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1.
J Cell Biol ; 223(3)2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38349334

RESUMO

The cell cycle is a highly regulated process in which proteins involved in cell cycle progression exhibit periodic expression patterns, controlled by specific mechanisms such as transcription, translation, and degradation. However, the precise mechanisms underlying the oscillations of mRNA levels in cell cycle regulators are not fully understood. In this study, we observed that the stability of cyclin D1 (CCND1) mRNA fluctuates during the cell cycle, with increased stability during interphase and decreased stability during the M phase. Additionally, we identified a key RNA binding protein, positive coactivator 4 (PC4), which plays a crucial role in stabilizing CCND1 mRNA and regulating its periodic expression. Moreover, the binding affinity of PC4 to CCND1 mRNA is modulated by two cell cycle-specific posttranslational modifications: ubiquitination of K68 enhances binding and stabilizes the CCND1 transcript during interphase, while phosphorylation of S17 inhibits binding during the M phase, leading to degradation of CCND1 mRNA. Remarkably, PC4 promotes the transition from G1 to S phase in the cell cycle, and depletion of PC4 enhances the efficacy of CDK4/6 inhibitors in hepatocellular carcinoma, suggesting that PC4 could serve as a potential therapeutic target. These findings provide valuable insights into the intricate regulation of cell cycle dynamics.


Assuntos
Ciclo Celular , Ciclina D1 , Estabilidade de RNA , Proteínas de Ligação a RNA , Ciclo Celular/genética , Divisão Celular , Ciclina D1/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina , Estabilidade de RNA/genética , RNA Mensageiro/genética , Masculino , Animais , Camundongos , Camundongos Endogâmicos BALB C , Humanos , Linhagem Celular Tumoral , Proteínas de Ligação a RNA/genética , Fosforilação , Ubiquitinação
2.
Plant Mol Biol ; 114(1): 18, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38353826

RESUMO

Microalgae represent a promising but yet underexplored production platform for biotechnology. The vast majority of studies on recombinant protein expression in algae have been conducted in a single species, the green alga Chlamydomonas reinhardtii. However, due to epigenetic silencing, transgene expression in Chlamydomonas is often inefficient. Here we have investigated parameters that govern efficient transgene expression in the red microalga Porphyridium purpureum. Porphyridium is unique in that the introduced transformation vectors are episomally maintained as autonomously replicating plasmids in the nucleus. We show that full codon optimization to the preferred codon usage in the Porphyridium genome confers superior transgene expression, not only at the level of protein accumulation, but also at the level of mRNA accumulation, indicating that high translation rates increase mRNA stability. Our optimized expression constructs resulted in YFP accumulation to unprecedented levels of up to 5% of the total soluble protein. We also designed expression cassettes that target foreign proteins to the secretory pathway and lead to efficient protein secretion into the culture medium, thus simplifying recombinant protein harvest and purification. Our study paves the way to the exploration of red microalgae as expression hosts in molecular farming for recombinant proteins and metabolites.


Assuntos
Chlamydomonas reinhardtii , Microalgas , Porphyridium , Porphyridium/genética , Biotecnologia , Estabilidade de RNA , Chlamydomonas reinhardtii/genética , Microalgas/genética , Proteínas Recombinantes/genética
3.
Int J Mol Sci ; 25(3)2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38339002

RESUMO

The ever-increasing applications of metabarcoding analyses for environmental samples demand a well-designed assessment of the stability of DNA and RNA contained in cells that are deposited or buried in marine sediments. We thus conducted a qPCR quantification of the DNA and RNA in the vegetative cells of three microalgae entrapped in facsimile marine sediments and found that >90% of DNA and up to 99% of RNA for all microalgal species were degraded within 60 days at 4 °C. A further examination of the potential interference of the relic DNA of the vegetative cells with resting cyst detection in sediments was performed via a metabarcoding analysis in artificial marine sediments spiked with the vegetative cells of two Kareniaceae dinoflagellates and the resting cysts of another three dinoflagellates. The results demonstrated a dramatic decrease in the relative abundances of the two Kareniaceae dinoflagellates in 120 days, while those of the three resting cysts increased dramatically. Together, our results suggest that a positive detection of microalgae via metabarcoding analysis in DNA or RNA extracted from marine sediments strongly indicates the presence of intact or viable cysts or spores due to the rapid decay of relic DNA/RNA. This study provides a solid basis for the data interpretation of metabarcoding surveys, particularly in resting cyst detection.


Assuntos
Dinoflagelados , Microalgas , Microalgas/genética , DNA , Dinoflagelados/genética , Código de Barras de DNA Taxonômico/métodos , RNA/genética , Estabilidade de RNA , Sedimentos Geológicos
4.
Methods Mol Biol ; 2741: 255-272, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38217658

RESUMO

Bacterial small RNAs (sRNAs) can be equipped at the 5' end with triphosphate (5'PPP) or monophosphate (5'P) groups, depending on whether they are primary transcripts, undergo dephosphorylation or originate via processing. Often, 5' groups hallmark RNAs for rapid decay, but whether this also applies to sRNAs is little explored. Moreover, the sRNA 5'P group could activate endoribonuclease RNase E to cleave the base-paired target RNA, but a tool for investigation in vivo was lacking. Here, we describe a two-plasmid system suitable for the generation of 5' monophosphorylated RNAs on demand inside the cell. The sRNA gene of interest is fused to the 3' end of a fragment of sRNA GlmZ and transcribed from a plasmid in an IPTG-inducible manner. The fusion RNA gets cleaved upon arabinose-controlled expression of rapZ, provided on a compatible plasmid. Adaptor protein RapZ binds the GlmZ aptamer and directs RNase E to release the sRNA of choice with 5'P ends. An isogenic plasmid generating the same sRNA with a 5'PPP end allows for direct comparison. The fates of the sRNA variants and target RNA(s) are monitored by Northern blotting. This tool is applicable to E. coli and likely other enteric bacteria.


Assuntos
Proteínas de Escherichia coli , Pequeno RNA não Traduzido , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fosforilação , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Estabilidade de RNA , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Regulação Bacteriana da Expressão Gênica
5.
BMC Cancer ; 24(1): 93, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38233795

RESUMO

BACKGROUND: Several publications suggest that UTP11 may be a promising gene engaged for involvement of hepatocellular carcinoma (HCC) pathology. However, there are extremely limited biological, mechanistic and clinical studies of UTP11 in HCC. METHODS: To anayze the UTP11 mRNA expression in HCC and normal clinical samples and further investigate the correlation between UTP11 expression and pathology and clinical prognosis via the Cancer Tissue Gene Atlas (TCGA) database. The protein levels of UTP11 were checked using the Human Protein Atlas (HPA) database. GO-KEGG enrichment was performed from Cancer Cell Line Encyclopedia (CCLE) database and TCGA dataset. The levels of UTP11 were tested with qRT-PCR and western blotting assays. Cell viability, immunofluorescence and flow cytometry assays and animal models were used to explore the potential involvement of UTP11 in regulating HCC growth in vitro and in vivo. The correlation of UTP11 and tumor stemness scores and stemness-associated proteins from TCGA database. The mRNA stability was treated with Actinomycin D, followed by testing the mRNA expression using qRT-PCR assay. RESULTS: UTP11 was highly expressed in HCC samples compared to normal tissues from TCGA database. Similarly, UTP11 protein expression levels were obviously elevated in HCC tissue samples from HPA database. Furthermore, UTP11 levels were correlated with poor prognosis in HCC patient samples in TCGA dataset. In addition, the UTP11 mRNA levels was notably enhanced in different HCC cell lines than in normal liver cells and knocking down UTP11 was obviously reduced the viability and cell death of HCC cells. UTP11 knockdown suppressed the tumor growth of HCC in vivo experiment and extended the mice survival time. GO-KEEG analysis from CCLE and TCGA database suggested that UTP11 might involve in RNA splicing and the stability of mRNA. Further, UTP11 was positively correlated with tumor stemness scores and stemness-associated proteins from TCGA database. Knockdown of UTP11 was reduced the expression of stem cell-related genes and regulated the mRNA stability of Oct4. CONCLUSIONS: UTP11 is potentially a diagnostic molecule and a therapeutic candidate for treatment of HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/patologia , Prognóstico , Estabilidade de RNA , RNA Mensageiro/genética
6.
Autoimmunity ; 57(1): 2304820, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38269483

RESUMO

Circular RNA (circRNA) has been found to be differentially expressed and involved in regulating the processes of human diseases, including thoracic aortic dissection (TAD). However, the role and mechanism of circNRIP1 in the TAD process are still unclear. GEO database was used to screen the differentially expressed circRNA and mRNA in type A TAD patients and age-matched normal donors. Angiotensin II (Ang II)-induced human aortic vascular smooth muscle cells (HA-VSMCs) were used to construct TAD cell models. The expression levels of circNRIP1, NRIP1, CXC-motif chemokine 5 (CXCL5) and IGF2BP1 were detected by quantitative real-time PCR. Cell proliferation and migration were determined by EdU assay, transwell assay and wound healing assay. The protein levels of synthetic phenotype markers, contractile phenotype markers, CXCL5 and IGF2BP1 were tested by western blot analysis. The interaction between IGF2BP1 and circNRIP1/CXCL5 was confirmed by RIP assay, and CXCL5 mRNA stability was assessed by actinomycin D assay. CircNRIP1 was upregulated in TAD patients and Ang II-induced HA-VSMCs. Knockdown of circNRIP1 suppressed Ang II-induced proliferation, migration and phenotypic switch of HA-VSMCs. Also, high expression of CXCL5 was observed in TAD patients, and its knockdown could inhibit Ang II-induced HA-VSMCs proliferation, migration and phenotypic switch. Moreover, CXCL5 overexpression reversed the regulation of circNRIP1 knockdown on Ang II-induced HA-VSMCs functions. Mechanistically, circNRIP1 could competitively bind to IGF2BP1 and subsequently enhance CXCL5 mRNA stability. CircNRIP1 might contribute to TAD progression by promoting CXCL5 mRNA stability via recruiting IGF2BP1.


Assuntos
Angiotensina II , Músculo Liso Vascular , Humanos , Angiotensina II/farmacologia , Proliferação de Células , Quimiocina CXCL5/genética , Fenótipo , Estabilidade de RNA , RNA Circular/genética
7.
J Virol ; 98(1): e0135023, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38169284

RESUMO

Epitranscriptomic RNA modifications can regulate the stability of mRNA and affect cellular and viral RNA functions. The N4-acetylcytidine (ac4C) modification in the RNA viral genome was recently found to promote viral replication; however, the mechanism by which RNA acetylation in the host mRNA regulates viral replication remains unclear. To help elucidate this mechanism, the roles of N-acetyltransferase 10 (NAT10) and ac4C during the infection and replication processes of the alphavirus, Sindbis virus (SINV), were investigated. Cellular NAT10 was upregulated, and ac4C modifications were promoted after alphavirus infection, while the loss of NAT10 or inhibition of its N-acetyltransferase activity reduced alphavirus replication. The NAT10 enhanced alphavirus replication as it helped to maintain the stability of lymphocyte antigen six family member E mRNA, which is a multifunctional interferon-stimulated gene that promotes alphavirus replication. The ac4C modification was thus found to have a non-conventional role in the virus life cycle through regulating host mRNA stability instead of viral mRNA, and its inhibition could be a potential target in the development of new alphavirus antivirals.IMPORTANCEThe role of N4-acetylcytidine (ac4C) modification in host mRNA and virus replication is not yet fully understood. In this study, the role of ac4C in the regulation of Sindbis virus (SINV), a prototype alphavirus infection, was investigated. SINV infection results in increased levels of N-acetyltransferase 10 (NAT10) and increases the ac4C modification level of cellular RNA. The NAT10 was found to positively regulate SINV infection in an N-acetyltransferase activity-dependent manner. Mechanistically, the NAT10 modifies lymphocyte antigen six family member E (LY6E) mRNA-the ac4C modification site within the 3'-untranslated region (UTR) of LY6E mRNA, which is essential for its translation and stability. The findings of this study demonstrate that NAT10 regulated mRNA stability and translation efficiency not only through the 5'-UTR or coding sequence but also via the 3'-UTR region. The ac4C modification of host mRNA stability instead of viral mRNA impacting the viral life cycle was thus identified, indicating that the inhibition of ac4C could be a potential target when developing alphavirus antivirals.


Assuntos
Infecções por Alphavirus , Antígenos de Superfície , Proteínas Ligadas por GPI , Acetiltransferases N-Terminal , Vírus Sindbis , Replicação Viral , Humanos , Infecções por Alphavirus/genética , Antígenos de Superfície/genética , Citidina/análogos & derivados , Proteínas Ligadas por GPI/genética , RNA Mensageiro/genética , Vírus Sindbis/fisiologia , Linhagem Celular , Acetiltransferases N-Terminal/genética , Estabilidade de RNA
8.
mBio ; 15(2): e0316823, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38236021

RESUMO

YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2) is a member of the YTH protein family that binds to N6-methyladenosine (m6A)-modified RNA, regulating RNA stability and restricting viral replication, including Epstein-Barr virus (EBV). PIAS1 is an E3 small ubiquitin-like modifier (SUMO) ligase known as an EBV restriction factor, but its role in YTHDF2 SUMOylation remains unclear. In this study, we investigated the functional regulation of YTHDF2 by PIAS1. We found that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues (K281, K571, and K572). Importantly, PIAS1 synergizes with wild-type YTHDF2, but not a SUMOylation-deficient mutant, to limit EBV lytic replication. Mechanistically, YTHDF2 lacking SUMOylation exhibits reduced binding to EBV transcripts, leading to increased viral mRNA stability. Furthermore, PIAS1 mediates SUMOylation of YTHDF2's paralogs, YTHDF1 and YTHDF3, to restrict EBV replication. These results collectively uncover a unique mechanism whereby YTHDF family proteins control EBV replication through PIAS1-mediated SUMOylation, highlighting the significance of SUMOylation in regulating viral mRNA stability and EBV replication.IMPORTANCEm6A RNA modification pathway plays important roles in diverse cellular processes and viral life cycle. Here, we investigated the relationship between PIAS1 and the m6A reader protein YTHDF2, which is involved in regulating RNA stability by binding to m6A-modified RNA. We found that both the N-terminal and C-terminal regions of YTHDF2 interact with PIAS1. We showed that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues. We also demonstrated that PIAS1 enhances the anti-EBV activity of YTHDF2. We further revealed that PIAS1 mediates the SUMOylation of other YTHDF family members, namely, YTHDF1 and YTHDF3, to limit EBV replication. These findings together illuminate an important regulatory mechanism of YTHDF proteins in controlling viral RNA decay and EBV replication through PIAS1-mediated SUMOylation.


Assuntos
Adenina/análogos & derivados , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Herpesvirus Humano 4/fisiologia , Sumoilação , RNA Viral/genética , RNA Viral/metabolismo , Lisina/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Estabilidade de RNA , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
9.
Redox Biol ; 69: 102993, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38104484

RESUMO

Resistance to chemotherapy is the main reason for treatment failure and poor prognosis in patients with triple-negative breast cancer (TNBC). Although the association of RNA N6-methyladenosine (m6A) modifications with therapy resistance is noticed, its role in the development of therapeutic resistance in TNBC is not well documented. This study aimed to investigate the potential mechanisms underlying reactive oxygen species (ROS) regulation in doxorubicin (DOX)-resistant TNBC. Here, we found that DOX-resistant TNBC cells displayed low ROS levels because of increased expression of superoxide dismutase (SOD2), thus maintaining cancer stem cells (CSCs) characteristics and DOX resistance. FOXO1 is a master regulator that reduces cellular ROS in DOX-resistant TNBC cells, and knockdown of FOXO1 significantly increased ROS levels by inhibiting SOD2 expression. Moreover, the m6A demethylase ALKBH5 promoted m6A demethylation of FOXO1 mRNA and increased FOXO1 mRNA stability in DOX-resistant TNBC cells. The analysis of clinical samples revealed that the increased expression levels of ALKBH5, FOXO1, and SOD2 were significantly positively correlated with chemoresistance and poor prognosis in patients with TNBC. To our knowledge, this is the first study to highlight that ALKBH5-mediated FOXO1 mRNA demethylation contributes to CSCs characteristics and DOX resistance in TNBC cells. Furthermore, pharmacological targeting of FOXO1 profoundly restored the response of DOX-resistant TNBC cells, both in vitro and in vivo. In conclusion, we demonstrated a critical function of ALKBH5-mediated m6A demethylation of FOXO1 mRNA in restoring redox balance, which in turn promoting CSCs characteristics and DOX resistance in TNBC, and suggested that targeting the ALKBH5/FOXO1 axis has therapeutic potential for patients with TNBC refractory to chemotherapy.


Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Espécies Reativas de Oxigênio , Doxorrubicina/farmacologia , RNA Mensageiro/genética , Estabilidade de RNA , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo
10.
J Biol Chem ; 300(1): 105567, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38103641

RESUMO

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.


Assuntos
Proteínas de Bactérias , Quadruplex G , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis , Biossíntese de Proteínas , RNA Bacteriano , RNA Mensageiro , Humanos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos/genética , Inflamação/microbiologia , Ligantes , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Estabilidade de RNA , RNA Bacteriano/genética , RNA Mensageiro/genética , Células THP-1 , Transcrição Gênica/efeitos dos fármacos
11.
RNA Biol ; 21(1): 1-15, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38111129

RESUMO

Inhibition of apoptosis is one of the hallmarks of cancer and is a target of various therapeutic interventions. BIRC5 is an inhibitor of apoptosis that is aberrantly expressed in cancer leading to sustained growth of tumours. Post-transcriptional control mechanisms involving RNA-binding proteins and AU-rich elements (AREs) are fundamental to many cellular processes and changes in the expression or function of these proteins can promote an aberrant and pathological phenotype. BIRC5 mRNA has an ARE in its 3' UTR making it a candidate for regulation by the RNA binding proteins tristetraprolin (TTP) and HuR (ELAVL1). In this study, we investigated the binding of TTP and HuR by RNA-immunoprecipitation assays and found that these proteins were associated with BIRC5 mRNA to varying extents. Consequently, BIRC5 expression decreased when TTP was overexpressed and apoptosis was induced. In the absence of TTP, BIRC5 mRNA was stabilized, protein expression increased and the number of apoptotic cells declined. As an ARE-mRNA stabilizing protein, recombinant HuR led to upregulation of BIRC5 expression, whereas HuR silencing was concomitant with downregulation of BIRC5 mRNA and protein and increased cell death. Survival analyses demonstrated that increased TTP and low BIRC5 expression predicted an overall better prognosis compared to dysregulated TTP and high BIRC5. Thus, the results present a novel target of ARE-mediated post-transcriptional regulation.


Assuntos
Neoplasias da Mama , Tristetraprolina , Humanos , Feminino , Tristetraprolina/genética , Tristetraprolina/metabolismo , Survivina/genética , Survivina/metabolismo , Neoplasias da Mama/genética , Regiões 3' não Traduzidas , Apoptose/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Estabilidade de RNA/genética
12.
Arch Biochem Biophys ; 752: 109876, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38141906

RESUMO

The metastasis of lung cancer poses a major clinical challenge, and m6A modification has been implicated in regulating the invasive capabilities of tumor cells. However, the mechanisms underlying m6A modification in lung cancer metastasis are not well understood. This study aims to explore the biological functions and molecular mechanisms of methyltransferase-like 3 (METTL3) in lung cancer. In this study, METTL3 were found to be downregulated in lung cancer tissues. Functionally, METTL3 inhibited the migration and invasion abilities of lung cancer cells in vitro. Furthermore, SH3 domain binding protein 5 (SH3BP5) was identified as a downstream target of METTL3. Overexpression of SH3BP5 suppressed the invasive capacity of lung cancer cells, and this regulation was m6A-dependent. Finally, we discovered that YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) mediated stability is responsible for maintaining the m6A modification of SH3BP5 mRNA. Overall, our study provides insights into the critical role of METTL3-mediated m6A modification and m6A-dependent regulatory mechanisms in the progression of human lung cancer. We demonstrated that METTL3 regulates the mRNA stability of SH3BP5 in a YTHDF1-dependent manner, thereby impacting the invasive capacity of lung cancer cells.


Assuntos
Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/genética , Adenina , Estabilidade de RNA , RNA Mensageiro , Metiltransferases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética
13.
Sci Rep ; 13(1): 23103, 2023 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-38158431

RESUMO

Glioma is the most common primary malignant brain tumor in adults and remains an incurable disease at present. Thus, there is an urgent need for progress in finding novel molecular mechanisms that control the progression of glioma which could be used as therapeutic targets for glioma patients. The RNA binding protein cytoplasmic polyadenylate element-binding protein 2 (CPEB2) is involved in the pathogenesis of several tumors. However, the role of CPEB2 in glioma progression is unknown. In this study, the functional characterization of the role and molecular mechanism of CPEB2 in glioma were examined using a series of biological and cellular approaches in vitro and in vivo. Our work shows CPEB2 is significantly downregulated in various glioma patient cohorts. Functional characterization of CPEB2 by overexpression and knockdown revealed that it inhibits glioma cell proliferation and promotes apoptosis. CPEB2 exerts an anti-tumor effect by increasing p21 mRNA stability and inducing G1 cell cycle arrest in glioma. Overall, this work stands as the first report of CPEB2 downregulation and involvement in glioma pathogenesis, and identifies CPEB2 as an important tumor suppressor gene through targeting p21 in glioma, which revealed that CPEB2 may become a promising predictive biomarker for prognosis in glioma patients.


Assuntos
Regulação Neoplásica da Expressão Gênica , Glioma , Proteína Oncogênica p21(ras) , Estabilidade de RNA , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/sangue , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proliferação de Células/genética , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Estabilidade de RNA/genética , Glioma/diagnóstico , Glioma/fisiopatologia , Técnicas de Silenciamento de Genes , Apoptose/genética , Regulação Neoplásica da Expressão Gênica/genética , Pontos de Checagem do Ciclo Celular/genética , Biomarcadores Tumorais/sangue , Regulação para Baixo/genética , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB C , Células HEK293 , Humanos , Feminino , Animais , Camundongos
14.
Sci Rep ; 13(1): 22870, 2023 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-38129448

RESUMO

A mutant deficient in polynucleotide phosphorylase (PNPase) activity was previously constructed in Enterococcus faecalis 14; a strain producing a leaderless two-peptide enterocin DD14 (EntDD14). Here, we examined the impact of the absence of PNPase on the expression and synthesis of EntDD14, at the transcriptional and functional levels. As result, EntDD14 synthesis augmented in line with the growth curve, reaching a two- to fourfold increase in the ΔpnpA mutant compared to the E. faecalis 14 wild-type strain (WT). EntDD14 synthesis has reached its highest level after 9 h of growth in both strains. Notably, high expression level of the ddABCDEFGHIJ cluster was registered in ΔpnpA mutant. Transcriptional and in silico analyses support the existence of ddAB and ddCDEFGHIJ independent transcripts, and analysis of the fate of ddAB and ddCDEFGHIJ mRNAs indicated that the differences in mRNA levels and the high EntDD14 activity are likely due to a better stability of the two transcripts in the ΔpnpA mutant, which should result in a higher translation efficiency of the ddAB EntDD14 structural genes and their other protein determinants. Consequently, this study shows a potential link between the mRNA stability and EntDD14 synthesis, secretion and immunity in a genetic background lacking PNPase.


Assuntos
Bacteriocinas , Bacteriocinas/genética , Bacteriocinas/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Estabilidade de RNA/genética
15.
Cell ; 186(24): 5254-5268.e26, 2023 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-37944513

RESUMO

A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size.


Assuntos
Tamanho Celular , RNA Polimerase II , Transcrição Gênica , Retroalimentação , RNA Polimerase II/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Methods Enzymol ; 692: 157-175, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37925178

RESUMO

Ribonuclease L (RNase L) is a mammalian endoribonuclease that initiates the mass degradation of cellular mRNAs in response to double-stranded RNA or viral infection. The kinetic rate of mRNA decay upon RNase L activation has been elusive because RNase L is heterogeneously activated with respect to time in individual cells. Herein, we describe a method using immunofluorescence combined with single-molecule fluorescence in situ hybridization (smFISH) to determine single-cell mRNA decay rates upon RNase L activation. Using these approaches, we deduce that the rate of mRNA decay upon RNase L activation is extremely rapid, whereby the half-life of stable mRNAs such as GAPDH mRNA is reduced to ∼15 minutes in individual cells. This allows for RNase L to degrade nearly every mRNA in a cell in less than 1 hour, which is much faster than the decay rate that would be derived using bulk measurement techniques for mRNA levels, such as qRT-PCR. These single-cell approaches can generally be employed to resolve mRNA decay kinetics in additional contexts.


Assuntos
Endorribonucleases , Estabilidade de RNA , Animais , Hibridização in Situ Fluorescente , Endorribonucleases/genética , Endorribonucleases/metabolismo , Análise de Célula Única , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mamíferos/genética
17.
Cell Death Dis ; 14(11): 767, 2023 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-38007473

RESUMO

Due to a lack of research on the critical non-coding RNAs in regulating ferroptosis, our study aimed to uncover the crucial ones involved in the process. We found that LINC01133 could make pancreatic cancer cells more resistant to ferroptosis. A higher expression of LINC01133 was associated with a higher IC50 of sorafenib in clinical samples. Furthermore, we discovered that LINC01133 induced this process through enhancing the mRNA stability of FSP1. CEBPB was the transcription factor to increase the expression of LINC01133. A higher CEBPB could also indicate a higher IC50 of sorafenib in patients with cancer. Moreover, we confirmed that LINC01133 could form a triple complex with FUS and FSP1 to increase the mRNA stability of FSP1.


Assuntos
Ferroptose , Neoplasias Pancreáticas , RNA Longo não Codificante , Humanos , Linhagem Celular Tumoral , Ferroptose/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Estabilidade de RNA/genética , RNA Longo não Codificante/genética , Proteína FUS de Ligação a RNA/metabolismo , Sorafenibe/farmacologia
18.
Methods Enzymol ; 691: 127-152, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37914443

RESUMO

RNA decay serves as a crucial mechanism for maintaining cellular homeostasis and regulating gene expression. Large-scale analyses indicate that altered rates of decay contribute significantly to changes in mRNA levels, with up to half of these changes attributed to decay. The regulation of RNA decay is, at least in part, through structured RNA elements, especially in the non-coding regions of the mRNAs. The development of next-generation sequencing, and in vivo chemical probing techniques has allowed for unprecedented understanding of RNA folding in vivo and genome-wide. To explore the RNA structure elements that are responsible for RNA cleavage, we need to capture the RNA structure before cleavage. In this method, we introduce a new experimental procedure called CAP-STRUCTURE-seq, a modified STRUCTURE-Seq approach combining with the enrichment of in intact mRNAs by the use of terminator exonuclease treatment (5'-Phosphate-Dependent Exonuclease) that digests RNA containing 5-monophosphate ends. This approach is designed to investigate the RNA structure for these intact RNAs, providing a means to study the impact of RNA structure on RNA decay in greater detail. This method can provide insights into the function of RNA structure in RNA decay and help advance our understanding of biological processes.


Assuntos
Exonucleases , RNA , RNA/genética , RNA/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Exonucleases/metabolismo , Estabilidade de RNA , Análise de Sequência de RNA/métodos
19.
Commun Biol ; 6(1): 1009, 2023 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-37794162

RESUMO

Regulated Ire1-dependent decay (RIDD) is a feedback mechanism in which the endoribonuclease Ire1 cleaves endoplasmic reticulum (ER)-localized mRNAs encoding secretory and membrane proteins in eukaryotic cells under ER stress. RIDD is artificially induced by chemicals that generate ER stress; however, its importance under physiological conditions remains unclear. Here, we demonstrate the occurrence of RIDD in filamentous fungus using Aspergillus oryzae as a model, which secretes copious amounts of amylases. α-Amylase mRNA was rapidly degraded by IreA, an Ire1 ortholog, depending on its ER-associated translation when mycelia were treated with dithiothreitol, an ER-stress inducer. The mRNA encoding maltose permease MalP, a prerequisite for the induction of amylolytic genes, was also identified as an RIDD target. Importantly, RIDD of malP mRNA is triggered by inducing amylase production without any artificial ER stress inducer. Our data provide the evidence that RIDD occurs in eukaryotic microorganisms under physiological ER stress.


Assuntos
Amilases , Aspergillus oryzae , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Estabilidade de RNA , RNA Mensageiro/metabolismo
20.
Am J Physiol Gastrointest Liver Physiol ; 325(6): G518-G527, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37788332

RESUMO

Gut barrier dysfunction occurs commonly in patients with critical disorders, leading to the translocation of luminal toxic substances and bacteria to the bloodstream. Connexin 43 (Cx43) acts as a gap junction protein and is crucial for intercellular communication and the diffusion of nutrients. The levels of cellular Cx43 are tightly regulated by multiple factors, including polyamines, but the exact mechanism underlying the control of Cx43 expression remains largely unknown. The RNA-binding protein HuR regulates the stability and translation of target mRNAs and is involved in many aspects of intestinal epithelial pathobiology. Here we show that HuR directly bound to Cx43 mRNA via its 3'-untranslated region in intestinal epithelial cells (IECs) and this interaction enhanced Cx43 expression by stabilizing Cx43 mRNA. Depletion of cellular polyamines inhibited the [HuR/Cx43 mRNA] complex and decreased the level of Cx43 protein by destabilizing its mRNA, but these changes were prevented by ectopic overexpression of HuR. Polyamine depletion caused intestinal epithelial barrier dysfunction, which was reversed by ectopic Cx43 overexpression. Moreover, overexpression of checkpoint kinase 2 in polyamine-deficient cells increased the [HuR/Cx43 mRNA] complex, elevated Cx43 levels, and promoted barrier function. These findings indicate that Cx43 mRNA is a novel target of HuR in IECs and that polyamines regulate Cx43 mRNA stability via HuR, thus playing a critical role in the maintenance of intestinal epithelial barrier function.NEW & NOTEWORTHY The current study shows that polyamines stabilize the Cx43 mRNA via HuR, thus enhancing the function of the Cx43-mediated gap junction. These findings suggest that induced Cx43 by HuR plays a critical role in the process by which polyamines regulate intestinal epithelial barrier.


Assuntos
Conexina 43 , Proteína Semelhante a ELAV 1 , Poliaminas , RNA Mensageiro , Humanos , Conexina 43/genética , Conexina 43/metabolismo , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Mucosa Intestinal/metabolismo , Poliaminas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estabilidade de RNA
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