RESUMO
ADP-ribosylation refers to the addition of one or more ADP-ribose groups onto proteins. The attached ADP-ribose monomers or polymers, commonly known as poly(ADP-ribose) (PAR), modulate the activities of the modified substrates or their binding affinities to other proteins. However, progress in this area is hindered by a lack of tools to investigate this protein modification. Here, we describe a new method named ELTA (enzymatic labeling of terminal ADP-ribose) for labeling free or protein-conjugated ADP-ribose monomers and polymers at their 2'-OH termini using the enzyme OAS1 and dATP. When coupled with various dATP analogs (e.g., radioactive, fluorescent, affinity tags), ELTA can be used to explore PAR biology with techniques routinely used to investigate DNA or RNA function. We demonstrate that ELTA enables the biophysical measurements of protein binding to PAR of a defined length, detection of PAR length from proteins and cells, and enrichment of sub-femtomole amounts of ADP-ribosylated peptides from cell lysates.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , ADP-Ribosilação , Adenosina Difosfato Ribose/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , Animais , Células HeLa , Humanos , Ligação Proteica , Domínios Proteicos , Células Sf9 , Ubiquitina-Proteína Ligases/genéticaRESUMO
In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs. Transcription initiation peaks associated with genes in the sense direction have a strong preference to produce either long or short capped RNAs, with one out of six peaks detected in the short capped RNA libraries only. Gene-associated short capped RNAs have highly specific 3' ends, typically overlapping splice sites. Enhancers also preferentially generate either short or long capped RNAs, with 10% of enhancers observed in the short capped RNA libraries only. Enhancers producing either short or long capped RNAs show enrichment for GWAS-associated disease SNPs. We conclude that deep sequencing of short capped RNAs reveals new families of noncoding RNAs and elucidates the diversity of transcripts generated at known and novel promoters and enhancers.
RESUMO
MOTIVATION: Cell type-specific activities of cis-regulatory elements (CRE) are central to understanding gene regulation and disease predisposition. Single-cell RNA 5'end sequencing (sc-end5-seq) captures the transcription start sites (TSS) which can be used as a proxy to measure the activity of transcribed CREs (tCREs). However, a substantial fraction of TSS identified from sc-end5-seq data may not be genuine due to various artifacts, hindering the use of sc-end5-seq for de novo discovery of tCREs. RESULTS: We developed SCAFE-Single-Cell Analysis of Five-prime Ends-a software suite that processes sc-end5-seq data to de novo identify TSS clusters based on multiple logistic regression. It annotates tCREs based on the identified TSS clusters and generates a tCRE-by-cell count matrix for downstream analyses. The software suite consists of a set of flexible tools that could either be run independently or as pre-configured workflows. AVAILABILITY AND IMPLEMENTATION: SCAFE is implemented in Perl and R. The source code and documentation are freely available for download under the MIT License from https://github.com/chung-lab/SCAFE. Docker images are available from https://hub.docker.com/r/cchon/scafe. The submitted software version and test data are archived at https://doi.org/10.5281/zenodo.7023163 and https://doi.org/10.5281/zenodo.7024060, respectively. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Sequências Reguladoras de Ácido Nucleico , Software , Fluxo de Trabalho , Sítio de Iniciação de TranscriçãoRESUMO
In this issue of Molecular Cell, Wu et al. (2013) report the identification of GW182-independent microRNA complexes that confer stronger repression upon serum starvation; interestingly, these complexes are associated with polyribosomes and the endoplasmic reticulum.
Assuntos
MicroRNAs/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Transdução de Sinais , AnimaisRESUMO
Over the past decade, modifications to microRNAs (miRNAs) via 3' end nucleotide addition have gone from a deep-sequencing curiosity to experimentally confirmed drivers of a range of regulatory activities. Here we overview the methods that have been deployed by researchers seeking to untangle these diverse functional roles and include characterizing not only the nucleotidyl transferases catalyzing the additions but also the nucleotides being added, and the timing of their addition during the miRNA pathway. These methods and their further development are key to clarifying the diverse and sometimes contradictory functional findings presently attributed to these nucleotide additions.
Assuntos
MicroRNAs/química , Biologia Computacional , Genoma Humano , Humanos , MicroRNAs/fisiologia , Processamento de Terminações 3' de RNA , Análise de Sequência de RNARESUMO
Next-generation sequencing experiments have shown that microRNAs (miRNAs) are expressed in many different isoforms (isomiRs), whose biological relevance is often unclear. We found that mature miR-21, the most widely researched miRNA because of its importance in human disease, is produced in two prevalent isomiR forms that differ by 1 nt at their 3' end, and moreover that the 3' end of miR-21 is posttranscriptionally adenylated by the noncanonical poly(A) polymerase PAPD5. PAPD5 knockdown caused an increase in the miR-21 expression level, suggesting that PAPD5-mediated adenylation of miR-21 leads to its degradation. Exoribonuclease knockdown experiments followed by small-RNA sequencing suggested that PARN degrades miR-21 in the 3'-to-5' direction. In accordance with this model, microarray expression profiling demonstrated that PAPD5 knockdown results in a down-regulation of miR-21 target mRNAs. We found that disruption of the miR-21 adenylation and degradation pathway is a general feature in tumors across a wide range of tissues, as evidenced by data from The Cancer Genome Atlas, as well as in the noncancerous proliferative disease psoriasis. We conclude that PAPD5 and PARN mediate degradation of oncogenic miRNA miR-21 through a tailing and trimming process, and that this pathway is disrupted in cancer and other proliferative diseases.
Assuntos
Adenina/metabolismo , MicroRNAs/metabolismo , Neoplasias/genética , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA , Sequência de Bases , Citosina/metabolismo , Exorribonucleases/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células MCF-7 , MicroRNAs/química , MicroRNAs/genética , Modelos Biológicos , Dados de Sequência Molecular , Neoplasias/patologia , Conformação de Ácido Nucleico , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ribonuclease III/metabolismoRESUMO
MicroRNAs are small non-coding RNAs that inhibit the translation of target mRNAs. In humans, most microRNAs are transcribed by RNA polymerase II as long primary transcripts and processed by sequential cleavage of the two RNase III enzymes, DROSHA and DICER, into precursor and mature microRNAs, respectively. Although the fundamental functions of microRNAs in RNA silencing have been gradually uncovered, less is known about the regulatory mechanisms of microRNA expression. Here, we report that telomerase reverse transcriptase (TERT) extensively affects the expression levels of mature microRNAs. Deep sequencing-based screens of short RNA populations revealed that the suppression of TERT resulted in the downregulation of microRNAs expressed in THP-1 cells and HeLa cells. Primary and precursor microRNA levels were also reduced under the suppression of TERT. Similar results were obtained with the suppression of either BRG1 (also called SMARCA4) or nucleostemin, which are proteins interacting with TERT and functioning beyond telomeres. These results suggest that TERT regulates microRNAs at the very early phases in their biogenesis, presumably through non-telomerase mechanism(s).
Assuntos
MicroRNAs/metabolismo , Telomerase/metabolismo , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação para Baixo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Telomerase/antagonistas & inibidores , Telomerase/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Recent research hints at an underappreciated complexity in pre-miRNA processing and regulation. Global profiling of pre-miRNA and its potential to increase understanding of the pre-miRNA landscape is impeded by overlap with highly expressed classes of other non coding (nc) RNA. Here, we present a data set excluding these RNA before sequencing through locked nucleic acids (LNA), greatly increasing pre-miRNA sequence counts with no discernable effect on pre-miRNA or mature miRNA sequencing. Analysis of profiles generated in total, nuclear and cytoplasmic cell fractions reveals that pre-miRNAs are subject to a wide range of regulatory processes involving loci-specific 3'- and 5'-end variation entailing complex cleavage patterns with co-occurring polyuridylation. Additionally, examination of nuclear-enriched flanking sequences of pre-miRNA, particularly those derived from polycistronic miRNA transcripts, provides insight into miRNA and miRNA-offset (moRNA) production, specifically identifying novel classes of RNA potentially functioning as moRNA precursors. Our findings point to particularly intricate regulation of the let-7 family in many ways reminiscent of DICER1-independent, pre-mir-451-like processing, introduce novel and unify known forms of pre-miRNA regulation and processing, and shed new light on overlooked products of miRNA processing pathways.
Assuntos
MicroRNAs/química , Oligonucleotídeos/química , Poli U/análise , Precursores de RNA/química , Processamento Pós-Transcricional do RNA , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/metabolismo , Motivos de Nucleotídeos , Oligodesoxirribonucleotídeos/química , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNA , Uridina/análise , Uridina/metabolismoRESUMO
The RNA-binding protein PARP13 is a primary factor in the innate antiviral response, which suppresses translation and drives decay of bound viral and host RNA. PARP13 interacts with many proteins encoded by interferon-stimulated genes (ISG) to activate antiviral pathways including co-translational addition of ISG15, or ISGylation. We performed enhanced crosslinking immunoprecipitation (eCLIP) and RNA-seq in human cells to investigate PARP13's role in transcriptome regulation for both basal and antiviral states. We find that the antiviral response shifts PARP13 target localization, but not its binding preferences, and that PARP13 supports the expression of ISGylation-related genes, including PARP13's cofactor, TRIM25. PARP13 associates with TRIM25 via RNA-protein interactions, and we elucidate a transcriptome-wide periodicity of PARP13 binding around TRIM25. Taken together, our study implicates PARP13 in creating and maintaining a cellular environment poised for an antiviral response through limiting PARP13 translation, regulating access to distinct mRNA pools, and elevating ISGylation machinery expression.
RESUMO
Several X-linked genes escape from X chromosome inactivation (XCI), while differences in escape across cell types and tissues are still poorly characterized. Here, we developed scLinaX for directly quantifying relative gene expression from the inactivated X chromosome with droplet-based single-cell RNA sequencing (scRNA-seq) data. The scLinaX and differentially expressed gene analyses with large-scale blood scRNA-seq datasets consistently identified the stronger escape in lymphocytes than in myeloid cells. An extension of scLinaX to a 10x multiome dataset (scLinaX-multi) suggested a stronger escape in lymphocytes than in myeloid cells at the chromatin-accessibility level. The scLinaX analysis of human multiple-organ scRNA-seq datasets also identified the relatively strong degree of escape from XCI in lymphoid tissues and lymphocytes. Finally, effect size comparisons of genome-wide association studies between sexes suggested the underlying impact of escape on the genotype-phenotype association. Overall, scLinaX and the quantified escape catalog identified the heterogeneity of escape across cell types and tissues.
Assuntos
Análise de Célula Única , Inativação do Cromossomo X , Inativação do Cromossomo X/genética , Humanos , Análise de Célula Única/métodos , Feminino , Linfócitos/metabolismo , Masculino , Estudo de Associação Genômica Ampla , Animais , Células Mieloides/metabolismo , Camundongos , Análise de Sequência de RNA/métodos , Especificidade de Órgãos , Genes Ligados ao Cromossomo X/genéticaRESUMO
Animal microRNA sequences are subject to 3' nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3' adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1-EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3' addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3' adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake.
Assuntos
Regiões 3' não Traduzidas/genética , Adenina/metabolismo , MicroRNAs/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Proteínas Argonautas , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Camundongos , MicroRNAs/química , MicroRNAs/genética , Monócitos , Nucleotidiltransferases/genética , Polinucleotídeo Adenililtransferase , Estabilidade de RNA , Fatores de Poliadenilação e Clivagem de mRNARESUMO
While rapid advancements in regenerative medicine strategies for spinal cord injury (SCI) have been made, most research in this field has focused on the early stages of incomplete injury. However, the majority of patients experience chronic severe injury; therefore, treatments for these situations are fundamentally important. Here, we hypothesized that environmental modulation via a clinically relevant hepatocyte growth factor (HGF)-releasing scaffold and human iPS cell-derived neural stem/progenitor cells (hNS/PCs) transplantation contributes to functional recovery after chronic complete transection SCI. Effective release of HGF from a collagen scaffold induced progressive axonal elongation and increased grafted cell viability by activating microglia/macrophages and meningeal cells, inhibiting inflammation, reducing scar formation, and enhancing vascularization. Furthermore, hNS/PCs transplantation enhanced endogenous neuronal regrowth, the extension of graft axons, and the formation of circuits around the lesion and lumbar enlargement between host and graft neurons, resulting in the restoration of locomotor and urinary function. This study presents an effective therapeutic strategy for severe chronic SCI and provides evidence for the feasibility of regenerative medicine strategies using clinically relevant materials.
Assuntos
Regeneração Nervosa , Traumatismos da Medula Espinal , Humanos , Traumatismos da Medula Espinal/patologia , Neurônios/metabolismo , Transplante de Células-Tronco/métodos , Medula Espinal/patologia , Axônios/patologia , Recuperação de Função FisiológicaRESUMO
Hydroxymethylcytosines (hmC), one of several reported cytosine modifications, was recently found to be enriched in embryonic stem cells and neuronal cells, and thought to play an important role in regulating gene expression and cell specification. However, unlike methylcytosines (mC), the fate of hmC beyond DNA replication is not well understood. Here, to monitor the status of hmC during DNA replication, we prepared a stable episomal vector-based monitoring system called MoCEV in 293T cells. The MoCEV system containing fully hydroxymethylated-cytosine fragments revealed a significant modification towards mC after several rounds of DNA replication. Strikingly this modification was specifically observed at the CpG sites (71.9% of cytosines), whereas only 1.1% of modified cytosines were detected at the non-CpG sites. Since the unmodified MoCEV did not undergo any DNA methylation during cell division, the results strongly suggest that somatic cells undergo hmC to mC specifically at the CpG sites during cell division.
Assuntos
5-Metilcitosina/metabolismo , Ilhas de CpG , Citosina/análogos & derivados , Metilação de DNA , Replicação do DNA , Reação em Cadeia da Polimerase/métodos , 5-Metilcitosina/análise , Sequência de Bases , Citosina/análise , Citosina/metabolismo , Vetores Genéticos , Células HEK293 , HumanosRESUMO
Since its discovery in 1963, poly(ADP-ribose) (pADPr) has been shown to play important functions in the nucleus of multicellular eukaryotes. Each of these functions centers upon DNA metabolism, including DNA-damage repair, chromatin remodeling, transcription and telomere functions. We recently described two novel functions for pADPr in the cytoplasm, both of which involve RNA metabolism - 1) the assembly of cytoplasmic stress granules, cellular macrostructures that aggregate translationally stalled mRNA/protein complexes, and 2) modulation of microRNA activities. Multiple stress granule-localized, post-transcriptional gene regulators, including microRNA-binding argonaute family members, are substrates for pADPr modification and are increasingly modified by pADPr upon stress. Interestingly, the cytoplasmic RNA regulatory functions for PARPs are likely mediated through activities of catalytically inactive PARP-13/ARTD13/ZC3HAV1/ZAP and mono/poly(ADP-ribose)-synthesizing enzymes, including PARP-5a/ARTD5/TNKS1, PARP-12/ARTD12/ZC3HDC1 and PARP-15/ARTD7/BAL3. These data are consistent with other recent work, which suggests that mono(ADP-ribosyl)ated residues can be poly(ADP-ribosyl)ated by different enzymes.
Assuntos
Citoplasma , Poli Adenosina Difosfato Ribose/fisiologia , Interferência de RNA , Grânulos Citoplasmáticos/metabolismo , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/fisiologia , Multimerização Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Cytotoxic CD4+ T cells (CD4-CTLs) show the presence of cytolytic granules, which include the enzymes granzyme and perforin. The cells have a pathogenic and protective role in various diseases, including cancer, viral infection, and autoimmune disease. In mice, cytotoxic CD4+ T cells express CD8αα+ and reside in the intestine (mouse CD4+CTLs; mCD4-CTLs). The population of cytotoxic CD4+ T cells in the human intestine is currently unknown. Moreover, it is unclear how cytotoxic CD4 T cells change in patients with inflammatory bowel disease (IBD). Here, we aimed to identify cytotoxic CD4+ T cells in the human intestine and analyze the characteristics of the population in patients with IBD using single-cell RNA-seq (scRNA-seq). In CD4+ T cells, granzyme and perforin expression was high in humanMAIT (hMAIT) cells and hCD4+CD8A+ T cell cluster. Both CD4 and CD8A were expressed in hTreg, hMAIT, and hCD4+CD8A+ T cell clusters. Next we performed fast gene set enrichment analysis to identify cell populations that showed homology to mCD4CTLs. The analysis identified the hCD4+CD8A+ T cell cluster (hCTL-like population; hCD4-CTL) similar to mouse CTLs. The percentage of CD4+CD8A+ T cells among the total CD4+ T cells in the inflamed intestine of the patients with Crohn's disease was significantly reduced compared with that in the noninflamed intestine of the patients. In summary, we identified cytotoxic CD4+CD8+ T cells in the small intestine of humans. The integration of the mouse and human sc-RNA-seq data analysis highlight an approach to identify human cell populations related to mouse cell populations, which may help determine the functional properties of several human cell populations in mice.
Assuntos
Linfócitos T CD8-Positivos , Doenças Inflamatórias Intestinais , Animais , Humanos , Camundongos , Linfócitos T CD4-Positivos , Granzimas/genética , Granzimas/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Perforina/genética , Perforina/metabolismo , Transcriptoma , Intestinos/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
BACKGROUND: DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs) and long double-stranded RNAs, generating microRNA (miRNA) duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts) in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown. RESULTS: In this study, we show that human recombinant DICER protein (rDICER) processes a hairpin RNA with 5' overhangs in vitro and generates an intermediate duplex with a 29 nt-5' strand and a 23 nt-3' strand, which was eventually cleaved into a canonical miRNA duplex via a two-step cleavage. The previously identified endogenous pre-miRNA with 5' overhangs, pre-mmu-mir-1982 RNA, is also determined to be a substrate of rDICER through the same two-step cleavage. CONCLUSIONS: The two-step cleavage of a hairpin RNA with 5' overhangs shows that DICER releases double-stranded RNAs after the first cleavage and binds them again in the inverse direction for a second cleavage. These findings have implications for how DICER may be able to interact with or process differing precursor structures.
Assuntos
RNA Helicases DEAD-box/metabolismo , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/metabolismo , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Humanos , MicroRNAs/metabolismo , RNA de Cadeia Dupla/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease III/química , Ribonuclease III/genéticaRESUMO
While several studies have focused on the relationship between individual miRNA loci or classes of small RNA with human Argonaute (AGO) proteins, a comprehensive, global analysis of the RNA content associating with different AGO proteins has yet to be performed. We have compared the content of deep sequenced RNA extracted from immunoprecipitation experiments with the AGO1, AGO2, and AGO3 proteins. Consistent with previous observations, sequence tags derived from miRNA loci globally associate in approximately equivalent amounts with AGO1, AGO2, and AGO3. Exceptions include miR-182, miR-222, and miR-223*, which could be coupled to processes targeting the loci for interaction with specific AGO proteins. A closer inspection of the data, however, supports the presence of an unusual sorting mechanism wherein a subset of miRNA loci give rise to distinct isomirs which preferentially associate with distinct AGO proteins in a significantly differential manner. We also identify the complete set of short RNA derived from non-miRNA sources including tRNA, snRNA, snoRNA, vRNA, and mRNA associating with the AGO proteins, many of which are predicted to play roles in post-transcriptional gene silencing. We also observe enrichment of tags mapping to promoter regions of genes, suggesting that a fraction of the recently-identified promoter-associated small RNAs in humans could function through interaction with AGO proteins. Finally, we observe antisense miRNA transcripts are frequently present in low copy numbers across a range of diverse miRNA loci and these transcripts appear to associate with AGO proteins.
Assuntos
Fatores de Iniciação em Eucariotos/genética , MicroRNAs/genética , Transporte de RNA , Proteínas Argonautas , Sequência de Bases , Mapeamento Cromossômico , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Biblioteca Gênica , Loci Gênicos , Humanos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Interferência de RNA , RNA Mensageiro/genética , RNA de Transferência/genética , Ribonuclease III/metabolismo , Análise de Sequência de RNARESUMO
The human body consists of 37 trillion single cells represented by over 50 organs that are stitched together to make us who we are, yet we still have very little understanding about the basic units of our body: what cell types and states make up our organs both compositionally and spatially. Previous efforts to profile a wide range of human cell types have been attempted by the FANTOM and GTEx consortia. Now, with the advancement in genomic technologies, profiling the human body at single-cell resolution is possible and will generate an unprecedented wealth of data that will accelerate basic and clinical research with tangible applications to future medicine. To date, several major organs have been profiled, but the challenges lie in ways to integrate single-cell genomics data in a meaningful way. In recent years, several consortia have begun to introduce harmonization and equity in data collection and analysis. Herein, we introduce existing and nascent single-cell genomics consortia, and present benefits to necessitate single-cell genomic consortia in a regional environment to achieve the universal human cell reference dataset.
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Genômica , Análise de Célula Única , Animais , Biologia Computacional/métodos , Bases de Dados Genéticas , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Humanos , Análise de Célula Única/métodos , NavegadorRESUMO
The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Here we report that hTERT is phosphorylated at threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathological analyses reveal that phosphorylation of hTERT at threonine 249 occurs more frequently in aggressive cancers. Using CRISPR/Cas9 genome editing, we introduce substitution mutations at threonine 249 in the endogenous hTERT locus and find that phosphorylation of threonine 249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but dispensable for reverse transcriptase and terminal transferase activities. Cap Analysis of Gene Expression (CAGE) demonstrates that hTERT phosphorylation at 249 regulates the expression of specific genes that are necessary for cancer cell proliferation and tumor formation. These observations indicate that phosphorylation at threonine 249 regulates hTERT RdRP and contributes to cancer progression in a telomere independent manner.
Assuntos
Proteína Quinase CDC2/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Telomerase/metabolismo , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Camundongos , Mitose , Mutação , Neoplasias/genética , Fosforilação , RNA Polimerase Dependente de RNA/metabolismo , Telomerase/genética , TreoninaRESUMO
Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.