RESUMEN
Developing an effective mRNA therapeutic often requires maximizing protein output per delivered mRNA molecule. We previously found that coding sequence (CDS) design can substantially affect protein output, with mRNA variants containing more optimal codons and higher secondary structure yielding the highest protein outputs due to their slow rates of mRNA decay. Here, we demonstrate that CDS-dependent differences in translation initiation and elongation rates lead to differences in translation- and deadenylation-dependent mRNA decay rates, thus explaining the effect of CDS on mRNA half-life. Surprisingly, the most stable and highest-expressing mRNAs in our test set have modest initiation/elongation rates and ribosome loads, leading to minimal translation-dependent mRNA decay. These findings are of potential interest for optimization of protein output from therapeutic mRNAs, which may be achieved by attenuating rather than maximizing ribosome load.
Asunto(s)
Biosíntesis de Proteínas , Estabilidad del ARN , ARN Mensajero , Ribosomas , Ribosomas/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , HumanosRESUMEN
Shortening of messenger RNA poly(A) tails, or deadenylation, is a rate-limiting step in mRNA decay and is highly regulated during gene expression. The incorporation of non-adenosines in poly(A) tails, or 'mixed tailing', has been observed in vertebrates and viruses. Here, to quantitate the effect of mixed tails, we mathematically modeled deadenylation reactions at single-nucleotide resolution using an in vitro deadenylation system reconstituted with the complete human CCR4-NOT complex. Applying this model, we assessed the disrupting impact of single guanosine, uridine or cytosine to be equivalent to approximately 6, 8 or 11 adenosines, respectively. CCR4-NOT stalls at the 0, -1 and -2 positions relative to the non-adenosine residue. CAF1 and CCR4 enzyme subunits commonly prefer adenosine but exhibit distinct sequence selectivities and stalling positions. Our study provides an analytical framework to monitor deadenylation and reveals the molecular basis of tail sequence-dependent regulation of mRNA stability.
Asunto(s)
Poli A , Estabilidad del ARN , ARN Mensajero , Humanos , Cinética , Poli A/metabolismo , Poli A/química , ARN Mensajero/metabolismo , ARN Mensajero/genética , ARN Mensajero/química , Adenosina/metabolismo , Receptores CCR4/metabolismo , Receptores CCR4/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/química , ARN NucleotidiltransferasasRESUMEN
NOT1, NOT10, and NOT11 form a conserved module in the CCR4-NOT complex, critical for post-transcriptional regulation in eukaryotes, but how this module contributes to the functions of the CCR4-NOT remains poorly understood. Here, we present cryo-EM structures of human and chicken NOT1:NOT10:NOT11 ternary complexes to sub-3 Å resolution, revealing an evolutionarily conserved, flexible structure. Through biochemical dissection studies, which include the Drosophila orthologs, we show that the module assembly is hierarchical, with NOT11 binding to NOT10, which then organizes it for binding to NOT1. A short proline-rich motif in NOT11 stabilizes the entire module assembly.
Asunto(s)
Ribonucleasas , Factores de Transcripción , Humanos , Unión Proteica , Receptores CCR4/metabolismo , Ribonucleasas/química , Factores de Transcripción/metabolismoRESUMEN
The CCR4-NOT complex acts as a central player in the control of mRNA turnover and mediates accelerated mRNA degradation upon HDAC inhibition. Here, we explored acetylation-induced changes in the composition of the CCR4-NOT complex by purification of the endogenously tagged scaffold subunit NOT1 and identified RNF219 as an acetylation-regulated cofactor. We demonstrate that RNF219 is an active RING-type E3 ligase which stably associates with CCR4-NOT via NOT9 through a short linear motif (SLiM) embedded within the C-terminal low-complexity region of RNF219. By using a reconstituted six-subunit human CCR4-NOT complex, we demonstrate that RNF219 inhibits deadenylation through the direct interaction of the α-helical SLiM with the NOT9 module. Transcriptome-wide mRNA half-life measurements reveal that RNF219 attenuates global mRNA turnover in cells, with differential requirement of its RING domain. Our results establish RNF219 as an inhibitor of CCR4-NOT-mediated deadenylation, whose loss upon HDAC inhibition contributes to accelerated mRNA turnover.
Asunto(s)
ARN Mensajero/metabolismo , Receptores CCR4/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Monofosfato/metabolismo , Células HeLa , Humanos , Unión Proteica , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/genética , Receptores CCR4/genética , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Pumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad coregulation of target mRNAs through the PUM-CNOT repression mechanism. Functional dissection of the domains of PUM1&2 identified a conserved amino-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the mRNA decapping enzyme, DCP2, has an important role in repression by PUM1&2 amino-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent mRNA decay pathway.
Asunto(s)
ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Receptores CCR4/genética , Factores de Transcripción/genética , Transcriptoma , Adenosina Monofosfato , Secuencia de Bases , Sitios de Unión , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Células HCT116 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Unión Proteica , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores CCR4/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Pumilio is an RNA-binding protein that represses a network of mRNAs to control embryogenesis, stem cell fate, fertility and neurological functions in Drosophila. We sought to identify the mechanism of Pumilio-mediated repression and find that it accelerates degradation of target mRNAs, mediated by three N-terminal Repression Domains (RDs), which are unique to Pumilio orthologs. We show that the repressive activities of the Pumilio RDs depend on specific subunits of the Ccr4-Not (CNOT) deadenylase complex. Depletion of Pop2, Not1, Not2, or Not3 subunits alleviates Pumilio RD-mediated repression of protein expression and mRNA decay, whereas depletion of other CNOT components had little or no effect. Moreover, the catalytic activity of Pop2 deadenylase is important for Pumilio RD activity. Further, we show that the Pumilio RDs directly bind to the CNOT complex. We also report that the decapping enzyme, Dcp2, participates in repression by the N-terminus of Pumilio. These results support a model wherein Pumilio utilizes CNOT deadenylase and decapping complexes to accelerate destruction of target mRNAs. Because the N-terminal RDs are conserved in mammalian Pumilio orthologs, the results of this work broadly enhance our understanding of Pumilio function and roles in diseases including cancer, neurodegeneration and epilepsy.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Unión al ARN/genética , Factores de Transcripción/genética , Animales , Drosophila melanogaster/genética , Unión Proteica , Dominios Proteicos/genética , Estabilidad del ARN/genética , ARN Mensajero/genéticaRESUMEN
XRN1 is the major cytoplasmic exoribonuclease in eukaryotes, which degrades deadenylated and decapped mRNAs in the last step of the 5'-3' mRNA decay pathway. Metazoan XRN1 interacts with decapping factors coupling the final stages of decay. Here, we reveal a direct interaction between XRN1 and the CCR4-NOT deadenylase complex mediated by a low-complexity region in XRN1, which we term the 'C-terminal interacting region' or CIR. The CIR represses reporter mRNA deadenylation in human cells when overexpressed and inhibits CCR4-NOT and isolated CAF1 deadenylase activity in vitro. Through complementation studies in an XRN1-null cell line, we dissect the specific contributions of XRN1 domains and regions toward decay of an mRNA reporter. We observe that XRN1 binding to the decapping activator EDC4 counteracts the dominant negative effect of CIR overexpression on decay. Another decapping activator PatL1 directly interacts with CIR and alleviates the CIR-mediated inhibition of CCR4-NOT activity in vitro. Ribosome profiling revealed that XRN1 loss impacts not only on mRNA levels but also on the translational efficiency of many cellular transcripts likely as a consequence of incomplete decay. Our findings reveal an additional layer of direct interactions in a tightly integrated network of factors mediating deadenylation, decapping and 5'-3' exonucleolytic decay.
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Proteínas de Unión al ADN/genética , Exorribonucleasas/genética , Proteínas Asociadas a Microtúbulos/genética , Caperuzas de ARN/genética , Estabilidad del ARN/genética , Endorribonucleasas/genética , Humanos , Complejos Multiproteicos/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Proteínas/genética , ARN Mensajero/química , ARN Mensajero/genética , Receptores CCR4/genética , Proteínas Represoras/genética , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
CCR4-NOT is a conserved multiprotein complex which regulates eukaryotic gene expression principally via shortening of poly(A) tails of messenger RNA or deadenylation. Here, we reconstitute a complete, recombinant human CCR4-NOT complex. Our reconstitution strategy permits strict compositional control to test mechanistic hypotheses with purified component variants. CCR4-NOT is more active and selective for poly(A) than the isolated exonucleases, CCR4a and CAF1, which have distinct deadenylation profiles in vitro. The exonucleases require at least two out of three conserved non-enzymatic modules (CAF40, NOT10:NOT11 or NOT) for full activity in CCR4-NOT. CAF40 and the NOT10:NOT11 module both bind RNA directly and stimulate deadenylation in a partially redundant manner. Linear motifs from different RNA-binding factors that recruit CCR4-NOT to specific mRNAs via protein-protein interactions with CAF40 can inhibit bulk deadenylation. We reveal an additional layer of regulatory complexity to the human deadenylation machinery, which may prime it either for general or target-specific degradation.
Asunto(s)
Exorribonucleasas/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Receptores CCR4/genética , Humanos , Complejos Multiproteicos/síntesis química , Complejos Multiproteicos/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Poli A/metabolismo , ARN Mensajero/genética , Receptores CCR4/metabolismo , Proteínas Recombinantes/genética , Ribonucleasas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Viruses from the genus Enterovirus are important human pathogens. Receptor binding or exposure to acidic pH in endosomes converts enterovirus particles to an activated state that is required for genome release. However, the mechanism of enterovirus uncoating is not well understood. Here, we use cryo-electron microscopy to visualize virions of human echovirus 18 in the process of genome release. We discover that the exit of the RNA from the particle of echovirus 18 results in a loss of one, two, or three adjacent capsid-protein pentamers. The opening in the capsid, which is more than 120 Å in diameter, enables the release of the genome without the need to unwind its putative double-stranded RNA segments. We also detect capsids lacking pentamers during genome release from echovirus 30. Thus, our findings uncover a mechanism of enterovirus genome release that could become target for antiviral drugs.
Asunto(s)
Cápside/ultraestructura , Enterovirus Humano B/ultraestructura , Genoma Viral , ARN Viral/genética , Virión/ultraestructura , Desencapsidación Viral/genética , Animales , Cápside/química , Chlorocebus aethiops , Microscopía por Crioelectrón , Enterovirus Humano B/genética , Células Epiteliales/ultraestructura , Células Epiteliales/virología , Humanos , Concentración de Iones de Hidrógeno , Simulación de Dinámica Molecular , ARN Bicatenario/química , ARN Bicatenario/genética , ARN Viral/química , Virión/genéticaRESUMEN
UNLABELLED: The western honeybee (Apis mellifera) is the most important commercial insect pollinator. However, bees are under pressure from habitat loss, environmental stress, and pathogens, including viruses that can cause lethal epidemics. Slow bee paralysis virus (SBPV) belongs to the Iflaviridae family of nonenveloped single-stranded RNA viruses. Here we present the structure of the SBPV virion determined from two crystal forms to resolutions of 3.4 Å and 2.6 Å. The overall structure of the virion resembles that of picornaviruses, with the three major capsid proteins VP1 to 3 organized into a pseudo-T3 icosahedral capsid. However, the SBPV capsid protein VP3 contains a C-terminal globular domain that has not been observed in other viruses from the order Picornavirales The protruding (P) domains form "crowns" on the virion surface around each 5-fold axis in one of the crystal forms. However, the P domains are shifted 36 Å toward the 3-fold axis in the other crystal form. Furthermore, the P domain contains the Ser-His-Asp triad within a surface patch of eight conserved residues that constitutes a putative catalytic or receptor-binding site. The movements of the domain might be required for efficient substrate cleavage or receptor binding during virus cell entry. In addition, capsid protein VP2 contains an RGD sequence that is exposed on the virion surface, indicating that integrins might be cellular receptors of SBPV. IMPORTANCE: Pollination by honeybees is needed to sustain agricultural productivity as well as the biodiversity of wild flora. However, honeybee populations in Europe and North America have been declining since the 1950s. Honeybee viruses from the Iflaviridae family are among the major causes of honeybee colony mortality. We determined the virion structure of an Iflavirus, slow bee paralysis virus (SBPV). SBPV exhibits unique structural features not observed in other picorna-like viruses. The SBPV capsid protein VP3 has a large C-terminal domain, five of which form highly prominent protruding "crowns" on the virion surface. However, the domains can change their positions depending on the conditions of the environment. The domain includes a putative catalytic or receptor binding site that might be important for SBPV cell entry.