNAs near EJCs. We demonstrate that eIF4A3 stimulates Alyref deposition not only on spliced RNAs close to EJC sites but also on single-exon transcripts. Our study reveals mechanistic insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome.