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1.
Mol Cell ; 84(8): 1475-1495.e18, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38521065

RESUMO

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.


Assuntos
Cromatina , Neoplasias , Animais , Humanos , Camundongos , Cromatina/genética , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo
2.
Mol Cell ; 82(6): 1107-1122.e7, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35303483

RESUMO

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Fator de Processamento U2AF , Grânulos de Estresse , Humanos , Leucemia Mieloide Aguda/genética , Mutação , Síndromes Mielodisplásicas/genética , Sítios de Splice de RNA , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Grânulos de Estresse/metabolismo
3.
Nature ; 622(7981): 173-179, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37731000

RESUMO

Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.


Assuntos
Acetilação , Cromatina , Lisina , Metilação , Processamento de Proteína Pós-Traducional , Sítio de Iniciação de Transcrição , Animais , Humanos , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Histona Desacetilases/metabolismo
4.
Mol Cell ; 81(21): 4398-4412.e7, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34520723

RESUMO

Despite the critical regulatory function of promoter-proximal pausing, the influence of pausing kinetics on transcriptional control remains an active area of investigation. Here, we present Start-TimeLapse-seq (STL-seq), a method that captures the genome-wide kinetics of short, capped RNA turnover and reveals principles of regulation at the pause site. By measuring the rates of release into elongation and premature termination through the inhibition of pause release, we determine that pause-release rates are highly variable, and most promoter-proximal paused RNA polymerase II molecules prematurely terminate (∼80%). The preferred regulatory mechanism upon a hormonal stimulus (20-hydroxyecdysone) is to influence pause-release rather than termination rates. Transcriptional shutdown occurs concurrently with the induction of promoter-proximal termination under hyperosmotic stress, but paused transcripts from TATA box-containing promoters remain stable, demonstrating an important role for cis-acting DNA elements in pausing. STL-seq dissects the kinetics of pause release and termination, providing an opportunity to identify mechanisms of transcriptional regulation.


Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Metilação de DNA , Ecdisterona/química , Perfilação da Expressão Gênica , Técnicas Genéticas , Genoma , Hormônios , Cinética , Mutação , Osmose , Ligação Proteica , Transdução de Sinais
5.
Mol Cell ; 81(3): 502-513.e4, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33400923

RESUMO

Stress-induced readthrough transcription results in the synthesis of downstream-of-gene (DoG)-containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse sequencing revealed widespread transcriptional repression upon hyperosmotic stress. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP sequencing confirmed that stress-induced redistribution of RNA polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While certain cleavage and polyadenylation factors remain Pol II associated, Integrator complex subunits dissociate from Pol II under stress leading to a genome-wide loss of Integrator on DNA. Depleting the catalytic subunit of Integrator using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription.


Assuntos
Endorribonucleases/metabolismo , Pressão Osmótica , RNA Polimerase II/metabolismo , RNA/biossíntese , Estresse Salino , Transcrição Gênica , Ativação Transcricional , Regulação para Baixo , Endorribonucleases/genética , Células HEK293 , Humanos , RNA/genética , RNA Polimerase II/genética , Fatores de Tempo
6.
bioRxiv ; 2023 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-37292657

RESUMO

RNA metabolic labeling using 4-thiouridine (s4U) captures the dynamics of RNA synthesis and decay. The power of this approach is dependent on appropriate quantification of labeled and unlabeled sequencing reads, which can be compromised by the apparent loss of s4U-labeled reads in a process we refer to as dropout. Here we show that s4U-containing transcripts can be selectively lost when RNA samples are handled under sub-optimal conditions, but that this loss can be minimized using an optimized protocol. We demonstrate a second cause of dropout in nucleotide recoding and RNA sequencing (NR-seq) experiments that is computational and downstream of library preparation. NR-seq experiments involve chemically converting s4U from a uridine analog to a cytidine analog and using the apparent T-to-C mutations to identify the populations of newly synthesized RNA. We show that high levels of T-to-C mutations can prevent read alignment with some computational pipelines, but that this bias can be overcome using improved alignment pipelines. Importantly, kinetic parameter estimates are affected by dropout independent of the NR chemistry employed, and all chemistries are practically indistinguishable in bulk, short-read RNA-seq experiments. Dropout is an avoidable problem that can be identified by including unlabeled controls, and mitigated through improved sample handing and read alignment that together improve the robustness and reproducibility of NR-seq experiments.

7.
Cell Rep ; 42(10): 113163, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37742191

RESUMO

N6-methyladenosine (m6A) RNA modification controls numerous cellular processes. To what extent these post-transcriptional regulatory mechanisms play a role in hematopoiesis has not been fully elucidated. We here show that the m6A demethylase alkB homolog 5 (ALKBH5) controls mitochondrial ATP production and modulates hematopoietic stem and progenitor cell (HSPC) fitness in an m6A-dependent manner. Loss of ALKBH5 results in increased RNA methylation and instability of oxoglutarate-dehydrogenase (Ogdh) messenger RNA and reduction of OGDH protein levels. Limited OGDH availability slows the tricarboxylic acid (TCA) cycle with accumulation of α-ketoglutarate (α-KG) and conversion of α-KG into L-2-hydroxyglutarate (L-2-HG). L-2-HG inhibits energy production in both murine and human hematopoietic cells in vitro. Impaired mitochondrial energy production confers competitive disadvantage to HSPCs and limits clonogenicity of Mll-AF9-induced leukemia. Our study uncovers a mechanism whereby the RNA m6A demethylase ALKBH5 regulates the stability of metabolic enzyme transcripts, thereby controlling energy metabolism in hematopoiesis and leukemia.


Assuntos
Leucemia , RNA , Animais , Humanos , Camundongos , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Metabolismo Energético , Células-Tronco Hematopoéticas/metabolismo , RNA/metabolismo , Estabilidade de RNA/genética
8.
Cell Rep ; 39(3): 110687, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35443176

RESUMO

The p53-induced long noncoding RNA (lncRNA) lincRNA-p21 is proposed to act in cis to promote p53-dependent expression of the neighboring cell cycle gene, Cdkn1a/p21. The molecular mechanism through which the transcribed lincRNA-p21 regulatory locus activates p21 expression remains poorly understood. To elucidate the functional elements of cis-regulation, we generate a series of genetic models that disrupt DNA regulatory elements, the transcription of lincRNA-p21, or the accumulation of mature lincRNA-p21. Unexpectedly, we determine that full-length transcription, splicing, and accumulation of lincRNA-p21 are dispensable for the chromatin organization of the locus and for cis-regulation. Instead, we find that production of lincRNA-p21 through conserved regions in exon 1 of lincRNA-p21 promotes cis-activation. These findings demonstrate that the activation of nascent transcription from this lncRNA locus, but not the generation or accumulation of a mature lncRNA transcript, is necessary to enact local gene expression control.


Assuntos
RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
9.
ACS Chem Biol ; 17(7): 1789-1798, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35749470

RESUMO

The RNA decapping scavenger protein, DcpS, has recently been identified as a dependency in acute myeloid leukemia (AML). The potent DcpS inhibitor RG3039 attenuates AML cell viability, and shRNA knockdown of DcpS is also antiproliferative. Importantly, DcpS was found to be non-essential in normal human hematopoietic cells, which opens a therapeutic window for AML treatment by DcpS modulation. Considering this strong DcpS dependence in AML cell lines, we explored PROTAC-mediated degradation as an alternative strategy to modulate DcpS activity. Herein, we report the development of JCS-1, a PROTAC exhibiting effective degradation of DcpS at nanomolar concentrations. JCS-1 non-covalently binds DcpS with a RG3039-based warhead and recruits the E3 ligase VHL, which induces potent, rapid, and sustained DcpS degradation in several AML cell lines. JCS-1 serves as a chemical biology tool to interrogate DcpS degradation and associated changes in RNA processes in different cellular contexts, which may be an attractive strategy for the treatment of AML and other DcpS-dependent genetic disorders.


Assuntos
Endorribonucleases , Leucemia Mieloide Aguda , Humanos , Endorribonucleases/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , RNA Interferente Pequeno , Proteína Supressora de Tumor Von Hippel-Lindau
10.
Virus Res ; 252: 48-57, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29782878

RESUMO

Many viruses make use of, and even direct, the ubiquitin-proteasome system to facilitate the generation of a cellular environment favorable for virus replication, while host cells use selected protein ubiquitylation pathways for antiviral defense. Relatively little information has been acquired, however, regarding the extent to which protein ubiquitylation determines the replication success of picornaviruses. Here we report that the ubiquitin-protein ligase E6AP/UBE3A, recently shown to be a participant in encephalomyocarditis virus (EMCV) 3C protease concentration regulation, also facilitates the early stages of EMCV replication, probably by a mechanism that does not involve 3C protease ubiquitylation. Using stably transfected E6AP knockdown cells, we found that reduced E6AP concentration extends the time required for infected cells to undergo the morphological changes caused by virally induced pathogenesis and to begin the production of infectious virions. This lag in virion production is accompanied by a corresponding delay in the appearance of detectable levels of viral proteins and RNA. We also found, by using both immunofluorescence microscopy and cell fractionation, that E6AP is partially redistributed from the nucleus to the cytoplasm in EMCV-infected cells, thereby increasing its availability to participate in cytoplasmic virus replication processes.


Assuntos
Vírus da Encefalomiocardite/fisiologia , Ubiquitina-Proteína Ligases/genética , Replicação Viral , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/virologia , Replicação do DNA , Imunofluorescência , Interações Hospedeiro-Patógeno , Camundongos , Células NIH 3T3 , Ubiquitinação
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