Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.345
Filtrar
1.
BMC Bioinformatics ; 23(1): 190, 2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35596139

RESUMO

BACKGROUND: Gene expression is the result of the balance between transcription and degradation. Recent experimental findings have shown fine and specific regulation of RNA degradation and the presence of various molecular machinery purposely devoted to this task, such as RNA binding proteins, non-coding RNAs, etc. A biological process can be studied by measuring time-courses of RNA abundance in response of internal and/or external stimuli, using recent technologies, such as the microarrays or the Next Generation Sequencing devices. Unfortunately, the picture provided by looking only at the transcriptome abundance may not gain insight into its dynamic regulation. By contrast, independent simultaneous measurement of RNA expression and half-lives could provide such valuable additional insight. A computational approach to the estimation of RNAs half-lives from RNA expression time profiles data, can be a low-cost alternative to its experimental measurement which may be also affected by various artifacts. RESULTS: Here we present a computational methodology, called StaRTrEK (STAbility Rates ThRough Expression Kinetics), able to estimate half-life values basing only on genome-wide gene expression time series without transcriptional inhibition. The StaRTrEK algorithm makes use of a simple first order kinetic model and of a [Formula: see text]-norm regularized least square optimization approach to find its parameter values. Estimates provided by StaRTrEK are validated using simulated data and three independent experimental datasets of two short (6 samples) and one long (48 samples) time-courses. CONCLUSIONS: We believe that our algorithm can be used as a fast valuable computational complement to time-course experimental gene expression studies by adding a relevant kinetic property, i.e. the RNA half-life, with a strong biological interpretation, thus providing a dynamic picture of what is going in a cell during the biological process under study.


Assuntos
Estabilidade de RNA , RNA , Genoma , Meia-Vida , RNA/genética , RNA/metabolismo , RNA Mensageiro/genética
2.
RNA Biol ; 19(1): 703-718, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35510877

RESUMO

Circadian clocks are an endogenous internal timekeeping mechanism that drives the rhythmic expression of genes, controlling the 24 h oscillatory pattern in behaviour and physiology. It has been recently shown that post-transcriptional mechanisms are essential for controlling rhythmic gene expression. Controlling the stability of mRNA through poly(A) tail length modulation is one such mechanism. In this study, we show that Cnot1, encoding the scaffold protein of the CCR4-NOT deadenylase complex, is highly expressed in the suprachiasmatic nucleus, the master timekeeper. CNOT1 deficiency in mice results in circadian period lengthening and alterations in the mRNA and protein expression patterns of various clock genes, mainly Per2. Per2 mRNA exhibited a longer poly(A) tail and increased mRNA stability in Cnot1+/- mice. CNOT1 is recruited to Per2 mRNA through BRF1 (ZFP36L1), which itself oscillates in antiphase with Per2 mRNA. Upon Brf1 knockdown, Per2 mRNA is stabilized leading to increased PER2 expression levels. This suggests that CNOT1 plays a role in tuning and regulating the mammalian circadian clock.


Assuntos
Ritmo Circadiano , Proteínas Circadianas Period , Animais , Ritmo Circadiano/genética , Mamíferos/genética , Camundongos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Núcleo Supraquiasmático/metabolismo
3.
Nucleic Acids Res ; 50(9): 5029-5046, 2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35524564

RESUMO

Bacterial mRNAs have short life cycles, in which transcription is rapidly followed by translation and degradation within seconds to minutes. The resulting diversity of mRNA molecules across different life-cycle stages impacts their functionality but has remained unresolved. Here we quantitatively map the 3' status of cellular RNAs in Escherichia coli during steady-state growth and report a large fraction of molecules (median>60%) that are fragments of canonical full-length mRNAs. The majority of RNA fragments are decay intermediates, whereas nascent RNAs contribute to a smaller fraction. Despite the prevalence of decay intermediates in total cellular RNA, these intermediates are underrepresented in the pool of ribosome-associated transcripts and can thus distort quantifications and differential expression analyses for the abundance of full-length, functional mRNAs. The large heterogeneity within mRNA molecules in vivo highlights the importance in discerning functional transcripts and provides a lens for studying the dynamic life cycle of mRNAs.


Assuntos
Escherichia coli , Transcriptoma , Escherichia coli/genética , Escherichia coli/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
4.
STAR Protoc ; 3(2): 101190, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35434657

RESUMO

Generated by RNA deprotection or cleavage, 5' monophosphates trigger RNA degradation in all organisms. Here we describe PABLO-QA (Phosphorylation Assay By Ligation of Oligonucleotides and Quantitative Amplification), a sensitive, low-cost procedure for determining the percentage of specific RNA 5' ends that are monophosphorylated from their ability to undergo ligation to an oligonucleotide. Comparison to a cognate internal standard and a fully monophosphorylated control allows precise quantification of monophosphorylated 5' termini by RT-PCR, enabling the analysis of transcripts undetectable by blotting. For complete details on the use and execution of this protocol, please refer to Richards and Belasco (2021).


Assuntos
Estabilidade de RNA , RNA , Fosforilação , RNA/genética
5.
Cell Death Dis ; 13(4): 312, 2022 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-35387981

RESUMO

Increasing research has uncovered the involvement of long noncoding RNAs (lncRNAs) in the progression of multiple cancers including lung adenocarcinoma (LUAD). RT-qPCR and western blot were done to measure RNAs and proteins. Functional assays assessed LUAD cell biological behaviors under knockdown or overexpression of LINC01468, SIX5, SERBP1 or SERPINE1, and the specific function of those genes in regulating LUAD progression was evaluated via animal experiments. Supported by bioinformatics analysis, the interaction among genes was verified via mechanism assays. Upregulation of LINC01468 in LUAD tissues and cells as well as its association with poor clinical outcome was predicted. LINC01468, transcriptionally activated by SIX5, could strengthen proliferative, migratory and invasive abilities of LUAD cells. The oncogenic role of LINC01468 was further validated via animal experiments. SIX5 was a positive transcription regulator of LINC01468 and could exacerbate LUAD cell malignant behaviors. LINC01468 could recruit SERBP1 to enhance SERPINE1 mRNA stability and interact with USP5 to affect PAI1 protein ubiquitination. The oncogenic role of SERBP1 and SERPINE1 was also confirmed. Rescue experiments finally verified LINC01468 modulated proliferation, migration and invasion of LUAD cells via upregulation of SERPINE1. Our observations could contribute to deeper understanding of LUAD.


Assuntos
Adenocarcinoma , Neoplasias Pulmonares , Adenocarcinoma/genética , Animais , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Pulmão/metabolismo , Neoplasias Pulmonares/patologia , Estabilidade de RNA/genética
6.
Nat Commun ; 13(1): 2077, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35440550

RESUMO

P-bodies are conserved mRNP complexes that are implicated in determining mRNA fate by affecting translation and mRNA decay. In this report, we identify RGG-motif containing translation repressor protein Sbp1 as a disassembly factor of P-bodies since disassembly of P-bodies is defective in Δsbp1. RGG-motif is necessary and sufficient to rescue the PB disassembly defect in Δsbp1. Binding studies using purified proteins revealed that Sbp1 physically interacts with Edc3 and Sbp1-Edc3 interaction competes with Edc3-Edc3 interaction. Purified Edc3 forms assemblies, promoted by the presence of RNA and NADH and the addition of purified Sbp1, but not the RGG-deletion mutant, leads to significantly decreased Edc3 assemblies. We further note that the aggregates of human EWSR1 protein, implicated in neurodegeneration, are more persistent in the absence of Sbp1 and overexpression of EWSR1 in Δsbp1 leads to a growth defect. Taken together, our observations suggest a role of Sbp1 in disassembly, which could apply to disease-relevant heterologous protein-aggregates.


Assuntos
Estabilidade de RNA , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Cell Rep ; 39(2): 110635, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35417690

RESUMO

Circadian genes such as Clock, Bmal1, Cryptochrome1/2, and Period1/2/3 constitute the precise circadian system. ClockΔ19 is a commonly used mouse model harboring a circadian clock gene mutation, which lacks the EXON-19-encoded 51 amino acids. Previous reports have shown that ClockΔ19 mice have severe metabolic abnormalities. Here, we report that the mitochondria of ClockΔ19 mice exhibit excessive fission and dysfunction. We also demonstrate that CLOCK binds to the RNA-binding protein PUF60 through its EXON 19. Further, we find that PUF60 directly maintains mitochondrial homeostasis through regulating Drp1 mRNA stability, while the association with CLOCK can competitively inhibit this function. In ClockΔ19 mice, CLOCKΔ19 releases PUF60, leading to enhanced Drp1 mRNA stability and persistent mitochondrial fission. Our results reveal a direct post-transcriptional role of CLOCK in regulating mitochondrial homeostasis via Drp1 mRNA stability and that the loss of EXON 19 of CLOCK in ClockΔ19 mice leads to severe mitochondrial homeostasis disorders.


Assuntos
Proteínas CLOCK , Relógios Circadianos , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relógios Circadianos/genética , Homeostase/genética , Camundongos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Estabilidade de RNA
8.
Nucleic Acids Res ; 50(7): 3693-3708, 2022 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-35380686

RESUMO

Periodic gene expression dynamics are key to cell and organism physiology. Studies of oscillatory expression have focused on networks with intuitive regulatory negative feedback loops, leaving unknown whether other common biochemical reactions can produce oscillations. Oscillation and noise have been proposed to support mammalian progenitor cells' capacity to restore heterogenous, multimodal expression from extreme subpopulations, but underlying networks and specific roles of noise remained elusive. We use mass-action-based models to show that regulated RNA degradation involving as few as two RNA species-applicable to nearly half of human protein-coding genes-can generate sustained oscillations without explicit feedback. Diverging oscillation periods synergize with noise to robustly restore cell populations' bimodal expression on timescales of days. The global bifurcation organizing this divergence relies on an oscillator and bistable switch which cannot be decomposed into two structural modules. Our work reveals surprisingly rich dynamics of post-transcriptional reactions and a potentially widespread mechanism underlying development, tissue regeneration, and cancer cell heterogeneity.


Assuntos
Retroalimentação Fisiológica , Estabilidade de RNA , Animais , Retroalimentação , Expressão Gênica , Redes Reguladoras de Genes , Humanos , Mamíferos , Modelos Biológicos
9.
Nucleic Acids Res ; 50(8): 4372-4388, 2022 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-35390159

RESUMO

The steady state levels of RNAs, often referred to as expression levels, result from a well-balanced combination of RNA transcription and decay. Alterations in RNA levels will therefore result from tight regulation of transcription rates, decay rates or both. Here, we explore the role of RNA stability in achieving balanced gene expression and present genome-wide RNA stabilities in Drosophila melanogaster male and female cells as well as male cells depleted of proteins essential for dosage compensation. We identify two distinct RNA-stability mediated responses involved in regulation of gene expression. The first of these responds to acute and global changes in transcription and thus counteracts potentially harmful gene mis-expression by shifting the RNA stability in the direction opposite to the transcriptional change. The second response enhances inter-individual differential gene expression by adjusting the RNA stability in the same direction as a transcriptional change. Both mechanisms are global, act on housekeeping as well as non-housekeeping genes and were observed in both flies and mammals. Additionally, we show that, in contrast to mammals, modulation of RNA stability does not detectably contribute to dosage compensation of the sex-chromosomes in D. melanogaster.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Compensação de Dosagem (Genética) , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Masculino , Mamíferos/genética , RNA/genética , Estabilidade de RNA/genética
10.
Protein Sci ; 31(5): e4312, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35481630

RESUMO

Human Suv3 is a unique homodimeric helicase that constitutes the major component of the mitochondrial degradosome to work cooperatively with exoribonuclease PNPase for efficient RNA decay. However, the molecular mechanism of how Suv3 is assembled into a homodimer to unwind RNA remains elusive. Here, we show that dimeric Suv3 preferentially binds to and unwinds DNA-DNA, DNA-RNA, and RNA-RNA duplexes with a long 3' overhang (≥10 nucleotides). The C-terminal tail (CTT)-truncated Suv3 (Suv3ΔC) becomes a monomeric protein that binds to and unwinds duplex substrates with ~six to sevenfold lower activities relative to dimeric Suv3. Only dimeric Suv3, but not monomeric Suv3ΔC, binds RNA independently of ATP or ADP, and is capable of interacting with PNPase, indicating that dimeric Suv3 assembly ensures its continuous association with RNA and PNPase during ATP hydrolysis cycles for efficient RNA degradation. We further determined the crystal structure of the apo-form of Suv3ΔC, and SAXS structures of dimeric Suv3 and PNPase-Suv3 complex, showing that dimeric Suv3 caps on the top of PNPase via interactions with S1 domains, and forms a dumbbell-shaped degradosome complex with PNPase. Overall, this study reveals that Suv3 is assembled into a dimeric helicase by its CTT for efficient and persistent RNA binding and unwinding to facilitate interactions with PNPase, promote RNA degradation, and maintain mitochondrial genome integrity and homeostasis.


Assuntos
Estabilidade de RNA , RNA , Trifosfato de Adenosina/metabolismo , DNA Helicases/metabolismo , Endorribonucleases , Humanos , Complexos Multienzimáticos , Polirribonucleotídeo Nucleotidiltransferase , RNA/química , RNA Helicases , RNA Mitocondrial , Espalhamento a Baixo Ângulo , Difração de Raios X
11.
Cell Metab ; 34(4): 564-580.e8, 2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35385705

RESUMO

Hepatokines, secretory proteins from the liver, mediate inter-organ communication to maintain a metabolic balance between food intake and energy expenditure. However, molecular mechanisms by which hepatokine levels are rapidly adjusted following stimuli are largely unknown. Here, we unravel how CNOT6L deadenylase switches off hepatokine expression after responding to stimuli (e.g., exercise and food) to orchestrate energy intake and expenditure. Mechanistically, CNOT6L inhibition stabilizes hepatic Gdf15 and Fgf21 mRNAs, increasing corresponding serum protein levels. The resulting upregulation of GDF15 stimulates the hindbrain to suppress appetite, while increased FGF21 affects the liver and adipose tissues to induce energy expenditure and lipid consumption. Despite the potential of hepatokines to treat metabolic disorders, their administration therapies have been challenging. Using small-molecule screening, we identified a CNOT6L inhibitor enhancing GDF15 and FGF21 hepatokine levels, which dramatically improves diet-induced metabolic syndrome. Our discovery, therefore, lays the foundation for an unprecedented strategy to treat metabolic syndrome.


Assuntos
Síndrome Metabólica , Estabilidade de RNA , Animais , Ingestão de Alimentos , Metabolismo Energético/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Fígado/metabolismo , Síndrome Metabólica/metabolismo , Camundongos , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , Ribonucleases/metabolismo
12.
Mol Cell ; 82(8): 1467-1476, 2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35452615

RESUMO

Messenger RNA (mRNA) translation by the ribosome represents the final step of a complicated molecular dance from DNA to protein. Although classically considered a decipherer that translates a 64-word genetic code into a proteome of astonishing complexity, the ribosome can also shape the transcriptome by controlling mRNA stability. Recent work has discovered that the ribosome is an arbiter of the general mRNA degradation pathway, wherein the ribosome transit rate serves as a major determinant of transcript half-lives. Specifically, members of the degradation complex sense ribosome translocation rates as a function of ribosome elongation rates. Central to this notion is the concept of codon optimality: although all codons impact translation rates, some are deciphered quickly, whereas others cause ribosome hesitation as a consequence of relative cognate tRNA concentration. These transient pauses induce a unique ribosome conformational state that is probed by the deadenylase complex, thereby inducing an orchestrated set of events that enhance both poly(A) shortening and cap removal. Together, these data imply that the coding region of an mRNA not only encodes for protein content but also impacts protein levels through determining the transcript's fate.


Assuntos
Biossíntese de Proteínas , Estabilidade de RNA , Códon/genética , Códon/metabolismo , Proteínas/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
13.
Bioengineered ; 13(3): 7034-7048, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35255776

RESUMO

N6-methyladenosine (m6A) plays a critical role in the tumorigenesis of cervical cancer (CC). Here, we aimed to investigate the potential role of methyltransferase-like 3 (METTL3) in CC. Gene expression was determined via real-time quantitative polymerase chain reaction. Cellular functions were detected using colony formation, 5-ethynyl-2'-deoxyuridine (EdU), and Transwell assays. The interactions among METTL3, insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), and apoptotic chromatin condensation inducer 1 (ACIN1) were confirmed using the MeRIP and RIP assays. An in vivo assay was performed to verify the role of METTL3 in CC development. METTL3 is overexpressed in CC, and therefore, its knockdown inhibits the proliferation and migration of CC cells. Silencing METTL3 inhibits tumor growth in vivo. Moreover, a positive association was observed between METTL3 and ACIN1. METTL3 interacts with IGF2BP3 to promote the mRNA stability of ACIN1, the overexpression of which induces the aggressiveness of CC cells. METTL3 promotes ACIN1 mRNA stability to accelerate CC progression, implying that METTL3 is a promising biomarker in CC.


Assuntos
Somatomedinas , Neoplasias do Colo do Útero , Cromatina/genética , Feminino , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Somatomedinas/genética , Neoplasias do Colo do Útero/genética
14.
STAR Protoc ; 3(1): 101231, 2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35284837

RESUMO

We developed a highly efficient, ultrashort immunohistochemistry-laser capture microdissection (IHC-LMD) protocol, which allows microdissection of up to 250 single cardiomyocytes. Before LMD, murine hearts are excised, snap-frozen, and cryosectioned. RNA isolated from LMD material is of high RNA quality, making it usable for gene expression analysis and RNA sequencing. Challenges and limitations of this protocol include visualization of the immunostaining and nuclei DAPI dye on the PEN slides, and timing and speed to limit RNA degradation as much as possible.


Assuntos
Miócitos Cardíacos , RNA , Animais , Imuno-Histoquímica , Microdissecção e Captura a Laser/métodos , Camundongos , Miócitos Cardíacos/química , RNA/genética , Estabilidade de RNA
15.
J Bacteriol ; 204(4): e0005322, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35311556

RESUMO

RNase J exerts both 5'-3' exoribonuclease and endoribonuclease activities and plays a major role in ribonucleotide metabolism in various bacteria; however, its gene regulation is not well understood. In this study, we investigated the regulation of rnj expression in Corynebacterium glutamicum. rnj mRNA expression was increased in a strain with an rnj mutation. Deletion of the genes encoding RNase E/G also resulted in increased rnj mRNA levels, although the effect was smaller than that of the rnj mutation. rnj mRNA was more stable in the rnj mutant strain than in wild-type cells. These results indicate that RNase J regulates its own gene by degrading its mRNA. The growth of rnj and pnp mutant cells was impaired at cold temperatures. The expression of rnj mRNA was transiently induced by cold shock; however, this induction was not observed in the rnj mutant strain, suggesting that autoregulation by self-degradation is responsible for inducing of rnj expression under cold-shock conditions. IMPORTANCE Corynebacterium glutamicum harbors one RNase E/G-type enzyme and one RNase J-type enzyme which are major ribonucleases in various bacteria. However, little is known about these gene regulations. Here, we show that RNase J autoregulates its own gene expression and RNase E/G is also involved in the rnj mRNA degradation. Furthermore, we show that transient induction of the rnj mRNA in the cold-shock condition is dependent on RNase J autoregulation. This study sheds light on the regulatory mechanism of RNase J in C. glutamicum.


Assuntos
Corynebacterium glutamicum , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Regulação Bacteriana da Expressão Gênica , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo
16.
Nature ; 604(7904): 167-174, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35355014

RESUMO

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate1-3. Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA-degradation complex known as the rixosome or RIX1 complex4-6. Whether RNA degradation also has a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5' monophosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosomal degradation of nascent RNA is conserved from fission yeast to human, with a primary role in RNA degradation at facultative heterochromatin in human cells.


Assuntos
Inativação Gênica , Heterocromatina , Complexo Repressor Polycomb 1 , Estabilidade de RNA , Exorribonucleases/genética , Heterocromatina/genética , Humanos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , Proteínas do Grupo Polycomb/genética , Schizosaccharomyces/genética
17.
mBio ; 13(2): e0040022, 2022 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-35311531

RESUMO

The Bacillus subtilis genome encodes four 3' exoribonucleases: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Previous work showed that PNPase, encoded by the pnpA gene, is the major 3' exonuclease involved in mRNA turnover; in a pnpA deletion strain, numerous mRNA decay intermediates accumulate. Whether B. subtilis mRNA decay occurs in the context of a degradosome complex is controversial. In this study, global mapping of mRNA decay intermediate 3' ends within coding sequences was performed in strains that were either deleted for or had an inactivating point mutation in the pnpA gene. The patterns of 3'-end accumulation in these strains were highly similar, which may have implications for the role of a degradosome in mRNA decay. A comparison with mapped 3' ends in a strain lacking CshA, the major RNA helicase, indicated that many mRNAs require both PNPase and CshA for efficient decay. Transcriptome sequencing (RNA-seq) analysis of strains lacking RNase R suggested that this enzyme did not play a major role in mRNA turnover in the wild-type strain. Strains were constructed that contained only one of the four known 3' exoribonucleases. When RNase R was the only 3' exonuclease present, it was able to degrade a model mRNA efficiently, showing processive decay even through a strong stem-loop structure that inhibits PNPase processivity. Strains containing only RNase PH or only YhaM were also insensitive to this RNA secondary structure, suggesting the existence of another, as-yet-unidentified, 3' exoribonuclease. IMPORTANCE The ability to rapidly change bacterial gene expression programs in response to environmental conditions is highly dependent on the efficient turnover of mRNA. While much is known about the regulation of gene expression at the transcriptional and translational levels, much less is known about the intermediate step of mRNA decay. Here, we mapped the 3' ends of mRNA decay intermediates in strains that were missing the major 3' exoribonuclease PNPase or the RNA helicase CshA. We also assessed the roles of three other B. subtilis 3' exonucleases in the mRNA decay process. The data confirm the primary role of PNPase in mRNA turnover and suggest the involvement of one or more unknown RNases.


Assuntos
Bacillus subtilis , Exorribonucleases , Bacillus subtilis/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
18.
Nat Commun ; 13(1): 1536, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35318324

RESUMO

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured "superfolder" mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.


Assuntos
COVID-19 , RNA , COVID-19/terapia , Humanos , Pseudouridina/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo
19.
Nucleic Acids Res ; 50(5): 2440-2451, 2022 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-35234905

RESUMO

CUGBP Elav-like family member 1 (CELF1), an RNA-binding protein (RBP), plays important roles in the pathogenesis of diseases such as myotonic dystrophy, liver fibrosis and cancers. However, targeting CELF1 is still a challenge, as RBPs are considered largely undruggable. Here, we discovered that compound 27 disrupted CELF1-RNA binding via structure-based virtual screening and biochemical assays. Compound 27 binds directly to CELF1 and competes with RNA for binding to CELF1. Compound 27 promotes IFN-γ secretion and suppresses TGF-ß1-induced hepatic stellate cell (HSC) activation by inhibiting CELF1-mediated IFN-γ mRNA decay. In vivo, compound 27 attenuates CCl4-induced murine liver fibrosis. Furthermore, the structure-activity relationship analysis was performed and compound 841, a derivative of compound 27, was identified as a selective CELF1 inhibitor. In conclusion, targeting CELF1 RNA-binding activity with small molecules was achieved, which provides a novel strategy for treating liver fibrosis and other CELF1-mediated diseases.


Assuntos
Proteínas de Ligação a RNA , RNA , Animais , Proteínas CELF1/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/genética , Camundongos , Estabilidade de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
20.
Bioengineered ; 13(4): 8581-8592, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35287546

RESUMO

The molecular etiology of esophageal squamous cell carcinoma (ESCC) has not been fully elucidated. Understanding the molecular mechanisms and finding new therapeutic targets for ESCC are of crucial importance. PolyC-RNA-binding protein 1 (PCBP1) is an RNA-binding protein. Here, we found overexpressed PCBP1 in esophageal cancer tissues by quantitative polymerase chain reaction (qPCR) and western blotting analysis. PCBP1 knockdown significantly attenuated migratory and invasion abilities of ESCC cells. Mechanistically, PCBP1 bound directly to tropomyosin 3 (TPM3) mRNA, which was verified by RNA-protein immunoprecipitation (RIP) assay. PCBP1 knockdown markedly reduced messenger RNA (mRNA) levels of TPM3. After inhibiting intracellular mRNA synthesis with actinomycin D (ActD), it was found that PCBP1 knockdown contributed to a significant decrease in TPM3 mRNA degradation. Furthermore, PCBP1 promoted migration and invasion of EC cells by directly binding to the 3'UTR of TPM3 mRNA, increasing TPM3 mRNA stability. Taken together, PCBP1 acting as a pro-oncogenic factor enhances TPM3 mRNA stability by directly binding to the 3'UTR of TPM3 mRNA in esophageal squamous cell carcinoma. Our findings provide a new perspective for understanding the molecular mechanism of esophageal carcinogenesis, and PCBP1 is a promising therapeutic target.


Assuntos
Proteínas de Ligação a DNA , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Proteínas de Ligação a RNA , Tropomiosina , Regiões 3' não Traduzidas , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Humanos , Estabilidade de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Tropomiosina/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...