RESUMO
Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.
Assuntos
Grânulos Citoplasmáticos/fisiologia , Estruturas Citoplasmáticas/fisiologia , Mapas de Interação de Proteínas/fisiologia , Fenômenos Biofísicos , Linhagem Celular Tumoral , Citoplasma/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Extração Líquido-Líquido/métodos , Organelas/química , RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/fisiologiaRESUMO
Proper defense against microbial infection depends on the controlled activation of the immune system. This is particularly important for the RIG-I-like receptors (RLRs), which recognize viral dsRNA and initiate antiviral innate immune responses with the potential of triggering systemic inflammation and immunopathology. Here, we show that stress granules (SGs), molecular condensates that form in response to various stresses including viral dsRNA, play key roles in the controlled activation of RLR signaling. Without the SG nucleators G3BP1/2 and UBAP2L, dsRNA triggers excessive inflammation and immune-mediated apoptosis. In addition to exogenous dsRNA, host-derived dsRNA generated in response to ADAR1 deficiency is also controlled by SG biology. Intriguingly, SGs can function beyond immune control by suppressing viral replication independently of the RLR pathway. These observations thus highlight the multi-functional nature of SGs as cellular "shock absorbers" that converge on protecting cell homeostasis by dampening both toxic immune response and viral replication.
Assuntos
DNA Helicases , RNA Helicases , Humanos , DNA Helicases/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Grânulos de Estresse , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Imunidade Inata , Inflamação/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.
Assuntos
Antígeno B7-H1/genética , Interferon gama/metabolismo , RNA Longo não Codificante/genética , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoterapia , Imunoterapia Adotiva/métodos , Interferon gama/genética , Interferons/genética , Interferons/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Proteína 2 Ligante de Morte Celular Programada 1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T CitotóxicosRESUMO
Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.
Assuntos
Células Epiteliais Alveolares/metabolismo , COVID-19/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , SARS-CoV-2/metabolismo , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , Antivirais , COVID-19/genética , COVID-19/patologia , Chlorocebus aethiops , Efeito Citopatogênico Viral , Citoesqueleto , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/virologia , Fosfoproteínas/genética , Transporte Proteico , Proteoma/genética , SARS-CoV-2/genética , Transdução de Sinais , Células Vero , Tratamento Farmacológico da COVID-19RESUMO
Emphysema and chronic obstructive pulmonary disease (COPD) most commonly result from the effects of environmental exposures in genetically susceptible individuals. Genome-wide association studies have implicated ADGRG6 in COPD and reduced lung function, and a limited number of studies have examined the role of ADGRG6 in cells representative of the airway. However, the ADGRG6 locus is also associated with DLCO/VA, an indicator of gas exchange efficiency and alveolar function. Here, we sought to evaluate the mechanistic contributions of ADGRG6 to homeostatic function and disease in type 2 alveolar epithelial cells. We applied an inducible CRISPR interference (CRISPRi) human induced pluripotent stem cell (iPSC) platform to explore ADGRG6 function in iPSC-derived AT2s (iAT2s). We demonstrate that ADGRG6 exerts pleiotropic effects on iAT2s including regulation of focal adhesions, cytoskeleton, tight junctions, and proliferation. Moreover, we find that ADGRG6 knockdown in cigarette smoke-exposed iAT2s alters cellular responses to injury, downregulating apical complexes in favor of proliferation. Our work functionally characterizes the COPD GWAS gene ADGRG6 in human alveolar epithelium.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença Pulmonar Obstrutiva Crônica , Receptores Acoplados a Proteínas G , Humanos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais/metabolismo , Estudo de Associação Genômica Ampla , Células-Tronco Pluripotentes Induzidas/metabolismo , Pulmão/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMO
Some chemotherapy drugs modulate the formation of stress granules (SGs), which are RNA-containing cytoplasmic foci contributing to stress response pathways. How SGs mechanistically contribute to pro-survival or pro-apoptotic functions must be better defined. The chemotherapy drug lomustine promotes SG formation by activating the stress-sensing eIF2α kinase HRI (encoded by the EIF2AK1 gene). Here, we applied a DNA microarray-based transcriptome analysis to determine the genes modulated by lomustine-induced stress and suggest roles for SGs in this process. We found that the expression of the pro-apoptotic EGR1 gene was specifically regulated in cells upon lomustine treatment. The appearance of EGR1-encoding mRNA in SGs correlated with a decrease in EGR1 mRNA translation. Specifically, EGR1 mRNA was sequestered to SGs upon lomustine treatment, probably preventing its ribosome translation and consequently limiting the degree of apoptosis. Our data support the model where SGs can selectively sequester specific mRNAs in a stress-specific manner, modulate their availability for translation, and thus determine the fate of a stressed cell.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce , Lomustina , RNA Mensageiro , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Lomustina/farmacologia , Grânulos de Estresse/metabolismo , Grânulos de Estresse/genética , Apoptose/efeitos dos fármacos , Antineoplásicos Alquilantes/farmacologiaRESUMO
G-Protein Pathway Suppressor 2 (GPS2) is an inhibitor of non-proteolytic K63 ubiquitination mediated by the E2 ubiquitin-conjugating enzyme Ubc13. Previous studies have associated GPS2-mediated restriction of ubiquitination with the regulation of insulin signaling, inflammatory responses and mitochondria-nuclear communication across different tissues and cell types. However, a detailed understanding of the targets of GPS2/Ubc13 activity is lacking. Here, we have dissected the GPS2-regulated K63 ubiquitome in mouse embryonic fibroblasts and human breast cancer cells, unexpectedly finding an enrichment for proteins involved in RNA binding and translation on the outer mitochondrial membrane. Validation of selected targets of GPS2-mediated regulation, including the RNA-binding protein PABPC1 and translation factors RPS1, RACK1 and eIF3M, revealed a mitochondrial-specific strategy for regulating the translation of nuclear-encoded mitochondrial proteins via non-proteolytic ubiquitination. Removal of GPS2-mediated inhibition, either via genetic deletion or stress-induced nuclear translocation, promotes the import-coupled translation of selected mRNAs leading to the increased expression of an adaptive antioxidant program. In light of GPS2 role in nuclear-mitochondria communication, these findings reveal an exquisite regulatory network for modulating mitochondrial gene expression through spatially coordinated transcription and translation.
Assuntos
Mitocôndrias , Biossíntese de Proteínas , Ubiquitinação , Animais , Humanos , Mitocôndrias/metabolismo , Camundongos , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Linhagem Celular Tumoral , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
The production of ribosomes is an energy-intensive process owing to the intricacy of these massive macromolecular machines. Each human ribosome contains 80 ribosomal proteins and four non-coding RNAs. Accurate assembly requires precise regulation of protein and RNA subunits. In response to stress, the integrated stress response (ISR) rapidly inhibits global translation. How rRNA is coordinately regulated with the rapid inhibition of ribosomal protein synthesis is not known. Here, we show that stress specifically inhibits the first step of rRNA processing. Unprocessed rRNA is stored within the nucleolus, and when stress resolves, it re-enters the ribosome biogenesis pathway. Retention of unprocessed rRNA within the nucleolus aids in the maintenance of this organelle. This response is independent of the ISR or inhibition of cellular translation but is independently regulated. Failure to coordinately control ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils how the rapid translational shut-off in response to stress coordinates with rRNA synthesis production to maintain nucleolar integrity.
Assuntos
RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos , Células HeLa , Humanos , Biogênese de Organelas , Processamento Pós-Transcricional do RNA , Ribossomos/genética , Ribossomos/metabolismo , Estresse Fisiológico , Transcrição GênicaRESUMO
The 3' untranslated regions (UTRs) of Ebola virus (EBOV) mRNAs are enriched in their AU content and therefore represent potential targets for RNA binding proteins targeting AU-rich elements (ARE-BPs). ARE-BPs are known to fine-tune RNA turnover and translational activity. We identified putative AREs within EBOV mRNA 3' UTRs and assessed whether they might modulate mRNA stability. Using mammalian and zebrafish embryo reporter assays, we show a conserved, ARE-BP-mediated stabilizing effect and increased reporter activity with the tested EBOV 3' UTRs. When coexpressed with the prototypic ARE-BP tristetraprolin (TTP, ZFP36) that mainly destabilizes its target mRNAs, the EBOV nucleoprotein (NP) 3' UTR resulted in decreased reporter gene activity. Coexpression of NP with TTP led to reduced NP protein expression and diminished EBOV minigenome activity. In conclusion, the enrichment of AU residues in EBOV 3' UTRs makes them possible targets for cellular ARE-BPs, leading to modulation of RNA stability and translational activity.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Animais , Regiões 3' não Traduzidas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ebolavirus/genética , Ebolavirus/metabolismo , Doença pelo Vírus Ebola/genética , Peixe-Zebra/metabolismo , Estabilidade de RNA/genética , MamíferosRESUMO
RNA provides the framework for the assembly of some of the most intricate macromolecular complexes within the cell, including the spliceosome and the mature ribosome. The assembly of these complexes relies on the coordinated association of RNA with hundreds of trans-acting protein factors. While some of these trans-acting factors are RNA-binding proteins (RBPs), others are adaptor proteins, and others still, function as both. Defects in the assembly of these complexes results in a number of human pathologies including neurodegeneration and cancer. Here, we demonstrate that Silencing Defective 2 (SDE2) is both an RNA binding protein and also a trans-acting adaptor protein that functions to regulate RNA splicing and ribosome biogenesis. SDE2 depletion leads to widespread changes in alternative splicing, defects in ribosome biogenesis and ultimately complete loss of cell viability. Our data highlight SDE2 as a previously uncharacterized essential gene required for the assembly and maturation of the complexes that carry out two of the most fundamental processes in mammalian cells.
Assuntos
Processamento Alternativo/genética , Proteínas de Ligação a DNA/genética , Splicing de RNA/genética , Ribossomos/genética , Genes Essenciais/genética , Humanos , Proteínas de Ligação a RNA/genética , Spliceossomos/genéticaRESUMO
As cells encounter adverse environmental conditions, such as hypoxia, oxidative stress or nutrient deprivation, they trigger stress response pathways to protect themselves until transient stresses have passed. Inhibition of translation is a key component of such cellular stress responses and mounting evidence has revealed the importance of a class of tRNA-derived small RNAs called tiRNAs in this process. The most potent of these small RNAs are those with the capability of assembling into tetrameric G-quadruplex (G4) structures. However, the mechanism by which these small RNAs inhibit translation has yet to be elucidated. Here we show that eIF4G, the major scaffolding protein in the translation initiation complex, directly binds G4s and this activity is required for tiRNA-mediated translation repression. Targeting of eIF4G results in an impairment of 40S ribosome scanning on mRNAs leading to the formation of eIF2α-independent stress granules. Our data reveals the mechanism by which tiRNAs inhibit translation and demonstrates novel activity for eIF4G in the regulation of translation.
Assuntos
Fator de Iniciação Eucariótico 4G/metabolismo , Quadruplex G , Biossíntese de Proteínas , RNA de Transferência/química , RNA de Transferência/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/química , Fator de Iniciação 4F em Eucariotos/metabolismo , Humanos , Iniciação Traducional da Cadeia Peptídica , Fosfoproteínas/metabolismo , Domínios Proteicos , RNA Mensageiro/metabolismo , RNA de Transferência/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismoRESUMO
Cancer cells show significant dysregulation of genes expression, which may favor their survival in the tumor environment. In this study, the cellular vault's components MVP (major vault protein), TEP1 (telomerase-associated protein 1) and vPARP (vault poly(ADP-ribose) polymerase) were transiently or completely inhibited in U2OS cells (human bone osteosarcoma epithelial cells) to evaluate their impact on the cell proliferative and migratory capacity as well as on the development of their resistance to the drug vinorelbine. Comparative analysis of MVP protein expression level in normal colon tissue, primary colorectal tumor, and metastasis showed that the expression of this protein does not increase significantly in the primary tumor, but its expression increases in metastatic cells. Further comparative molecular analysis using the whole transcriptome microarrays for MVP-positive and MVP-negative cells showed that MVP is involved in regulating proliferation and migration of cancer cells. MVP may facilitate metastasis of colon cancer due to its impact on cell migration. Moreover, two vault proteins, MVP and TEP1, contribute the resistance to vinorelbine, while vPARP does not.
Assuntos
Neoplasias Colorretais/genética , Metástase Neoplásica/genética , Poli(ADP-Ribose) Polimerases/genética , Proteínas de Ligação a RNA/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Adulto , Idoso , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/patologia , Transcriptoma/genéticaRESUMO
Cells have developed different mechanisms to respond to stress, including the formation of cytoplasmic foci known as stress granules (SGs). SGs are dynamic and formed as a result of stress-induced inhibition of translation. Despite enormous interest in SGs due to their contribution to the pathogenesis of several human diseases, many aspects of SG formation are poorly understood. SGs induced by different stresses are generally assumed to be uniform, although some studies suggest that different SG subtypes and SG-like cytoplasmic foci exist. Here, we investigated the molecular mechanisms of SG assembly and characterized their composition when induced by various stresses. Our data revealed stress-specific differences in composition, assembly and dynamics of SGs and SG-like cytoplasmic foci. Using a set of genetically modified haploid human cells, we determined the molecular circuitry of stress-specific translation inhibition upstream of SG formation and its relation to cell survival. Finally, our studies characterize cytoplasmic stress-induced foci related to, but distinct from, canonical SGs, and also introduce haploid cells as a valuable resource to study RNA granules and translation control mechanisms.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Estresse Fisiológico , Animais , Arsenitos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Inativação de Genes , Humanos , Camundongos , Mutação/genética , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Compostos de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacosRESUMO
Stress-induced angiogenin (ANG)-mediated tRNA cleavage promotes a cascade of cellular events that starts with production of tRNA-derived stress-induced RNAs (tiRNAs) and culminates with enhanced cell survival. This stress response program relies on a subset tiRNAs that inhibit translation initiation and induce the assembly of stress granules (SGs), cytoplasmic ribonucleoprotein complexes with cytoprotective and pro-survival properties. SG-promoting tiRNAs bear oligoguanine motifs at their 5'-ends, assemble G-quadruplex-like structures and interact with the translational silencer YB-1. We used CRISPR/Cas9-based genetic manipulations and biochemical approaches to examine the role of YB-1 in tiRNA-mediated translational repression and SG assembly. We found that YB-1 directly binds to tiRNAs via its cold shock domain. This interaction is required for packaging of tiRNA-repressed mRNAs into SGs but is dispensable for tiRNA-mediated translational repression. Our studies reveal the functional role of YB-1 in the ANG-mediated stress response program.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Estresse Fisiológico , Proteína 1 de Ligação a Y-Box/metabolismo , Sequência de Aminoácidos , Arsenitos/farmacologia , Sequência de Bases , Sistemas CRISPR-Cas/genética , Proliferação de Células , Sobrevivência Celular , Grânulos Citoplasmáticos/efeitos dos fármacos , Fator de Iniciação 4F em Eucariotos/metabolismo , Deleção de Genes , Técnicas de Inativação de Genes , Heterogeneidade Genética , Humanos , Células MCF-7 , Modelos Moleculares , Biossíntese de Proteínas/efeitos dos fármacos , RNA Guia de Cinetoplastídeos/metabolismo , RNA de Transferência/genética , Compostos de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Proteína 1 de Ligação a Y-Box/química , Proteína 1 de Ligação a Y-Box/genéticaRESUMO
Histone proteins are synthesized in large amounts during S-phase to package the newly replicated DNA, and are among the most stable proteins in the cell. The replication-dependent (RD)-histone mRNAs expressed during S-phase end in a conserved stem-loop rather than a polyA tail. In addition, there are replication-independent (RI)-histone genes that encode histone variants as polyadenylated mRNAs. Most variants have specific functions in chromatin, but H3.3 also serves as a replacement histone for damaged histones in long-lived terminally differentiated cells. There are no reported replacement histone genes for histones H2A, H2B or H4. We report that a subset of RD-histone genes are expressed in terminally differentiated tissues as polyadenylated mRNAs, likely serving as replacement histone genes in long-lived non-dividing cells. Expression of two genes, HIST2H2AA3 and HIST1H2BC, is conserved in mammals. They are expressed as polyadenylated mRNAs in fibroblasts differentiated in vitro, but not in serum starved fibroblasts, suggesting that their expression is part of the terminal differentiation program. There are two histone H4 genes and an H3 gene that encode mRNAs that are polyadenylated and expressed at 5- to 10-fold lower levels than the mRNAs from H2A and H2B genes, which may be replacement genes for the H3.1 and H4 proteins.
Assuntos
Expressão Gênica , Histonas/genética , RNA Mensageiro/genética , Animais , Sequência de Bases , Ciclo Celular/genética , Linhagem Celular , Humanos , Fígado/metabolismo , Camundongos , Especificidade de Órgãos/genética , Poli A , Estabilidade de RNA , RNA Mensageiro/química , Transcrição GênicaRESUMO
The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem-loop (SL) structure. This SL structure interacts with the stem-loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3'hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3' end of RD-histone mRNAs together with SLBP.
Assuntos
Histonas/genética , RNA Mensageiro/genética , Animais , Sítios de Ligação/genética , Linhagem Celular , Linhagem Celular Tumoral , Replicação do DNA/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Ligação Proteica/genética , Proteínas de Ligação a RNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Angiogenin (ANG) is a secreted ribonuclease best known for its ability to promote formation of blood vessels. Extensive research over many years has elucidated its structure and biophysical properties, although our knowledge of molecular mechanisms underlying ANG-associated biologic processes remains limited. Intriguingly, many of processes require the ribonuclease activity of ANG, thus highlighting the importance of identifying and characterizing RNA targets and intermediates of ANG-mediated endonucleolytic cleavage. While ANG demonstrates ribonuclease activity toward many RNA substrates in vitro, specific target of ANG, namely mature tRNA, was only recently identified in vivo. ANG-mediated tRNA cleavage is an unorthodox manner of generating non-coding RNAs with diverse biologic activities. In addition, the ribonuclease activity of ANG has been reported to be crucial for rRNA transcription. Here we critically discuss various aspects of ANG biology related to its RNase activity and discuss areas in need of further investigation.
Assuntos
RNA/genética , RNA/metabolismo , Ribonuclease Pancreático/metabolismo , Adaptação Biológica , Animais , Suscetibilidade a Doenças , Ativação Enzimática , Humanos , Neovascularização Fisiológica/genética , Ligação Proteica , RNA/química , Clivagem do RNA , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonucleases/metabolismo , Ribossomos/metabolismo , Transdução de Sinais , Especificidade por SubstratoRESUMO
Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that lack a poly(A) tail, ending instead in a conserved stem-loop sequence, which is bound to the stem-loop binding protein (SLBP) on the histone mRNP. Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S phase in mammalian cells. Rapid degradation of histone mRNAs is initiated by oligouridylation of the 3' end of histone mRNAs and requires the cytoplasmic Lsm1-7 complex, which can bind to the oligo(U) tail. An exonuclease, 3'hExo, forms a ternary complex with SLBP and the stem-loop and is required for the initiation of histone mRNA degradation. The Lsm1-7 complex is also involved in degradation of polyadenylated mRNAs. It binds to the oligo(A) tail remaining after deadenylation, inhibiting translation and recruiting the enzymes required for decapping. Whether the Lsm1-7 complex interacts directly with other components of the mRNP is not known. We report here that the C-terminal extension of Lsm4 interacts directly with the histone mRNP, contacting both SLBP and 3'hExo. Mutants in the C-terminal tail of Lsm4 that prevent SLBP and 3'hExo binding reduce the rate of histone mRNA degradation when DNA synthesis is inhibited.
Assuntos
Região 3'-Flanqueadora , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Estabilidade de RNA , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Células HeLa , Histonas/genética , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologia , RNA Mensageiro/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Fatores de Poliadenilação e Clivagem de mRNA/químicaRESUMO
We present RNA-mechanically induced trapping of molecular interactions (RNA-MITOMI), a microfluidic platform that allows integrated synthesis and functional assays for programmable RNA libraries. The interaction of a comprehensive library of RNA mutants with stem-loop-binding protein precisely defined the RNA structural and sequence features that govern affinity. The functional motif reconstructed in a single experiment on our platform uncovers new binding specificities and enriches interpretation of phylogenetic data.