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1.
Nat Commun ; 11(1): 5441, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116138

RESUMO

With global warming and climate change, breeding crop plants tolerant to high-temperature stress is of immense significance. tRNA 2-thiolation is a highly conserved form of tRNA modification among living organisms. Here, we report the identification of SLG1 (Slender Guy 1), which encodes the cytosolic tRNA 2-thiolation protein 2 (RCTU2) in rice. SLG1 plays a key role in the response of rice plants to high-temperature stress at both seedling and reproductive stages. Dysfunction of SLG1 results in plants with thermosensitive phenotype, while overexpression of SLG1 enhances the tolerance of plants to high temperature. SLG1 is differentiated between the two Asian cultivated rice subspecies, indica and japonica, and the variations at both promoter and coding regions lead to an increased level of thiolated tRNA and enhanced thermotolerance of indica rice varieties. Our results demonstrate that the allelic differentiation of SLG1 confers indica rice to high-temperature tolerance, and tRNA thiolation pathway might be a potential target in the next generation rice breeding for the warming globe.


Assuntos
Genes de Plantas , Oryza/genética , Oryza/fisiologia , Termotolerância/genética , Termotolerância/fisiologia , Variação Genética , Aquecimento Global , Modelos Biológicos , Melhoramento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA/genética , Processamento Pós-Transcricional do RNA/fisiologia , RNA de Plantas/metabolismo , RNA de Transferência/metabolismo , Tionucleotídeos/metabolismo
2.
Chemosphere ; 261: 127757, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32726721

RESUMO

N6-methyladenosine (m6A) is one of the most common RNA modifications in eukaryotes involved in the regulation of post-transcriptional gene expression, as well as the occurrence and development of diseases related to environmental exposures. Adverse factors produced by environmental exposures, such as reactive oxygen species, inflammation, and cyclobutane pyrimidine dimers, mediate m6A modification, thereby regulating downstream gene and protein expression, and signaling pathways, such as FTO/m6A RNA/p53 axis, PI3K/AKT/mTOR pathway, and PARP/METTL3/m6A RNA/Pol κ pathway. Moreover, an imbalance in m6A methylation levels directly mediates disease pathogenesis. To date, some studies have detailed the mechanisms underlying environmental exposure-mediated global changes in RNA m6A methylation. Based on our current understanding, we aimed to elaborate on the molecular mechanisms through which RNA m6A methylation regulates gene expression under environmental exposures. In this review, we outline the biogenesis and functions of RNA m6A modification. Furthermore, we focus on the effects of environmental exposures on m6A levels and highlight the relationships between environmental exposures (doses and time) and m6A levels. Although the molecular mechanisms regulating gene expression remains to be elucidated, m6A has potential applications as a disease biomarker.


Assuntos
Adenosina/análogos & derivados , Exposição Ambiental , Poluição Ambiental/efeitos adversos , Regulação da Expressão Gênica , Processamento Pós-Transcricional do RNA/genética , RNA/genética , Adenosina/genética , Células Eucarióticas/efeitos dos fármacos , Humanos , Metilação , Transdução de Sinais/genética
3.
Am J Hum Genet ; 107(3): 544-554, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32730804

RESUMO

RNA polymerase II interacts with various other complexes and factors to ensure correct initiation, elongation, and termination of mRNA transcription. One of these proteins is SR-related CTD-associated factor 4 (SCAF4), which is important for correct usage of polyA sites for mRNA termination. Using exome sequencing and international matchmaking, we identified nine likely pathogenic germline variants in SCAF4 including two splice-site and seven truncating variants, all residing in the N-terminal two thirds of the protein. Eight of these variants occurred de novo, and one was inherited. Affected individuals demonstrated a variable neurodevelopmental disorder characterized by mild intellectual disability, seizures, behavioral abnormalities, and various skeletal and structural anomalies. Paired-end RNA sequencing on blood lymphocytes of SCAF4-deficient individuals revealed a broad deregulation of more than 9,000 genes and significant differential splicing of more than 2,900 genes, indicating an important role of SCAF4 in mRNA processing. Knockdown of the SCAF4 ortholog CG4266 in the model organism Drosophila melanogaster resulted in impaired locomotor function, learning, and short-term memory. Furthermore, we observed an increased number of active zones in larval neuromuscular junctions, representing large glutamatergic synapses. These observations indicate a role of CG4266 in nervous system development and function and support the implication of SCAF4 in neurodevelopmental phenotypes. In summary, our data show that heterozygous, likely gene-disrupting variants in SCAF4 are causative for a variable neurodevelopmental disorder associated with impaired mRNA processing.


Assuntos
Deficiência Intelectual/genética , Transtornos do Neurodesenvolvimento/genética , Convulsões/genética , Fatores de Processamento de Serina-Arginina/genética , Animais , Criança , Drosophila melanogaster/genética , Feminino , Técnicas de Silenciamento de Genes , Variação Genética/genética , Heterozigoto , Humanos , Deficiência Intelectual/fisiopatologia , Locomoção/genética , Masculino , Mutação/genética , Transtornos do Neurodesenvolvimento/fisiopatologia , RNA Polimerase II/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Convulsões/fisiopatologia , Sequenciamento Completo do Exoma
4.
Mol Cell ; 78(5): 876-889.e6, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32502422

RESUMO

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.


Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , MicroRNAs/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores Estrogênicos/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Sequências Repetidas Invertidas/genética , Sequências Repetidas Invertidas/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , MicroRNAs/genética , Proteínas Associadas à Matriz Nuclear/genética , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Estrogênicos/genética
5.
Nat Rev Mol Cell Biol ; 21(9): 542-556, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32483315

RESUMO

RNA tailing, or the addition of non-templated nucleotides to the 3' end of RNA, is the most frequent and conserved type of RNA modification. The addition of tails and their composition reflect RNA maturation stages and have important roles in determining the fate of the modified RNAs. Apart from canonical poly(A) polymerases, which add poly(A) tails to mRNAs in a transcription-coupled manner, a family of terminal nucleotidyltransferases (TENTs), including terminal uridylyltransferases (TUTs), modify RNAs post-transcriptionally to control RNA stability and activity. The human genome encodes 11 different TENTs with distinct substrate specificity, intracellular localization and tissue distribution. In this Review, we discuss recent advances in our understanding of non-canonical RNA tails, with a focus on the functions of human TENTs, which include uridylation, mixed tailing and post-transcriptional polyadenylation of mRNAs, microRNAs and other types of non-coding RNA.


Assuntos
Regulação da Expressão Gênica/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , RNA/genética , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/fisiologia , Animais , Regulação da Expressão Gênica/genética , Humanos , MicroRNAs/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Poliadenilação , RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética
6.
RNA ; 26(10): 1345-1359, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32554553

RESUMO

Metazoan replication-dependent histone pre-mRNAs are cleaved at the 3' end by U7 snRNP, an RNA-guided endonuclease that contains U7 snRNA, seven proteins of the Sm ring, FLASH, and four polyadenylation factors: symplekin, CPSF73, CPSF100, and CstF64. A fully recombinant U7 snRNP was recently reconstituted from all 13 components for functional and structural studies and shown to accurately cleave histone pre-mRNAs. Here, we analyzed the activity of recombinant U7 snRNP in more detail. We demonstrate that in addition to cleaving histone pre-mRNAs endonucleolytically, reconstituted U7 snRNP acts as a 5'-3' exonuclease that degrades the downstream product generated from histone pre-mRNAs as a result of the endonucleolytic cleavage. Surprisingly, recombinant U7 snRNP also acts as an endonuclease on single-stranded DNA substrates. All these activities depend on the ability of U7 snRNA to base-pair with the substrate and on the presence of the amino-terminal domain (NTD) of symplekin in either cis or trans, and are abolished by mutations within the catalytic center of CPSF73, or by binding of the NTD to the SSU72 phosphatase of RNA polymerase II. Altogether, our results demonstrate that recombinant U7 snRNP functionally mimics its endogenous counterpart and provide evidence that CPSF73 is both an endonuclease and a 5'-3' exonuclease, consistent with the activity of other members of the ß-CASP family. Our results also raise the intriguing possibility that CPSF73 may be involved in some aspects of DNA metabolism in vivo.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/genética , Endonucleases/genética , Exonucleases/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U7/genética , Animais , Histonas/genética , Camundongos , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA/genética
7.
Nat Commun ; 11(1): 2834, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503981

RESUMO

Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m5C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m5C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m5C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m5C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Animais , Linhagem Celular Tumoral , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Resistencia a Medicamentos Antineoplásicos/genética , Técnicas de Silenciamento de Genes , Humanos , Metilação , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
J Biotechnol ; 318: 20-30, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32387451

RESUMO

Niemann-Pick type C (NPC) is an autosomal recessive lysosomal storage disorder due to mutations in NPC1 (95 % cases) or NPC2 genes, encoding NPC1 and NPC2 proteins, respectively. Both NPC1 and NPC2 proteins are involved in transport of intracellular cholesterol and their alteration leads to the accumulation of unesterified cholesterol and other lipids within the lysosomes. The disease is characterized by visceral, neurological and psychiatric symptoms. However, the pathogenic mechanisms that lead to the fatal neurodegeneration are still unclear. To date, several mutations leading to the generation of aberrant splicing variants or mRNA degradation in NPC1 and NPC2 genes have been reported. In addition, different lines of experimental evidence have highlighted the possible role of RNA-binding proteins and RNA-metabolism, in the onset and progression of many neurodegenerative disorders, that could explain NPC neurological features and in general, the disease pathogenesis. In this review, we will provide an overview of the impact of mRNA processing and metabolism on NPC disease pathology.


Assuntos
Doença de Niemann-Pick Tipo C/patologia , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/metabolismo , Colesterol/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , RNA/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
10.
Nucleic Acids Res ; 48(12): 6839-6854, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32449937

RESUMO

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.


Assuntos
Proteínas de Arabidopsis/genética , MicroRNAs/genética , RNA Helicases/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genética , Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/genética , Exossomos/genética , Regulação da Expressão Gênica de Plantas/genética , Mutação/genética , Precursores de RNA/genética , Estabilidade de RNA/genética , Ribonuclease III/genética
11.
Nucleic Acids Res ; 48(12): 6874-6888, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32427329

RESUMO

MicroRNAs (miRNAs) are predicted to regulate the expression of >60% of mammalian genes and play fundamental roles in most biological processes. Deregulation of miRNA expression is a hallmark of most cancers and further investigation of mechanisms controlling miRNA biogenesis is needed. The double stranded RNA-binding protein, NF90 has been shown to act as a competitor of Microprocessor for a limited number of primary miRNAs (pri-miRNAs). Here, we show that NF90 has a more widespread effect on pri-miRNA biogenesis than previously thought. Genome-wide approaches revealed that NF90 is associated with the stem region of 38 pri-miRNAs, in a manner that is largely exclusive of Microprocessor. Following loss of NF90, 22 NF90-bound pri-miRNAs showed increased abundance of mature miRNA products. NF90-targeted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. Mutations leading to less stable structures reduced NF90 binding while increasing pri-miRNA stability led to acquisition of NF90 association, as determined by RNA electrophoretic mobility shift assay (EMSA). NF90-bound and downregulated pri-miRNAs are embedded in introns of host genes and expression of several host genes is concomitantly reduced. These data suggest that NF90 controls the processing of a subset of highly stable, intronic miRNAs.


Assuntos
Sequências Repetidas Invertidas/genética , MicroRNAs/genética , Neoplasias/genética , Proteínas do Fator Nuclear 90/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Humanos , MicroRNAs/biossíntese , Proteínas do Fator Nuclear 90/antagonistas & inibidores , Processamento Pós-Transcricional do RNA/genética
12.
Nat Rev Cancer ; 20(6): 303-322, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32300195

RESUMO

Specific chemical modifications of biological molecules are an efficient way of regulating molecular function, and a plethora of downstream signalling pathways are influenced by the modification of DNA and proteins. Many of the enzymes responsible for regulating protein and DNA modifications are targets of current cancer therapies. RNA epitranscriptomics, the study of RNA modifications, is the new frontier of this arena. Despite being known since the 1970s, eukaryotic RNA modifications were mostly identified on transfer RNA and ribosomal RNA until the last decade, when they have been identified and characterized on mRNA and various non-coding RNAs. Increasing evidence suggests that RNA modification pathways are also misregulated in human cancers and may be ideal targets of cancer therapy. In this Review we highlight the RNA epitranscriptomic pathways implicated in cancer, describing their biological functions and their connections to the disease.


Assuntos
Epigênese Genética/genética , Neoplasias/genética , Neoplasias/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , RNA/metabolismo , Epigênese Genética/fisiologia , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , RNA/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(19): 10520-10529, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32332166

RESUMO

In the opportunistic pathogen Pseudomonas aeruginosa, RsmA is an RNA-binding protein that plays critical roles in the control of virulence, interbacterial interactions, and biofilm formation. Although RsmA is thought to exert its regulatory effects by binding full-length transcripts, the extent to which RsmA binds nascent transcripts has not been addressed. Moreover, which transcripts are direct targets of this key posttranscriptional regulator is largely unknown. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, with cells grown in the presence and absence of the RNA polymerase inhibitor rifampicin, we identify hundreds of nascent transcripts that RsmA associates with in P. aeruginosa We also find that the RNA chaperone Hfq targets a subset of those nascent transcripts that RsmA associates with and that the two RNA-binding proteins can exert regulatory effects on common targets. Our findings establish that RsmA associates with many transcripts as they are being synthesized in P. aeruginosa, identify the transcripts targeted by RsmA, and suggest that RsmA and Hfq may act in a combinatorial fashion on certain transcripts. The binding of posttranscriptional regulators to nascent transcripts may be commonplace in bacteria where distinct regulators can function alone or in concert to achieve control over the translation of transcripts as soon as they emerge from RNA polymerase.


Assuntos
Pseudomonas aeruginosa/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Pseudomonas aeruginosa/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Virulência
14.
PLoS One ; 15(4): e0227172, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240200

RESUMO

Cold stenothermal insects living in glacier-fed streams are stressed by temperature variations resulting from glacial retreat during global warming. The molecular aspects of insect response to environmental stresses remain largely unexplored. The aim of this study was to expand our knowledge of how a cold stenothermal organism controls gene expression at the transcriptional, translational, and protein level under warming conditions. Using the chironomid Diamesa tonsa as target species and a combination of RACE, qPCR, polysomal profiling, western blotting, and bioinformatics techniques, we discovered a new molecular pathway leading to previously overlooked adaptive strategies to stress. We obtained and characterized the complete cDNA sequences of three heat shock inducible 70 (hsp70) and two members of heat-shock cognate 70 (hsc70). Strikingly, we showed that a novel pseudo-hsp70 gene encoding a putative long noncoding RNA (lncRNA) which is transcribed during thermal stress, acting as a ribosome sponge to provide post-transcriptional control of HSP70 protein levels. The expression of the pseudo-hsp70 gene and its function suggest the existence of a new and unexpected mechanism to cope with thermal stress: lowering the pace of protein production to save energy and optimize resources for recovery.


Assuntos
Chironomidae/genética , Proteínas de Choque Térmico HSP70/genética , Processamento Pós-Transcricional do RNA/genética , RNA Longo não Codificante/genética , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Resposta ao Choque Térmico/genética , Polirribossomos/genética , Estresse Fisiológico/genética
15.
Nat Commun ; 11(1): 1685, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245947

RESUMO

N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic RNAs. The biological importance of m6A relies on m6A readers, which control mRNA fate and function. However, it remains unexplored whether additional regulatory subunits of m6A readers are involved in the m6A recognition on RNAs. Here we discover that the long noncoding RNA (lncRNA) LINC00266-1 encodes a 71-amino acid peptide. The peptide mainly interacts with the RNA-binding proteins, including the m6A reader IGF2BP1, and is thus named "RNA-binding regulatory peptide" (RBRP). RBRP binds to IGF2BP1 and strengthens m6A recognition by IGF2BP1 on RNAs, such as c-Myc mRNA, to increase the mRNA stability and expression of c-Myc, thereby promoting tumorigenesis. Cancer patients with RBRPhigh have a poor prognosis. Thus, the oncopeptide RBRP encoded by LINC00266-1 is a regulatory subunit of m6A readers and strengthens m6A recognition on the target RNAs by the m6A reader to exert its oncogenic functions.


Assuntos
Carcinogênese/genética , Neoplasias Colorretais/genética , Processamento Pós-Transcricional do RNA/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Metilação , Camundongos , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , Estabilidade de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Mol Cell ; 78(2): 289-302.e6, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302541

RESUMO

Microprocessor initiates the processing of microRNAs (miRNAs) from the hairpin regions of primary transcripts (pri-miRNAs). Pri-miRNAs often contain multiple miRNA hairpins, and this clustered arrangement can assist in the processing of otherwise defective hairpins. We find that miR-451, which derives from a hairpin with a suboptimal terminal loop and a suboptimal stem length, accumulates to 40-fold higher levels when clustered with a helper hairpin. This phenomenon tolerates changes in hairpin order, linker lengths, and the identities of the helper hairpin, the recipient hairpin, the linker-sequence, and the RNA polymerase that transcribes the hairpins. It can act reciprocally and need not occur co-transcriptionally. It requires Microprocessor recognition of the helper hairpin and linkage of the two hairpins, yet predominantly manifests after helper-hairpin processing. It also requires enhancer of rudimentary homolog (ERH), which copurifies with Microprocessor and can dimerize and interact with other proteins that can dimerize, suggesting a model in which one Microprocessor recruits another Microprocessor.


Assuntos
Proteínas de Ciclo Celular/genética , MicroRNAs/genética , RNA Polimerase III/genética , Fatores de Transcrição/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Genética
17.
Mol Cell ; 78(2): 303-316.e4, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302542

RESUMO

Nuclear processing of most miRNAs is mediated by Microprocessor, comprised of RNase III enzyme Drosha and its cofactor DGCR8. Here, we uncover a hidden layer of Microprocessor regulation via studies of Dicer-independent mir-451, which is clustered with canonical mir-144. Although mir-451 is fully dependent on Drosha/DGCR8, its short stem and small terminal loop render it an intrinsically weak Microprocessor substrate. Thus, it must reside within a cluster for normal biogenesis, although the identity and orientation of its neighbor are flexible. We use DGCR8 tethering assays and operon structure-function assays to demonstrate that local recruitment and transfer of Microprocessor enhances suboptimal substrate processing. This principle applies more broadly since genomic analysis indicates suboptimal canonical miRNAs are enriched in operons, and we validate several of these experimentally. Proximity-based enhancement of suboptimal hairpin processing provides a rationale for genomic retention of certain miRNA operons and may explain preferential evolutionary emergence of miRNA operons.


Assuntos
Genômica , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Núcleo Celular/genética , Humanos , Processamento Pós-Transcricional do RNA/genética
18.
RNA ; 26(7): 866-877, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32213618

RESUMO

Ribosomal subunits are assembled on a precursor rRNA that includes four spacers in addition to mature rRNA sequences. The 5' external transcribed spacer (5' ETS) is the most prominent one that recruits U3 snoRNA and a plethora of proteins during the early assembly of 90S small subunit preribosomes. Here, we have conducted a comprehensive mutational analysis of 5' ETS by monitoring the processing and assembly of a plasmid-expressed pre-18S RNA. Remarkably, nearly half of the 5' ETS sequences, when depleted individually, are dispensable for 18S rRNA processing. The dispensable elements largely bind at the surface of the 90S structure. Defective assembly of 5' ETS completely blocks the last stage of 90S formation yet has little effect on the early assembly of 5' and central domains of 18S rRNA. Our study reveals the functional regions of 5' ETS and provides new insight into the assembly hierarchy of 90S preribosomes.


Assuntos
Precursores de RNA/genética , RNA Fúngico/genética , RNA Ribossômico 18S/genética , Leveduras/genética , Sítios de Ligação/genética , Nucléolo Celular/genética , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico/genética , RNA Nucleolar Pequeno , Ribossomos/genética
19.
Development ; 147(6)2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32098764

RESUMO

Neocortex development during embryonic stages requires the precise control of mRNA metabolism. Human antigen R (HuR) is a well-studied mRNA-binding protein that regulates mRNA metabolism, and it is highly expressed in the neocortex during developmental stages. Deletion of HuR does not impair neural progenitor cell proliferation or differentiation, but it disturbs the laminar structure of the neocortex. We report that HuR is expressed in postmitotic projection neurons during mouse brain development. Specifically, depletion of HuR in these neurons led to a mislocalization of CDP+ neurons in deeper layers of the cortex. Time-lapse microscopy showed that HuR was required for the promotion of cell motility in migrating neurons. PCR array identified profilin 1 (Pfn1) mRNA as a major binding partner of HuR in neurons. HuR positively mediated the stability of Pfn1 mRNA and influenced actin polymerization. Overexpression of Pfn1 successfully rescued the migration defects of HuR-deleted neurons. Our data reveal a post-transcriptional mechanism that maintains actin dynamics during neuronal migration.


Assuntos
Movimento Celular , Proteína Semelhante a ELAV 1/fisiologia , Neurônios/fisiologia , RNA Mensageiro/metabolismo , Animais , Padronização Corporal/genética , Movimento Celular/genética , Células Cultivadas , Embrião de Mamíferos , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/fisiologia , Neurogênese/genética , Gravidez , Profilinas/fisiologia , Processamento Pós-Transcricional do RNA/genética
20.
Nucleic Acids Res ; 48(6): 3181-3194, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32047918

RESUMO

N 6-Threonylcarbamoyladenosine (t6A) is a universal tRNA modification essential for translational accuracy and fidelity. In human mitochondria, YrdC synthesises an l-threonylcarbamoyl adenylate (TC-AMP) intermediate, and OSGEPL1 transfers the TC-moiety to five tRNAs, including human mitochondrial tRNAThr (hmtRNAThr). Mutation of hmtRNAs, YrdC and OSGEPL1, affecting efficient t6A modification, has been implicated in various human diseases. However, little is known about the tRNA recognition mechanism in t6A formation in human mitochondria. Herein, we showed that OSGEPL1 is a monomer and is unique in utilising C34 as an anti-determinant by studying the contributions of individual bases in the anticodon loop of hmtRNAThr to t6A modification. OSGEPL1 activity was greatly enhanced by introducing G38A in hmtRNAIle or the A28:U42 base pair in a chimeric tRNA containing the anticodon stem of hmtRNASer(AGY), suggesting that sequences of specific hmtRNAs are fine-tuned for different modification levels. Moreover, using purified OSGEPL1, we identified multiple acetylation sites, and OSGEPL1 activity was readily affected by acetylation via multiple mechanisms in vitro and in vivo. Collectively, we systematically elucidated the nucleotide requirement in the anticodon loop of hmtRNAs, and revealed mechanisms involving tRNA sequence optimisation and post-translational protein modification that determine t6A modification levels.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Mitocôndrias/genética , Biossíntese de Proteínas , RNA de Transferência de Treonina/genética , Acetilação , Adenosina/análogos & derivados , Adenosina/genética , Anticódon/genética , Escherichia coli/genética , Proteínas de Ligação ao GTP/genética , Humanos , Mutação , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Proteínas de Ligação a RNA/genética
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