RESUMO
Poly(UG) or 'pUG' dinucleotide repeats direct gene silencing in Caenorhabditis elegans by adopting an unusual quadruplex structure. Humans have thousands of pUG sequences and proteins that interact with them. The pUG fold reveals new aspects of gene regulation and RNA folding, highlighting how a simple sequence can encode a complex structure.
Assuntos
Caenorhabditis elegans , Quadruplex G , Inativação Gênica , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Humanos , RNA/metabolismo , RNA/genética , RNA/químicaRESUMO
Spliceosome activation is the process of creating the catalytic site for RNA splicing and occurs de novo on each intron following spliceosome assembly. Dozens of factors bind to or are released from the activating spliceosome including the Lsm2-8 heteroheptameric ring that binds the U6 small nuclear RNA 3'-end. Lsm2-8 must be released to permit active site stabilization by the Prp19-containing complex (NineTeen Complex, NTC); however, little is known about the temporal order of events and dynamic interactions that lead up to and follow Lsm2-8 release. We have used colocalization single molecule spectroscopy (CoSMoS) to visualize Lsm2-8 dynamics during activation of Saccharomyces cerevisiae spliceosomes in vitro. Lsm2-8 is recruited as a component of the tri-snRNP and is released after integration of the Prp19-containing complex (NTC). Despite Lsm2-8 and the NTC being mutually exclusive in existing cryo-EM structures of yeast B complex spliceosomes, we identify a transient intermediate containing both ([Formula: see text]) and provide a kinetic framework for its formation and transformation during activation. Prior to [Formula: see text] assembly, the NTC rapidly and reversibly samples the spliceosome suggesting a mechanism for preventing NTC sequestration by defective spliceosomes that fail to properly activate. In complementary ensemble assays, we show that a base-pairing-dependent ternary complex can form between Lsm2-8 and U2 and U6 helix II RNAs. We propose that this interaction may play a role in formation of transient spliceosome intermediates formed during activation.
Assuntos
Proteínas de Saccharomyces cerevisiae , Spliceossomos , Spliceossomos/genética , Imagem Individual de Molécula , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Microscopia de Fluorescência , Fatores de Processamento de RNA/metabolismoRESUMO
RNA stability is meticulously controlled. Here, we sought to determine whether an essential post-transcriptional regulatory mechanism plays a role in pain. Nonsense-mediated decay (NMD) safeguards against translation of mRNAs that harbor premature termination codons and controls the stability of â¼10% of typical protein-coding mRNAs. It hinges on the activity of the conserved kinase SMG1. Both SMG1 and its target, UPF1, are expressed in murine DRG sensory neurons. SMG1 protein is present in both the DRG and sciatic nerve. Using high-throughput sequencing, we examined changes in mRNA abundance following inhibition of SMG1. We confirmed multiple NMD stability targets in sensory neurons, including ATF4. ATF4 is preferentially translated during the integrated stress response (ISR). This led us to ask whether suspension of NMD induces the ISR. Inhibition of NMD increased eIF2-α phosphorylation and reduced the abundance of the eIF2-α phosphatase constitutive repressor of eIF2-α phosphorylation. Finally, we examined the effects of SMG1 inhibition on pain-associated behaviors. Peripheral inhibition of SMG1 results in mechanical hypersensitivity in males and females that persists for several days and priming to a subthreshold dose of PGE2. Priming was fully rescued by a small-molecule inhibitor of the ISR. Collectively, our results indicate that suspension of NMD promotes pain through stimulation of the ISR.SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in their plasticity which may contribute to chronic pain. Translational regulation has emerged as a dominant mechanism in pain. Here, we investigate the role of a major pathway of RNA surveillance called nonsense-mediated decay (NMD). Modulation of NMD is potentially beneficial for a broad array of diseases caused by frameshift or nonsense mutations. Our results suggest that inhibition of the rate-limiting step of NMD drives behaviors associated with pain through activation of the ISR. This work reveals complex interconnectivity between RNA stability and translational regulation and suggests an important consideration in harnessing the salubrious benefits of NMD disruption.
Assuntos
Fator de Iniciação 2 em Eucariotos , Nociceptividade , Masculino , Feminino , Humanos , Camundongos , Animais , Fator de Iniciação 2 em Eucariotos/genética , Degradação do RNAm Mediada por Códon sem Sentido , Fosforilação , Dor , RNA Helicases/genética , RNA Helicases/metabolismo , Transativadores/genéticaRESUMO
HIV-1 virion production is driven by Gag and Gag-Pol (GP) proteins, with Gag forming the bulk of the capsid and driving budding, while GP binds Gag to deliver the essential virion enzymes protease, reverse transcriptase, and integrase. Virion GP levels are traditionally thought to reflect the relative abundances of GP and Gag in cells (â¼1:20), dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event occurring in gag-pol mRNAs. Here, we exploited a panel of PRF mutant viruses to show that mechanisms in addition to PRF regulate GP incorporation into virions. First, we show that GP is enriched â¼3-fold in virions relative to cells, with viral infectivity being better maintained at subphysiological levels of GP than when GP levels are too high. Second, we report that GP is more efficiently incorporated into virions when Gag and GP are synthesized in cis (i.e., from the same gag-pol mRNA) than in trans, suggesting that Gag/GP translation and assembly are spatially coupled processes. Third, we show that, surprisingly, virions exhibit a strong upper limit to trans-delivered GP incorporation; an adaptation that appears to allow the virus to temper defects to GP/Gag cleavage that may negatively impact reverse transcription. Taking these results together, we propose a "weighted Goldilocks" scenario for HIV-1 GP incorporation, wherein combined mechanisms of GP enrichment and exclusion buffer virion infectivity over a broad range of local GP concentrations. These results provide new insights into the HIV-1 virion assembly pathway relevant to the anticipated efficacy of PRF-targeted antiviral strategies. IMPORTANCE HIV-1 infectivity requires incorporation of the Gag-Pol (GP) precursor polyprotein into virions during the process of virus particle assembly. Mechanisms dictating GP incorporation into assembling virions are poorly defined, with GP levels in virions traditionally thought to solely reflect relative levels of Gag and GP expressed in cells, dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event that occurs in gag-pol mRNAs. Herein, we provide experimental support for a "weighted Goldilocks" scenario for GP incorporation, wherein the virus exploits both random and nonrandom mechanisms to buffer infectivity over a wide range of GP expression levels. These mechanistic data are relevant to ongoing efforts to develop antiviral strategies targeting PRF frequency and/or HIV-1 virion maturation.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , HIV-1/fisiologia , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Humanos , Sequências Repetidas Invertidas , Modelos Biológicos , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , Vírion , Replicação ViralRESUMO
Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2',3' cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3' end and removal of the Lsm1 carboxy-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.
Assuntos
Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Sítios de Ligação/genética , Ligação Proteica/genética , RNA/genética , Proteínas de Ligação ao Cap de RNA/genética , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Saccharomyces cerevisiae/genética , Spliceossomos/genéticaRESUMO
U6 snRNA undergoes post-transcriptional 3' end modification prior to incorporation into the active site of spliceosomes. The responsible exoribonuclease is Usb1, which removes nucleotides from the 3' end of U6 and, in humans, leaves a 2',3' cyclic phosphate that is recognized by the Lsm2-8 complex. Saccharomycescerevisiae Usb1 has additional 2',3' cyclic phosphodiesterase (CPDase) activity, which converts the cyclic phosphate into a 3' phosphate group. Here we investigate the molecular basis for the evolution of Usb1 CPDase activity. We examine the structure and function of Usb1 from Kluyveromyces marxianus, which shares 25 and 19% sequence identity to the S. cerevisiae and Homo sapiens orthologs of Usb1, respectively. We show that K. marxianus Usb1 enzyme has CPDase activity and determined its structure, free and bound to the substrate analog uridine 5'-monophosphate. We find that the origin of CPDase activity is related to a loop structure that is conserved in yeast and forms a distinct penultimate (n - 1) nucleotide binding site. These data provide structural and mechanistic insight into the evolutionary divergence of Usb1 catalysis.
Assuntos
Evolução Molecular , Proteínas Mitocondriais/genética , Diester Fosfórico Hidrolases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sítios de Ligação/genética , Domínio Catalítico/genética , Humanos , Kluyveromyces/química , Proteínas Mitocondriais/química , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleotídeos/química , Nucleotídeos/genética , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/química , Splicing de RNA/genética , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Spliceossomos/química , Spliceossomos/genéticaRESUMO
A presumed RNA cloverleaf (5'CL), located at the 5'-most end of the noncoding region of the enterovirus genome, is the primary established site for initiation of genomic replication. Stem-loop B (SLB) and stem-loop D (SLD), the two largest stem-loops within the 5'CL, serve as recognition sites for protein interactions that are essential for replication. Here we present the solution structure of rhinovirus serotype 14 5'CL using a combination of nuclear magnetic resonance spectroscopy and small-angle X-ray scattering. In the absence of magnesium, the structure adopts an open, somewhat extended conformation. In the presence of magnesium, the structure compacts, bringing SLB and SLD into close contact, a geometry that creates an extensive accessible major groove surface, and permits interaction between the proteins that target each stem-loop.
Assuntos
Enterovirus/genética , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , Transcrição Gênica , Regulação Viral da Expressão Gênica , Magnésio/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Relação Estrutura-Atividade , Replicação ViralRESUMO
There are few methods available for the rapid discovery of multitarget drugs. Herein, we describe the template-assisted, target-guided discovery of small molecules that recognize d(CTG) in the expanded d(CTG·CAG) sequence and its r(CUG) transcript that cause myotonic dystrophy type 1. A positive cross-selection was performed using a small library of 30 monomeric alkyne- and azide-containing ligands capable of producing >5000 possible di- and trimeric click products. The monomers were incubated with d(CTG)16 or r(CUG)16 under physiological conditions, and both sequences showed selectivity in the proximity-accelerated azide-alkyne [3+2] cycloaddition click reaction. The limited number of click products formed in both selections and the even smaller number of common products suggests that this method is a useful tool for the discovery of single-target and multitarget lead therapeutic agents.
Assuntos
DNA/antagonistas & inibidores , Distrofia Miotônica/tratamento farmacológico , Distrofia Miotônica/genética , RNA/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Expansão das Repetições de Trinucleotídeos/efeitos dos fármacos , Células Cultivadas , DNA/genética , DNA/metabolismo , Humanos , Distrofia Miotônica/patologia , RNA/genética , RNA/metabolismo , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Removal of introns from precursor messenger RNA (pre-mRNA) and some noncoding transcripts is an essential step in eukaryotic gene expression. In the nucleus, this process of RNA splicing is carried out by the spliceosome, a multi-megaDalton macromolecular machine whose core components are conserved from yeast to humans. In addition to many proteins, the spliceosome contains five uridine-rich small nuclear RNAs (snRNAs) that undergo an elaborate series of conformational changes to correctly recognize the splice sites and catalyze intron removal. Decades of biochemical and genetic data, along with recent cryo-EM structures, unequivocally demonstrate that U6 snRNA forms much of the catalytic core of the spliceosome and is highly dynamic, interacting with three snRNAs, the pre-mRNA substrate, and >25 protein partners throughout the splicing cycle. This review summarizes the current state of knowledge on how U6 snRNA is synthesized, modified, incorporated into snRNPs and spliceosomes, recycled, and degraded.
Assuntos
Sítios de Splice de RNA/genética , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Spliceossomos/metabolismo , Humanos , Conformação de Ácido Nucleico , RNA Polimerase III/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Nuclear Pequeno/biossíntese , Saccharomyces cerevisiae/genéticaRESUMO
Post-transcriptional modification of snRNA is central to spliceosome function. Usb1 is an exoribonuclease that shortens the oligo-uridine tail of U6 snRNA, resulting in a terminal 2',3' cyclic phosphate group in most eukaryotes, including humans. Loss of function mutations in human Usb1 cause the rare disorder poikiloderma with neutropenia (PN), and result in U6 snRNAs with elongated 3' ends that are aberrantly adenylated. Here, we show that human Usb1 removes 3' adenosines with 20-fold greater efficiency than uridines, which explains the presence of adenylated U6 snRNAs in cells lacking Usb1. We determined three high-resolution co-crystal structures of Usb1: wild-type Usb1 bound to the substrate analog adenosine 5'-monophosphate, and an inactive mutant bound to RNAs with a 3' terminal adenosine and uridine. These structures, along with QM/MM MD simulations of the catalytic mechanism, illuminate the molecular basis for preferential deadenylation of U6 snRNA. The extent of Usb1 processing is influenced by the secondary structure of U6 snRNA.
Assuntos
Adenosina/metabolismo , Diester Fosfórico Hidrolases/metabolismo , RNA Nuclear Pequeno/metabolismo , Uridina/metabolismo , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Sequência de Bases , Cristalografia por Raios X , Predisposição Genética para Doença/genética , Humanos , Modelos Moleculares , Mutação , Neutropenia/genética , Neutropenia/metabolismo , Conformação de Ácido Nucleico , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Ligação Proteica , Conformação Proteica , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Anormalidades da Pele/genética , Anormalidades da Pele/metabolismo , Especificidade por SubstratoRESUMO
Escherichia coli DksA is a transcription factor that binds to RNA polymerase (RNAP) without binding to DNA, destabilizing RNAP-promoter interactions, sensitizing RNAP to the global regulator ppGpp, and regulating transcription of several hundred target genes, including those encoding rRNA. Previously, we described promoter sequences and kinetic properties that account for DksA's promoter specificity, but how DksA exerts its effects on RNAP has remained unclear. To better understand DksA's mechanism of action, we incorporated benzoyl-phenylalanine at specific positions in DksA and mapped its cross-links to RNAP, constraining computational docking of the two proteins. The resulting evidence-based model of the DksA-RNAP complex as well as additional genetic and biochemical approaches confirmed that DksA binds to the RNAP secondary channel, defined the orientation of DksA in the channel, and predicted a network of DksA interactions with RNAP that includes the rim helices and the mobile trigger loop (TL) domain. Engineered cysteine substitutions in the TL and DksA coiled-coil tip generated a disulfide bond between them, and the interacting residues were absolutely required for DksA function. We suggest that DksA traps the TL in a conformation that destabilizes promoter complexes, an interaction explaining the requirement for the DksA tip and its effects on transcription.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
Base-pairing of U4 and U6 snRNAs during di-snRNP assembly requires large-scale remodeling of RNA structure that is chaperoned by the U6 snRNP protein Prp24. We investigated the mechanism of U4/U6 annealing in vitro using an assay that enables visualization of ribonucleoprotein complexes and faithfully recapitulates known in vivo determinants for the process. We find that annealing, but not U6 RNA binding, is highly dependent on the electropositive character of a 20 Å-wide groove on the surface of Prp24. During annealing, we observe the formation of a stable ternary complex between U4 and U6 RNAs and Prp24, indicating that displacement of Prp24 in vivo requires additional factors. Mutations that stabilize the U6 'telestem' helix increase annealing rates by up to 15-fold, suggesting that telestem formation is rate-limiting for U4/U6 pairing. The Lsm2-8 complex, which binds adjacent to the telestem at the 3' end of U6, provides a comparable rate enhancement. Collectively, these data identify domains of the U6 snRNP that are critical for one of the first steps in assembly of the megaDalton U4/U6.U5 tri-snRNP complex, and lead to a dynamic model for U4/U6 pairing that involves a striking degree of evolved cooperativity between protein and RNA.
Assuntos
RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Ligação Competitiva , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability.
Assuntos
RNA Fúngico/química , RNA Nuclear Pequeno/química , Saccharomyces cerevisiae/fisiologia , Pareamento de Bases , Cinética , Estabilidade de RNA , RNA de Cadeia Dupla/químicaRESUMO
RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots â¼90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.
Assuntos
Bactérias/enzimologia , Cronobacter/enzimologia , DNA/química , RecQ Helicases/química , Trifosfato de Adenosina/química , Anisotropia , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Análise Mutacional de DNA , DNA de Cadeia Simples/química , Escherichia coli/enzimologia , Genoma Bacteriano , Hidrólise , Ligação ProteicaRESUMO
The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame.
Assuntos
Dicistroviridae/genética , Sítios Internos de Entrada Ribossomal/genética , Fases de Leitura Aberta/genética , Biossíntese de Proteínas , RNA de Transferência/química , RNA de Transferência/genética , Animais , Sequência de Bases , Bovinos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Fatores de Terminação de Peptídeos , Ribossomos/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The cycle of spliceosome assembly, intron excision, and spliceosome disassembly involves large-scale structural rearrangements of U6 snRNA that are functionally important. U6 enters the splicing pathway bound to the Prp24 protein, which chaperones annealing of U6 to U4 RNA to form a U4/U6 di-snRNP. During catalytic activation of the assembled spliceosome, U4 snRNP is released and U6 is paired to U2 snRNA. Here we show that point mutations in U4 and U6 that decrease U4/U6 base-pairing in vivo are lethal in combination. However, this synthetic phenotype is rescued by a mutation in U6 that alters a U6-Prp24 contact and stabilizes U2/U6. Remarkably, the resulting viable triple mutant strain lacks detectable U4/U6 base-pairing and U4/U6 di-snRNP. Instead, this strain accumulates free U4 snRNP, protein-free U6 RNA, and a novel complex containing U2/U6 di-snRNP. Further mutational analysis indicates that disruption of the U6-Prp24 interaction rather than stabilization of U2/U6 renders stable U4/U6 di-snRNP assembly nonessential. We propose that an essential function of U4/U6 pairing is to displace Prp24 from U6 RNA, and thus a destabilized U6-Prp24 complex renders stable U4/U6 pairing nonessential.
Assuntos
RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Pareamento de Bases , Sequência de Bases , Epistasia Genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Conformação de Ácido Nucleico , Multimerização Proteica , Splicing de RNA/genética , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Saccharomyces cerevisiae/genéticaRESUMO
UNLABELLED: Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus- or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-polymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication. IMPORTANCE: HIV, like many retroviruses, utilizes a -1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies.
Assuntos
Mutação da Fase de Leitura/genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , Proteínas de Fusão gag-pol/metabolismo , Infecções por HIV/virologia , HIV-1/genética , RNA Viral/genética , Vírion/fisiologia , Pareamento de Bases , Sequência de Bases , Regulação Viral da Expressão Gênica , Células HEK293 , Infecções por HIV/genética , HIV-1/química , HIV-1/metabolismo , Humanos , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Viral/química , RNA Viral/metabolismo , Montagem de Vírus , Replicação ViralRESUMO
NMR spectroscopy is a powerful technique for determining structural and functional features of biomolecules in physiological solution as well as for observing their intermolecular interactions in real-time. However, complex steps associated with its practice have made the approach daunting for non-specialists. We introduce an NMR platform that makes biomolecular NMR spectroscopy much more accessible by integrating tools, databases, web services, and video tutorials that can be launched by simple installation of NMRFAM software packages or using a cross-platform virtual machine that can be run on any standard laptop or desktop computer. The software package can be downloaded freely from the NMRFAM software download page ( http://pine.nmrfam.wisc.edu/download_packages.html ), and detailed instructions are available from the Integrative NMR Video Tutorial page ( http://pine.nmrfam.wisc.edu/integrative.html ).
Assuntos
Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Ácidos Nucleicos/química , Proteínas/química , Pesquisa , Software , NavegadorRESUMO
Viruses maintain compact genomes that must be packaged within capsids typically less than 200 nanometers in diameter. Therefore, instead of coding for a full set of genes needed for replication, viruses have evolved remarkable strategies for co-opting the host cellular machinery. Additionally, viruses often increase the coding capacity of their own genomes by employing overlapping open reading frames (ORFs). Some overlapping viral ORFs involve recoding events that are programmed by the viral RNA. During these programmed recoding events, the ribosome is directed to translate in an alternative reading frame. Here we describe how the Dicistroviridae family of viruses utilize an internal ribosome entry site (IRES) in order to recruit ribosomes to initiate translation at a non-AUG codon. The IRES accomplishes this in part by mimicking the structure of a tRNA. Recently, we showed that the Israeli Acute Paralysis Virus (IAPV) member of the Dicistroviridae family utilizes its IRES to initiate translation in 2 different reading frames. Thus, IAPV has evolved an apparently novel recoding mechanism that reveals important insights into translation. Finally, we compare the IAPV structure to other systems that utilize tRNA mimicry in translation.
Assuntos
Dicistroviridae/genética , Sítios Internos de Entrada Ribossomal , RNA de Transferência/química , Ribossomos/metabolismo , Código Genético , Genoma Viral , Modelos Moleculares , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA de Transferência/genéticaRESUMO
The HIV-1 frameshift site (FS) plays a critical role in viral replication. During translation, the HIV-1 FS transitions from a 3-helix to a 2-helix junction RNA secondary structure. The 2-helix junction structure contains a GGA bulge, and purine-rich bulges are common motifs in RNA secondary structure. Here, we investigate the dynamics of the HIV-1 FS 2-helix junction RNA. Interhelical motions were studied under different ionic conditions using NMR order tensor analysis of residual dipolar couplings. In 150 mM potassium, the RNA adopts a 43°(±4°) interhelical bend angle (ß) and displays large amplitude, anisotropic interhelical motions characterized by a 0.52(±0.04) internal generalized degree of order (GDOint) and distinct order tensor asymmetries for its two helices (η = 0.26(±0.04) and 0.5(±0.1)). These motions are effectively quenched by addition of 2 mM magnesium (GDOint = 0.87(±0.06)), which promotes a near-coaxial conformation (ß = 15°(±6°)) of the two helices. Base stacking in the bulge was investigated using the fluorescent purine analog 2-aminopurine. These results indicate that magnesium stabilizes extrahelical conformations of the bulge nucleotides, thereby promoting coaxial stacking of helices. These results are highly similar to previous studies of the HIV transactivation response RNA, despite a complete lack of sequence similarity between the two RNAs. Thus, the conformational space of these RNAs is largely determined by the topology of their interhelical junctions.