RESUMO
Many long noncoding RNAs (lncRNAs) are unstable and rapidly degraded in the nucleus by the nuclear exosome. An exosome adaptor complex called NEXT (nuclear exosome targeting) functions to facilitate turnover of some of these lncRNAs. Here we show that knockdown of one NEXT subunit, Mtr4, but neither of the other two subunits, resulted in accumulation of two types of lncRNAs: prematurely terminated RNAs (ptRNAs) and upstream antisense RNAs (uaRNAs). This suggested a NEXT-independent Mtr4 function, and, consistent with this, we isolated a distinct complex containing Mtr4 and the zinc finger protein ZFC3H1. Strikingly, knockdown of either protein not only increased pt/uaRNA levels but also led to their accumulation in the cytoplasm. Furthermore, all pt/uaRNAs examined associated with active ribosomes, but, paradoxically, this correlated with a global reduction in heavy polysomes and overall repression of translation. Our findings highlight a critical role for Mtr4/ZFC3H1 in nuclear surveillance of naturally unstable lncRNAs to prevent their accumulation, transport to the cytoplasm, and resultant disruption of protein synthesis.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Citoplasma/metabolismo , Regulação da Expressão Gênica/genética , RNA Helicases/metabolismo , RNA Nuclear/metabolismo , Fatores de Transcrição/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , RNA Helicases/genética , Estabilidade de RNA , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Combinatorial gene regulation by multiple microRNAs (miRNAs) is widespread and closely spaced target sites often act cooperatively to achieve stronger repression ("neighborhood" miRNA cotargeting). While miRNA cotarget sites are suggested to be more conserved and implicated in developmental control, the pathological significance of miRNA cotargeting remains elusive. RESULTS: Here, we report the pathogenic impacts of combinatorial miRNA regulation on inflammation in systemic lupus erythematosus (SLE). In the SLE mouse model, we identified the downregulation of two miRNAs, miR-128 and miR-148a, by TLR7 stimulation in plasmacytoid dendritic cells. Functional analyses using human cell lines demonstrated that miR-128 and miR-148a additively target KLF4 via extensively overlapping target sites ("seed overlap" miRNA cotargeting) and suppress the inflammatory responses. At the transcriptome level, "seed overlap" miRNA cotargeting increases susceptibility to downregulation by two miRNAs, consistent with additive but not cooperative recruitment of two miRNAs. Systematic characterization further revealed that extensive "seed overlap" is a prevalent feature among broadly conserved miRNAs. Highly conserved target sites of broadly conserved miRNAs are largely divided into two classes-those conserved among eutherian mammals and from human to Coelacanth, and the latter, including KLF4-cotargeting sites, has a stronger association with both "seed overlap" and "neighborhood" miRNA cotargeting. Furthermore, a deeply conserved miRNA target class has a higher probability of haplo-insufficient genes. CONCLUSIONS: Our study collectively suggests the complexity of distinct modes of miRNA cotargeting and the importance of their perturbations in human diseases.
Assuntos
Lúpus Eritematoso Sistêmico , MicroRNAs , Humanos , Animais , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação da Expressão Gênica , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Regulação para Baixo , Transcriptoma , Mamíferos/genéticaRESUMO
Polyadenylation of mRNA precursors is mediated by a large multisubunit protein complex. Here we show that RBBP6 (retinoblastoma-binding protein 6), identified initially as an Rb- and p53-binding protein, is a component of this complex and functions in 3' processing in vitro and in vivo. RBBP6 associates with other core factors, and this interaction is mediated by an unusual ubiquitin-like domain, DWNN ("domain with no name"), that is required for 3' processing activity. The DWNN is also expressed, via alternative RNA processing, as a small single-domain protein (isoform 3 [iso3]). Importantly, we show that iso3, known to be down-regulated in several cancers, competes with RBBP6 for binding to the core machinery, thereby inhibiting 3' processing. Genome-wide analyses following RBBP6 knockdown revealed decreased transcript levels, especially of mRNAs with AU-rich 3' untranslated regions (UTRs) such as c-Fos and c-Jun, and increased usage of distal poly(A) sites. Our results implicate RBBP6 and iso3 as novel regulators of 3' processing, especially of RNAs with AU-rich 3' UTRs.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , RNA Mensageiro/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Poliadenilação/genética , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ubiquitina-Proteína LigasesRESUMO
MicroRNA-122 (miR-122) is highly expressed in hepatocytes, where it plays an important role in regulating cholesterol and fatty acid metabolism, and it is also a host factor required for hepatitis C virus replication. miR-122 is selectively stabilized by 3' adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2 (also known as PAPD4 or TENT2). However, it is unclear how GLD-2 specifically stabilizes miR-122. Here, we show that QKI7 KH domain-containing RNA binding (QKI-7), one of three isoforms of the QKI proteins, which are members of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, is involved in miR-122 stabilization. QKI down-regulation specifically decreased the steady-state level of mature miR-122, but did not affect the pre-miR-122 level. We also found that QKI-7 uses its C-terminal region to interact with GLD-2 and its QUA2 domain to associate with the RNA-induced silencing complex protein Argonaute 2 (Ago2), indicating that the GLD-2-QKI-7 interaction recruits GLD-2 to Ago2. QKI-7 exhibited specific affinity to miR-122 and significantly promoted GLD-2-mediated 3' adenylation of miR-122 in vitro Taken together, our findings indicate that miR-122 binds Ago2-interacting QKI-7, which recruits GLD-2 for 3' adenylation and stabilization of miR-122.
Assuntos
MicroRNAs/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Proteínas Argonautas/metabolismo , Linhagem Celular Tumoral , Humanos , Poliadenilação , Mapas de Interação de Proteínas , Estabilidade de RNARESUMO
The genome is pervasively transcribed across various species, yielding numerous non-coding RNAs. As a counterbalance for pervasive transcription, various organisms have a nuclear RNA exosome complex, whose structure is well conserved between yeast and mammalian cells. The RNA exosome not only regulates the processing of stable RNA species, such as rRNAs, tRNAs, small nucleolar RNAs, and small nuclear RNAs, but also plays a central role in RNA surveillance by degrading many unstable RNAs and misprocessed pre-mRNAs. In addition, associated cofactors of RNA exosome direct the exosome to distinct classes of RNA substrates, suggesting divergent and/or multi-layer control of RNA quality in the cell. While the RNA exosome is essential for cell viability and influences various cellular processes, mutations and alterations in the RNA exosome components are linked to the collection of rare diseases and various diseases including cancer, respectively. The present review summarizes the relationships between pervasive transcription and RNA exosome, including evolutionary crosstalk, mechanisms of RNA exosome-mediated RNA surveillance, and physiopathological effects of perturbation of RNA exosome.
Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/fisiologia , Estabilidade de RNA/fisiologia , Transcrição Gênica/genética , Animais , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Genoma/genética , Humanos , RNA/genética , RNA/metabolismo , Estabilidade de RNA/genética , RNA Nuclear/genética , RNA Nuclear/metabolismoRESUMO
MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate gene expression at post-transcriptional level via translational repression and/or mRNA degradation. miRNAs are associated with many cellular processes, and down-regulation of miRNAs causes numerous diseases including cancer, neurological disorders, inflammation, and cardiovascular diseases, for which miRNA replacement therapy has emerged as a promising approach. This approach aims to restore down-regulated miRNAs using synthetic miRNA mimics. However, it remains a critical issue that miRNA mimics are unstable and transient in cells. Here, we first show that miRNA mimics are rapidly degraded by a mechanism different from Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay, which degrades endogenous miRNAs, and newly identified 2'-5'-oligoadenylate synthetase (OAS)/RNase L as key factors responsible for the degradation of miRNA mimics in human cells. Our results suggest that the OAS1 recognizes miRNA mimics and produces 2'-5'-oligoadenylates (2-5A), which leads to the activation of latent endoribonuclease RNase L to degrade miRNA mimics. A small-molecule inhibitor that blocks RNase L can stabilize miRNA mimics. These findings provide a promising method for the stabilization of miRNA mimics, as well as for the efficient miRNA replacement therapy.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Endorribonucleases/metabolismo , MicroRNAs/metabolismo , Estabilidade de RNA , Células HeLa , Humanos , MicroRNAs/químicaRESUMO
MicroRNAs are small noncoding RNAs that regulate translation and mRNA stability by binding target mRNAs in complex with Argonaute (AGO) proteins. AGO interacts with a member of the TNRC6 family proteins to form a microRNP complex, which recruits the CCR4-NOT complex to accelerate deadenylation and inhibits translation. MicroRNAs primarily repress translation of target mRNAs but have been shown to enhance translation of a specific type of target reporter mRNAs in various experimental systems: G0 quiescent mammalian cells, Xenopus laevis oocytes, Drosophila embryo extracts, and HeLa cells. In all of the cases mentioned, a common feature of the activated target mRNAs is the lack of a poly(A) tail. Here, we show let-7-microRNP-mediated translational activation of nonadenylated target mRNAs in a mammalian cell-free system, which contains over-expressed AGO2, TNRC6B, and PAPD7 (TUTase5, TRF4-1). Importantly, translation of nonadenylated mRNAs was activated also by tethered TNRC6B silencing domain (SD), in the presence of PAPD7. Deletion of the poly(A)-binding protein (PABP) interacting motif (PAM2) from the TNRC6B-SD abolished the translational activation, suggesting the involvement of PABP in the process. Similar results were also obtained in cultured HEK293T cells. This work may provide novel insights into microRNP-mediated mRNA regulation.
Assuntos
Sistema Livre de Células , MicroRNAs/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Células HEK293 , Humanos , MicroRNAs/genética , Proteína I de Ligação a Poli(A)/genética , Proteína I de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/metabolismo , Poliadenilação , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The RNA helicase Mtr4 is a versatile protein that is a crucial component of several distinct RNA surveillance complexes. Here we describe a novel complex that contains Mtr4, but has a role distinct from any of those previously described. We found that Mtr4 association with the human homolog of fission yeast Nrl1, NRDE-2, defines a novel function for Mtr4 in the DNA damage response pathway. We provide biochemical evidence that Mtr4 and NRDE-2 are part of the same complex and show that both proteins play a role in the DNA damage response by maintaining low DNA double-strand break levels. Importantly, the DNA damage response function of the Mtr4/NRDE-2 complex does not depend on the formation of R loops. We show however that NRDE-2 and Mtr4 can affect R-loop signals at a subset of distinct genes, possibly regulating their expression. Our work not only expands the wide range of Mtr4 functions, but also elucidates an important role of the less characterized human NRDE-2 protein.
Assuntos
Quebras de DNA de Cadeia Dupla , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Estrutura Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , RNA Helicases/química , RNA Helicases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Tob is a member of the anti-proliferative protein family, which functions in transcription and mRNA decay. We have previously demonstrated that Tob is involved in the general mechanism of mRNA decay by mediating mRNA deadenylation through interaction with Caf1 and a general RNA-binding protein, PABPC1. Here, we focus on the role of Tob in the regulation of specific mRNA. We show that Tob binds directly to a sequence-specific RNA-binding protein, cytoplasmic polyadenylation element-binding protein 3 (CPEB3). CPEB3 negatively regulates the expression of a target by accelerating deadenylation and decay of its mRNA, which it achieves by tethering to the mRNA. The carboxyl-terminal RNA-binding domain of CPEB3 binds to the carboxyl-terminal unstructured region of Tob. Tob then binds Caf1 deadenylase and recruits it to CPEB3 to form a ternary complex. The CPEB3-accelerated deadenylation was abrogated by a dominant-negative mutant of either Caf1 or Tob. Together, these results indicate that Tob mediates the recruitment of Caf1 to the target of CPEB3 and elicits deadenylation and decay of the mRNA. Our results provide an explanation of how Tob regulates specific biological processes.
Assuntos
Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismoRESUMO
Non-canonical poly(A) polymerases (ncPAPs) catalyze the addition of poly(A) tail to the 3' end of RNA to play pivotal roles in the regulation of gene expression and also in quality control. Here we identified a novel isoform of the 7th member of ncPAPs: PAPD7 (PAPD7 l), which contains 230 extra amino acids at the amino terminus of the previously identified PAPD7 (PAPD7 s). In sharp contrast to the inactive PAPD7 s, PAPD7 l showed robust nucleotidyl transferase activity when tethered to an RNA. A region required for the activity was localized to 187-219 aa, and this region was also required for the nuclear retention of PAPD7 l. Western blot analysis revealed that 94 kDa band (corresponding to PAPD7 l) but not 62 kDa band (corresponding to PAPD7 s) detected by PAPD7 antibody was specifically depleted by treatment with PAPD7 siRNA in both HeLa and U2OS cells. These results suggest that PAPD7 l is the major and active isoform of PAPD7 expressed in cells.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Sequência de Aminoácidos , Núcleo Celular/enzimologia , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Clonagem Molecular , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Mutação , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Estrutura Terciária de Proteína , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismoRESUMO
Poly(A) tail metabolism contributes to post-transcriptional regulation of gene expression. Here, we present a protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing, which excludes truncated RNAs from the measurement. We describe steps for preparing recombinant eIF4E mutant protein, purifying m7G- capped RNAs, library preparation, and sequencing. Resulting data can be used not only for expression profiling and poly(A) tail length estimation but also for detecting alternative splicing and polyadenylation events and RNA base modification. For complete details on the use and execution of this protocol, please refer to Ogami et al. (2022).1.
RESUMO
During early embryonic development, the RNA-binding protein CPEB mediates cytoplasmic polyadenylation and translational activation through a combinatorial code defined by the cy-toplasmic polyadenylation element (CPE) present in maternal mRNAs. However, in non-neuronal somatic cells, CPEB accelerates deadenylation to repress translation of the target, including c-myc mRNA, through an ill-defined cis-regulatory mechanism. Using RNA mutagenesis and electrophoretic mobility shift assays, we demonstrated that a combination of tandemly arranged consensus (cCPE) and non-consensus (ncCPE) cytoplasmic polyadenylation elements (CPEs) constituted a combinatorial code for CPEB-mediated c-myc mRNA decay. CPEB binds to cCPEs with high affinity (Kd = ~250 nM), whereas it binds to ncCPEs with low affinity (Kd > ~900 nM). CPEB binding to a cCPE enhances CPEB binding to the proximal ncCPE. In contrast, while a cCPE did not activate mRNA degradation, an ncCPE was essential for the induction of degradation, and a combination of a cCPE and ncCPEs further promoted degradation. Based on these findings, we propose a model in which the high-affinity binding of CPEB to the cCPE accelerates the binding of the second CPEB to the ncCPEs, resulting in the recruitment of deadenylases, acceleration of deadenylation, and repression of c-myc mRNAs.
Assuntos
Oócitos , Fatores de Poliadenilação e Clivagem de mRNA , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Oócitos/metabolismo , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , HumanosRESUMO
Oncogene-induced DNA replication stress (RS) and consequent pathogenic R-loop formation are known to impede S phase progression. Nonetheless, cancer cells continuously proliferate under such high-stressed conditions through incompletely understood mechanisms. Here, we report taurine upregulated gene 1 (TUG1) long noncoding RNA (lncRNA), which is highly expressed in many types of cancers, as an important regulator of intrinsic R-loop in cancer cells. Under RS conditions, TUG1 is rapidly upregulated via activation of the ATR-CHK1 signaling pathway, interacts with RPA and DHX9, and engages in resolving R-loops at certain loci, particularly at the CA repeat microsatellite loci. Depletion of TUG1 leads to overabundant R-loops and enhanced RS, leading to substantial inhibition of tumor growth. Our data reveal a role of TUG1 as molecule important for resolving R-loop accumulation in cancer cells and suggest targeting TUG1 as a potent therapeutic approach for cancer treatment.
Assuntos
Neoplasias , Estruturas R-Loop , Humanos , Replicação do DNA/genética , Proliferação de Células/genética , Neoplasias/genética , Repetições de Microssatélites/genética , TaurinaRESUMO
Translation of 5' terminal oligopyrimidine (TOP) mRNAs encoding the protein synthesis machinery is strictly regulated by an amino-acid-sensing mTOR pathway. However, its regulatory mechanism remains elusive. Here, we demonstrate that TOP mRNA translation positively correlates with its poly(A) tail length under mTOR active/amino-acid-rich conditions, suggesting that TOP mRNAs are post-transcriptionally controlled by poly(A) tail-length regulation. Consistent with this, the tail length of TOP mRNAs dynamically fluctuates in response to amino acid availability. The poly(A) tail shortens under mTOR active/amino-acid-rich conditions, whereas the long-tailed TOP mRNAs accumulate under mTOR inactive/amino-acid-starved (AAS) conditions. An RNA-binding protein, LARP1, is indispensable for the process. LARP1 interacts with non-canonical poly(A) polymerases and induces post-transcriptional polyadenylation of the target. Our findings illustrate that LARP1 contributes to the selective accumulation of TOP mRNAs with long poly(A) tails under AAS, resulting in accelerated ribosomal loading onto TOP mRNAs for the resumption of translation after AAS.
Assuntos
Autoantígenos , Ribonucleoproteínas , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Autoantígenos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Ribossomos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Polinucleotídeo Adenililtransferase/genética , Aminoácidos/metabolismo , Biossíntese de ProteínasRESUMO
Endothelial cells (ECs) and endothelial progenitor cells (EPCs) play crucial roles in maintaining vascular health and homeostasis. Both cell types have been used in regenerative therapy as well as in various in vitro models; however, the properties of primary human ECs and EPCs are dissimilar owing to differences in genetic backgrounds and sampling techniques. Human induced pluripotent stem cells (hiPSCs) are an alternative cell source of ECs and EPCs. However, owing to the low purity of differentiated cells from hiPSCs, purification via an antigen-antibody reaction, which damages the cells, is indispensable. Besides, owing to limited expandability, it is difficult to produce these cells in large numbers. Here we report the development of relatively simple differentiation and purification methods for hiPSC-derived EPCs (iEPCs). Furthermore, we discovered that a combination of three small molecules, that is, Y-27632 (a selective inhibitor of Rho-associated, coiled-coil containing protein kinase [ROCK]), A 83-01 (a receptor-like kinase inhibitor of transforming growth factor beta [TGF-ß]), and CHIR-99021 (a selective inhibitor of glycogen synthase kinase-3ß [GSK3ß] that also activates Wnt), dramatically stimulated protein synthesis-related pathways and enhanced the proliferative capacity of iEPCs. These findings will help to establish a supply system of EPCs at an industrial scale.
RESUMO
The 2'-5'-oligoadenylate synthetase (OAS)/RNase L system protects hosts against pathogenic viruses through cleavage of the exogenous single-stranded RNA. In this system, an evolutionally conserved RNA quality control factor Dom34 (known as Pelota (Pelo) in higher eukaryotes) forms a surveillance complex with RNase L to recognize and eliminate the exogenous RNA in a manner dependent on translation. Here, we newly identified that ATP-binding cassette sub-family E member 1 (ABCE1), which is also known as RNase L inhibitor (RLI), is involved in the regulation of exogenous RNA decay. ABCE1 directly binds to form a complex with RNase L and accelerates RNase L dimer formation in the absence of 2'-5' oligoadenylates (2-5A). Depletion of ABCE1 represses 2-5A-induced RNase L activation and stabilizes exogenous RNA to a level comparable to that seen in RNase L depletion. The increased half-life of the RNA by the single depletion of either protein is not significantly affected by the double depletion of both proteins, suggesting that RNase L and ABCE1 act together to eliminate exogenous RNA. Our results indicate that ABCE1 functions as a positive regulator of exogenous RNA decay rather than an inhibitor of RNase L.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Regulação da Expressão Gênica , Estabilidade de RNA , Endorribonucleases/metabolismo , Células HeLa , Humanos , Ligação ProteicaRESUMO
Most eukaryotic genes produce alternative polyadenylation (APA) isoforms. Here we report that, unlike previously characterized cell lineages, differentiation of syncytiotrophoblast (SCT), a cell type critical for hormone production and secretion during pregnancy, elicits widespread transcript shortening through APA in 3'UTRs and in introns. This global APA change is observed in multiple in vitro trophoblast differentiation models, and in single cells from placentas at different stages of pregnancy. Strikingly, the transcript shortening is unrelated to cell proliferation, a feature previously associated with APA control, but instead accompanies increased secretory functions. We show that 3'UTR shortening leads to transcripts with higher mRNA stability, which augments transcriptional activation, especially for genes involved in secretion. Moreover, this mechanism, named secretion-coupled APA (SCAP), is also executed in B cell differentiation to plasma cells. Together, our data indicate that SCAP tailors the transcriptome during formation of secretory cells, boosting their protein production and secretion capacity.
Assuntos
Diferenciação Celular/fisiologia , Poliadenilação/fisiologia , Transporte Proteico/fisiologia , Transcriptoma , Regiões 3' não Traduzidas , Diferenciação Celular/genética , Linhagem da Célula , Proliferação de Células , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Isoformas de Proteínas , Transporte Proteico/genética , Estabilidade de RNA , RNA Mensageiro/metabolismoRESUMO
The nuclear RNA exosome is an essential and versatile machinery that regulates maturation and degradation of a huge plethora of RNA species. The past two decades have witnessed remarkable progress in understanding the whole picture of its RNA substrates and the structural basis of its functions. In addition to the exosome itself, recent studies focusing on associated co-factors have been elucidating how the exosome is directed towards specific substrates. Moreover, it has been gradually realized that loss-of-function of exosome subunits affect multiple biological processes such as the DNA damage response, R-loop resolution, maintenance of genome integrity, RNA export, translation and cell differentiation. In this review, we summarize the current knowledge of the mechanisms of nuclear exosome-mediated RNA metabolism and discuss their physiological significance.