RESUMO
Nonsense-mediated mRNA decay (NMD) is one of the best characterized and most evolutionarily conserved cellular quality control mechanisms. Although NMD was first found to target one-third of mutated, disease-causing mRNAs, it is now known to also target ~10% of unmutated mammalian mRNAs to facilitate appropriate cellular responses - adaptation, differentiation or death - to environmental changes. Mutations in NMD genes in humans are associated with intellectual disability and cancer. In this Review, we discuss how NMD serves multiple purposes in human cells by degrading both mutated mRNAs to protect the integrity of the transcriptome and normal mRNAs to control the quantities of unmutated transcripts.
Assuntos
Regulação Neoplásica da Expressão Gênica , Deficiência Intelectual/metabolismo , Mutação , Neoplasias/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Transcriptoma , Animais , Humanos , Deficiência Intelectual/genética , Neoplasias/genética , RNA Mensageiro/genética , RNA Neoplásico/genéticaRESUMO
The HTML version of the article displayed the wrong Figure 3 (while the PDF version was correct); the HTML has now been corrected and we apologize for any confusion it may have created.
RESUMO
Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA quality control and regulatory process that plays direct roles in human health and disease. In this Minireview, we discuss how understanding the molecular events that trigger NMD can facilitate strategic targeting of genes via CRISPR/Cas9 technologies and also inform disease diagnostics and treatments.
Assuntos
Engenharia Genética , Degradação do RNAm Mediada por Códon sem Sentido , Medicina de Precisão , Sistemas CRISPR-Cas , Marcação de Genes , Genoma , Humanos , RNA MensageiroRESUMO
PGC-1α is well established as a metazoan transcriptional coactivator of cellular adaptation in response to stress. However, the mechanisms by which PGC-1α activates gene transcription are incompletely understood. Here, we report that PGC-1α serves as a scaffold protein that physically and functionally connects the DNA-binding protein estrogen-related receptor α (ERRα), cap-binding protein 80 (CBP80), and Mediator to overcome promoter-proximal pausing of RNAPII and transcriptionally activate stress-response genes. We show that PGC-1α promotes pausing release in a two-arm mechanism (1) by recruiting the positive transcription elongation factor b (P-TEFb) and (2) by outcompeting the premature transcription termination complex Integrator. Using mice homozygous for five amino acid changes in the CBP80-binding motif (CBM) of PGC-1α that destroy CBM function, we show that efficient differentiation of primary myoblasts to myofibers and timely skeletal muscle regeneration after injury require PGC-1α binding to CBP80. Our findings reveal how PGC-1α activates stress-response gene transcription in a previously unanticipated pre-mRNA quality-control pathway.
Assuntos
Precursores de RNA , Fatores de Transcrição , Animais , Camundongos , Proteínas de Ligação a DNA/genética , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Regiões Promotoras Genéticas , Proteínas de Ligação ao Cap de RNA/genética , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Proper regulation of mRNA production in the nucleus is critical for the maintenance of cellular homoeostasis during adaptation to internal and environmental cues. Over the past 25 years, it has become clear that the nuclear machineries governing gene transcription, pre-mRNA processing, pre-mRNA and mRNA decay, and mRNA export to the cytoplasm are inextricably linked to control the quality and quantity of mRNAs available for translation. More recently, an ever-expanding diversity of new mechanisms by which nuclear RNA decay factors finely tune the expression of protein-encoding genes have been uncovered. Here, we review the current understanding of how mammalian cells shape their protein-encoding potential by regulating the decay of pre-mRNAs and mRNAs in the nucleus.
RESUMO
How fragile X syndrome protein (FMRP) binds mRNAs and regulates mRNA metabolism remains unclear. Our previous work using human neuronal cells focused on mRNAs targeted for nonsense-mediated mRNA decay (NMD), which we showed are generally bound by FMRP and destabilized upon FMRP loss. Here, we identify >400 high-confidence FMRP-bound mRNAs, only â¼35% of which are NMD targets. Integrative transcriptomics together with SILAC-LC-MS/MS reveal that FMRP loss generally results in mRNA destabilization and more protein produced per FMRP target. We use our established RIP-seq technology to show that FMRP footprints are independent of protein-coding potential, target GC-rich and structured sequences, and are densest in 5' UTRs. Regardless of where within an mRNA FMRP binds, we find that FMRP protects mRNAs from deadenylation and directly binds the cytoplasmic poly(A)-binding protein. Our results reveal how FMRP sequesters polyadenylated mRNAs into stabilized and translationally repressed complexes, whose regulation is critical for neurogenesis and synaptic plasticity.
Assuntos
Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil , Humanos , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cromatografia Líquida , Espectrometria de Massas em Tandem , Síndrome do Cromossomo X Frágil/genéticaRESUMO
Despite a long appreciation for the role of nonsense-mediated mRNA decay (NMD) in destroying faulty, disease-causing mRNAs and maintaining normal, physiologic mRNA abundance, additional effectors that regulate NMD activity in mammalian cells continue to be identified. Here, we describe a haploid-cell genetic screen for NMD effectors that has unexpectedly identified 13 proteins constituting the AKT signaling pathway. We show that AKT supersedes UPF2 in exon-junction complexes (EJCs) that are devoid of RNPS1 but contain CASC3, defining an unanticipated insulin-stimulated EJC. Without altering UPF1 RNA binding or ATPase activity, AKT-mediated phosphorylation of the UPF1 CH domain at T151 augments UPF1 helicase activity, which is critical for NMD and also decreases the dependence of helicase activity on ATP. We demonstrate that upregulation of AKT signaling contributes to the hyperactivation of NMD that typifies Fragile X syndrome, as exemplified using FMR1-KO neural stem cells derived from induced pluripotent stem cells.
Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , Proteínas Proto-Oncogênicas c-akt , Animais , Códon sem Sentido/genética , Éxons/genética , Mamíferos/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The largely nuclear cap-binding complex (CBC) binds to the 5' caps of RNA polymerase II (RNAPII)-synthesized transcripts and serves as a dynamic interaction platform for a myriad of RNA processing factors that regulate gene expression. While influence of the CBC can extend into the cytoplasm, here we review the roles of the CBC in the nucleus, with a focus on protein-coding genes. We discuss differences between CBC function in yeast and mammals, covering the steps of transcription initiation, release of RNAPII from pausing, transcription elongation, cotranscriptional pre-mRNA splicing, transcription termination, and consequences of spurious transcription. We describe parameters known to control the binding of generic or gene-specific cofactors that regulate CBC activities depending on the process(es) targeted, illustrating how the CBC is an ever-changing choreographer of gene expression.
Assuntos
Regulação da Expressão Gênica , Proteínas de Ligação ao Cap de RNA/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , Animais , Núcleo Celular/metabolismo , Humanos , Fases de Leitura Aberta/genética , Saccharomyces cerevisiaeRESUMO
Although peroxisome proliferator-activated receptor-γ (PPARγ) coactivator 1α (PGC-1α) is a well-established transcriptional coactivator for the metabolic adaptation of mammalian cells to diverse physiological stresses, the molecular mechanism by which it functions is incompletely understood. Here we used in vitro binding assays, X-ray crystallography, and immunoprecipitations of mouse myoblast cell lysates to define a previously unknown cap-binding protein 80 (CBP80)-binding motif (CBM) in the C terminus of PGC-1α. We show that the CBM, which consists of a nine-amino-acid α helix, is critical for the association of PGC-1α with CBP80 at the 5' cap of target transcripts. Results from RNA sequencing demonstrate that the PGC-1α CBM promotes RNA synthesis from promyogenic genes. Our findings reveal a new conduit between DNA-associated and RNA-associated proteins that functions in a cap-binding protein surveillance mechanism, without which efficient differentiation of myoblasts to myotubes fails to occur.
Assuntos
Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/química , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ativação Transcricional , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Diferenciação Celular , Humanos , Células MCF-7 , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/metabolismo , Proteínas de Ligação a RNA , Transcrição GênicaRESUMO
Eukaryotic cells have divided the steps of gene expression between their nucleus and cytoplasm. Protein-encoding genes generate mRNAs in the nucleus and mRNAs undergo transport to the cytoplasm for the purpose of producing proteins. Cap-binding protein (CBP)20 and its binding partner CBP80 have been thought to constitute the cap-binding complex (CBC) that is acquired co-transcriptionally by the precursors of all mRNAs. However, this principle has recently been challenged by studies of nuclear cap-binding protein 3 (NCBP3). Here we submit how NCBP3, as an alternative to CBP20, an accessory to the canonical CBP20-CBP80 CBC, and/or an RNA-binding protein - possibly in association with the exon-junction complex (EJC) - expands the capacity of cells to regulate gene expression.
Assuntos
Núcleo Celular , Proteínas de Ligação a RNA , Expressão Gênica , Proteínas de Ligação ao Cap de RNA/genética , RNA Mensageiro , Proteínas de Ligação a RNA/genéticaRESUMO
In mammalian cells, newly synthesized mRNAs undergo a pioneer round of translation that is important for mRNA quality control. Following maturation of messenger ribonucleoprotein particles during and after the pioneer round, steady-state cycles of mRNA translation generate most of the cell's proteins. Translation factors, RNA-binding proteins, and targets of signaling pathways that are particular to newly synthesized mRNAs regulate critical functions of the pioneer round.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Animais , Fenômenos Fisiológicos Celulares , Humanos , Estabilidade de RNA , RNA Mensageiro/genética , Ribonucleoproteínas/metabolismo , Ribossomos/metabolismoRESUMO
While microRNAs (miRNAs) regulate the vast majority of protein-encoding transcripts, little is known about how miRNAs themselves are degraded. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway in which the nuclease TSN promotes the decay of miRNAs that contain CA and/or UA dinucleotides. While TSN-mediated degradation of either protein-free or AGO2-loaded miRNAs does not require the ATP-dependent RNA helicase UPF1 in vitro, we report here that cellular TumiD requires UPF1. Results from experiments using AGO2-loaded miRNAs in duplex with target mRNAs indicate that UPF1 can dissociate miRNAs from their mRNA targets, making the miRNAs susceptible to TumiD. miR-seq (deep sequencing of miRNAs) data reveal that the degradation of â¼50% of candidate TumiD targets in T24 human urinary bladder cancer cells is augmented by UPF1. We illustrate the physiological relevance by demonstrating that UPF1-augmented TumiD promotes the invasion of T24 cells in part by degrading anti-invasive miRNAs so as to up-regulate the expression of proinvasive proteins.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , MicroRNAs/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA , Transativadores/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/química , Análise de Sequência de RNA , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismoRESUMO
How does a mammalian cell determine when newly synthesized mRNAs are fully processed and appropriate for nuclear export? Müller-McNicoll and colleagues (pp. 553-566) expand on mechanisms known to be mediated by nuclear export factor 1 (NXF1) by describing SR proteins as NXF1 adaptors that flag alternatively spliced and polyadenylated mRNA isoforms as cargo ready for the cytoplasm.
Assuntos
Núcleo Celular/metabolismo , Processamento de Terminações 3' de RNA , Precursores de RNA/metabolismo , Splicing de RNA , Transporte de RNA , Animais , Humanos , Camundongos , Ligação ProteicaRESUMO
In mammalian cells, nonsense-mediated mRNA decay (NMD) generally requires that translation terminates sufficiently upstream of a post-splicing exon junction complex (EJC) during a pioneer round of translation. The subsequent binding of Upf1 to the EJC triggers Upf1 phosphorylation. We provide evidence that phospho-Upf1 functions after nonsense codon recognition during steps that involve the translation initiation factor eIF3 and mRNA decay factors. Phospho-Upf1 interacts directly with eIF3 and inhibits the eIF3-dependent conversion of 40S/Met-tRNA(i)(Met)/mRNA to translationally competent 80S/Met-tRNA(i)(Met)/mRNA initiation complexes to repress continued translation initiation. Consistent with phospho-Upf1 impairing eIF3 function, NMD fails to detectably target nonsense-containing transcripts that initiate translation independently of eIF3 from the CrPV IRES. There is growing evidence that translational repression is a key transition that precedes mRNA delivery to the degradation machinery. Our results uncover a critical step during NMD that converts a pioneer translation initiation complex to a translationally compromised mRNP.
Assuntos
Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Animais , Células COS , Chlorocebus aethiops , Códon sem Sentido , Células HeLa , Hepacivirus/metabolismo , Humanos , Fosforilação , RNA Helicases , Ribonucleoproteínas/metabolismoRESUMO
A subset of messenger RNAs (mRNAs) that contain inverted Alu elements in their 3' untranslated region are inefficiently exported to the cytoplasm and retained in subnuclear bodies called paraspeckles. The arginine methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1) promotes the nuclear export of these mRNAs by methylating the paraspeckle component p54(nrb), which reduces the binding of p54(nrb) to the inverted Alu elements, and down-regulating synthesis of another paraspeckle component, the long noncoding RNA NEAT1, which inhibits paraspeckle formation.
Assuntos
Núcleo Celular/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Animais , HumanosRESUMO
Viruses have evolved in tandem with the organisms that they infect. Afflictions of the plant and animal kingdoms with viral infections have forced the host organism to evolve new or exploit existing systems to develop the countermeasures needed to offset viral insults. As one example, nonsense-mediated mRNA decay, a cellular quality-control mechanism ensuring the translational fidelity of mRNA transcripts, has been used to restrict virus replication in both plants and animals. In response, viruses have developed a slew of means to disrupt or become insensitive to NMD, providing researchers with potential new reagents that can be used to more fully understand the NMD mechanism.
Assuntos
Interações Hospedeiro-Patógeno , Degradação do RNAm Mediada por Códon sem Sentido , Vírus/metabolismo , Animais , Humanos , Plantas/virologia , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Virais/genética , Fenômenos Fisiológicos Virais , Vírus/classificação , Vírus/genética , Vírus/crescimento & desenvolvimentoRESUMO
Staufen1 (STAU1) is an RNA-binding protein (RBP) that interacts with double-stranded RNA structures and has been implicated in regulating different aspects of mRNA metabolism. Previous studies have indicated that STAU1 interacts extensively with RNA structures in coding regions (CDSs) and 3'-untranslated regions (3'UTRs). In particular, duplex structures formed within 3'UTRs by inverted-repeat Alu elements (IRAlus) interact with STAU1 through its double-stranded RNA-binding domains (dsRBDs). Using 3' region extraction and deep sequencing coupled to ribonucleoprotein immunoprecipitation (3'READS + RIP), together with reanalyzing previous STAU1 binding and RNA structure data, we delineate STAU1 interactions transcriptome-wide, including binding differences between alternative polyadenylation (APA) isoforms. Consistent with previous reports, RNA structures are dominant features for STAU1 binding to CDSs and 3'UTRs. Overall, relative to short 3'UTR counterparts, longer 3'UTR isoforms of genes have stronger STAU1 binding, most likely due to a higher frequency of RNA structures, including specific IRAlus sequences. Nevertheless, a sizable fraction of genes express transcripts showing the opposite trend, attributable to AU-rich sequences in their alternative 3'UTRs that may recruit antagonistic RBPs and/or destabilize RNA structures. Using STAU1-knockout cells, we show that strong STAU1 binding to mRNA 3'UTRs generally enhances polysome association. However, IRAlus generally have little impact on STAU1-mediated polysome association despite having strong interactions with the protein. Taken together, our work reveals complex interactions of STAU1 with its cognate RNA substrates. Our data also shed light on distinct post-transcriptional fates for the widespread APA isoforms in mammalian cells.
Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Polirribossomos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Processamento Alternativo , Elementos Alu , Proteínas do Citoesqueleto/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoprecipitação , Conformação Molecular , Motivos de Ligação ao RNA , Proteínas de Ligação a RNA/genéticaRESUMO
Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase upframeshift 1 (UPF1). Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for premature termination codon (PTC)-containing reporter mRNAs when compared with their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1)-binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD target marker. p-UPF1 is enriched on NMD target 3' untranslated regions (UTRs) along with suppressor with morphogenic effect on genitalia 5 (SMG5) and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with Förster resonance energy transfer (FRET) experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3' UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3' UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay.
Assuntos
Estabilidade de RNA/genética , Transativadores/genética , Transativadores/metabolismo , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , RNA Helicases/metabolismo , Transcriptoma , Regulação para CimaRESUMO
Cells use messenger RNAs (mRNAs) to ensure the accurate dissemination of genetic information encoded by DNA. Given that mRNAs largely direct the synthesis of a critical effector of cellular phenotype, i.e., proteins, tight regulation of both the quality and quantity of mRNA is a prerequisite for effective cellular homeostasis. Here, we review nonsense-mediated mRNA decay (NMD), which is the best-characterized posttranscriptional quality control mechanism that cells have evolved in their cytoplasm to ensure transcriptome fidelity. We use protein quality control as a conceptual framework to organize what is known about NMD, highlighting overarching similarities between these two polymer quality control pathways, where the protein quality control and NMD pathways intersect, and how protein quality control can suggest new avenues for research into mRNA quality control.