Pseudouridine(Ψ),a crucial modified nucleoside in RNA,holds significant application value in biomedical fields.However,its industrial-scale production still faces challenges because of the low efficiency and the high...Pseudouridine(Ψ),a crucial modified nucleoside in RNA,holds significant application value in biomedical fields.However,its industrial-scale production still faces challenges because of the low efficiency and the high costs.In this study,a Ψ high-producing strain was constructed from the E.coli BL21(DE3)through metabolic engineering and dynamic regulation strategies.To do this,the key enzymes,i.e.,pseudouridine 5′-monophosphate glycosidase(Ψ MP glycosidase)and phosphatase,were firstly optimized to obtain the best co-expression system for Ψ biosynthesis,and the results indicated that co-expression of Ψ MP glycosidase(i.e.,EcPsuG)and phosphatase(EcYjjG)from E.coli showed the highest Ψ production and the resultant strain Tran-B4 produced 1.09±0.035 g/L of Ψ.Subsequently,the supply of R5P was enhanced by overexpressing the rate-limiting enzymes in the PP pathway and dynamically regulating the expression of pgi gene using a growth-phase-dependent promoter(i.e.,P_(fliC)),and the resultant strain Tran-D4 produced 2.35±0.085 g/L of Ψ.Lastly,the transmembrane transports and the competitive metabolic pathway were rationally modified to increase Ψ production,and the resultant strain Tran-F2 produced 2.81±0.098 g/L of Ψ in shake-flask fermentation.The final strain Tran-F2 could produce 29.5 g/L of Ψ with a productivity of 0.670 g/(L·h)and the molar conversion ratio of uracil was 90.4%in fed-batch culture.As far as we know,this is the best efficiency of Ψ production using glucose and uracil as feedstock.This study provides an efficient and cost-effective biosynthetic strategy for the Ψ production in industry.展开更多
Colorectal cancer(CRC)is a prevalent malignancy characterized by rising incidence and mortality rates worldwide.The involvement of RNA pseudouridine synthase enzymes in CRC development is well-documented,yet the trans...Colorectal cancer(CRC)is a prevalent malignancy characterized by rising incidence and mortality rates worldwide.The involvement of RNA pseudouridine synthase enzymes in CRC development is well-documented,yet the transcriptomic profile of pseudouridine(Ψ)in CRC remains largely unexplored.This study explored the transcriptomic landscape of Ψ in CRC by analyzing 21 biopsies and their corresponding adjacent healthy tissues,along with blood samples from 10 patients using PRAISE,a quantitative pseudouridine sequencing technology at base resolution.Our findings revealed significant differences in Ψ distribution and levels between CRC samples and adjacent normal tissues,correlating with the expression levels of DKC1,PUS7L,and PUS10.Notably,distinct Ψ levels of sno RNAs could serve as potential tumor biomarkers.Furthermore,we assessed the clinical utility of Ψ in both tumor and blood samples.Differentiated Ψ levels showed promising correlations with clinical markers such as cancer antigen 125,cancer antigen 153,and cancer antigen 199 in tumors,whereas Ψ sites in blood revealed enhanced correlations with routine blood indicators like white blood cells(WBCs)and alpha-fetoprotein(AFP).These findings underscore the significant role of RNA Ψ in CRC,providing valuable insights into its potential applications for clinical diagnosis and treatment.展开更多
Spliceosomal RNAs are a family of small nuclear RNAs(snRNAs)that are essential for pre-mRNA splicing.All vertebrate spliceosomal snRNAs are extensively pseudouridylated after transcription.Pseudouridines in spliceosom...Spliceosomal RNAs are a family of small nuclear RNAs(snRNAs)that are essential for pre-mRNA splicing.All vertebrate spliceosomal snRNAs are extensively pseudouridylated after transcription.Pseudouridines in spliceosomal snRNAs are generally clustered in regions that are functionally important during splicing.Many of these modified nucleotides are conserved across species lines.Recent studies have demonstrated that spliceosomal snRNA pseudouridylation is catalyzed by two different mechanisms:an RNA-dependent mechanism and an RNA-independent mechanism.The functions of the pseudouridines in spliceosomal snRNAs(U2 snRNA in particular)have also been extensively studied.Experimental data indicate that virtually all pseudouridines in U2 snRNA are functionally important.Besides the currently known pseudouridines(constitutive modifications),recent work has also indicated that pseudouridylation can be induced at novel positions under stress conditions,thus strongly suggesting that pseudouridylation is also a regulatory modification.展开更多
Urinary and serum pseudouridine concentrations were determined by high-performance liquid chromatography in 80 patients with primary liver cancer, 32 with benign space occupying lesions of the liver, 42 with liver cir...Urinary and serum pseudouridine concentrations were determined by high-performance liquid chromatography in 80 patients with primary liver cancer, 32 with benign space occupying lesions of the liver, 42 with liver cirrhosis and 40 healthy subjects. Their mean urinary and serum pseudouridine levels were 39.2=11.5 nmol / /μmol creatinine and 3.4 ± 1.3 μmol / L, 24.5 = 5.4 nmol / μmol creatinine and 2.5 = 0.5 μmol / L, 22.8 ± 7.8 nmol / μmol creatinine and 2.3 = 0.4 μmol / L, 26.4 ± 4.6 nmol / μmol creatinine and 2.3 = 0.4 μmol / L, respectively. Exceeding the mean plus 2SD of pseudouridine of healthy control was considered as positive value for the diagnosis of primary liver cancer. Thus the positivity of urinary and serum pseudouridine in hepatoma was 71.3% and 70.0%, respectively. The positive rate of combined pseudouridine and alpha-fetoprotein assay was 91.3% in patients with hepatoma. Besides, pseudouridine levels could elevate before positive localization and reduce to normal levels after tumor resection. The results showed that the determination of pseudouridine is of clinical significance in the diagnosis and monitoring of primary liver cancer.展开更多
The functional impact of modifications of cellular RNAs,including m RNAs,mi RNAs and lnc RNAs,is a field of intense study.The role of such modifications in cancer has started to be elucidated.Diverse and sometimes opp...The functional impact of modifications of cellular RNAs,including m RNAs,mi RNAs and lnc RNAs,is a field of intense study.The role of such modifications in cancer has started to be elucidated.Diverse and sometimes opposite effects of RNA modifications have been reported.Some RNA modifications promote,while ot-hers decrease the growth and invasiveness of cancer.The present manuscript reviews the current knowledge on the potential impacts of N6-Methyladenosine,Pse-udouridine,Inosine,2’O-methylation or methylcytidine in cancer’s RNA.It also highlights the remaining qu-estions and provides hints on research avenues and potential therapeutic applications,whereby modulating dynamic RNA modifications may be a new method to treat cancer.展开更多
Pseudouridines(Ψs) are the most abundant and highly conserved modified nucleotides found in various stable RNAs of all organisms. Most Ψs are clustered in regions that are functionally important for pre-m RNA splici...Pseudouridines(Ψs) are the most abundant and highly conserved modified nucleotides found in various stable RNAs of all organisms. Most Ψs are clustered in regions that are functionally important for pre-m RNA splicing. Ψ has an extra hydrogen bond donor that endows RNA molecules with distinct properties that contribute significantly to RNA-mediated cellular processes. Experimental data indicate that spliceosomal sn RNA pseudouridylation can be catalyzed by both RNA-dependent and RNA-independent mechanisms. Recent work has also demonstrated that pseudouridylation can be induced at novel positions under stress conditions, suggesting a regulatory role for Ψ.展开更多
RNA modifications encompass a series of dynamic chemical changes and editing events on RNA molecules,playing a pivotal role in essential physiological processes such as embryonic development,immune response,and the ma...RNA modifications encompass a series of dynamic chemical changes and editing events on RNA molecules,playing a pivotal role in essential physiological processes such as embryonic development,immune response,and the maintenance of cell homeostasis.By influencing RNA stability,splicing,translation,and intermolecular interactions,RNA modifications serve as crucial mechanisms regulating gene expression at the post-transcriptional level.Dysregulation of the modification machineries or aberrant modification patterns is closely associated with the onset and progression of various diseases,including tumors,metabolic disorders,cardiovascular diseases,and neurological and immune conditions,making them potential biomarkers for disease diagnosis,prognosis,and treatment.In this review,we summarize the molecular mechanisms of major RNA modifications,emphasize their functions in health and disease,and discuss their diagnostic and therapeutic value in pathological contexts.展开更多
The advent of high-throughput sequencing technol- ogies coupled with new detection methods of RNA modifica- tions has enabled investigation of a new layer of gene regulation - the epitranscriptome. With over loo known...The advent of high-throughput sequencing technol- ogies coupled with new detection methods of RNA modifica- tions has enabled investigation of a new layer of gene regulation - the epitranscriptome. With over loo known RNA modifications, understanding the repertoire of RNA modifications is a huge undertaking. This review summarizes what is known about RNA modifications with an emphasis on discoveries in plants. RNA ribose modifications, base methyl- ations and pseudouridylation are required for normal develop- ment in Arabidopsis, as mutations in the enzymes modifying them have diverse effects on plant development and stress responses. These modifications can regulate RNA structure, turnover and translation. Transfer RNA and ribosomal RNA modifications have been mapped extensively and their functions investigated in many organisms, including plants. Recent work exploring the locations, functions and targeting of N6-methyladenosine （m^6A）, 5-methylcytosine （m^5C）, pseudour- idine （up）, and additional modifications in mRNAs and ncRNAs are highlighted, as well as those previously known on tRNAs and rRNAs. Many questions remain as to the exact mechanisms of targeting and functions of specific modified sites and whether these modifications have distinct functions in the different classes of RNAs.展开更多
More than 160 types of post-transcriptional RNA modifications have been reported;there is substantial variation in modification type,abundance,site,and function across species,tissues,and RNA type.The recent developme...More than 160 types of post-transcriptional RNA modifications have been reported;there is substantial variation in modification type,abundance,site,and function across species,tissues,and RNA type.The recent development of high-throughput detection technology has enabled identification of diverse dynamic and reversible RNA modifications,including N6,2′-O-dimethyladenosine(m6Am),N1-methyladenosine(m1A),5-methylcytosine(m5C),N6-methyladenosine(m6A),pseudouridine(Ψ),and inosine(I).In this review,we focus on eukaryotic mRNA modifications.We summarize their biogenesis,regulatory mechanisms,and biological functions,as well as highthroughput methods for detection of mRNA modifications.We also discuss challenges that must be addressed in mRNA modification research.展开更多
基金supported by the National Natural Science Foundation of China(Grant number 32271534)the Class I project of independent innovation of agricultural science and technology in Jiangsu Province(grant numbers CX(22)1012)+1 种基金the Green Bio-fabrication Program of China(Grant number 2021YFC2100900)the Top-Notch Academic Programs Project of Jiangsu Higher Education Institutions,the 111 project(Grant number 111-2-06).
文摘Pseudouridine(Ψ),a crucial modified nucleoside in RNA,holds significant application value in biomedical fields.However,its industrial-scale production still faces challenges because of the low efficiency and the high costs.In this study,a Ψ high-producing strain was constructed from the E.coli BL21(DE3)through metabolic engineering and dynamic regulation strategies.To do this,the key enzymes,i.e.,pseudouridine 5′-monophosphate glycosidase(Ψ MP glycosidase)and phosphatase,were firstly optimized to obtain the best co-expression system for Ψ biosynthesis,and the results indicated that co-expression of Ψ MP glycosidase(i.e.,EcPsuG)and phosphatase(EcYjjG)from E.coli showed the highest Ψ production and the resultant strain Tran-B4 produced 1.09±0.035 g/L of Ψ.Subsequently,the supply of R5P was enhanced by overexpressing the rate-limiting enzymes in the PP pathway and dynamically regulating the expression of pgi gene using a growth-phase-dependent promoter(i.e.,P_(fliC)),and the resultant strain Tran-D4 produced 2.35±0.085 g/L of Ψ.Lastly,the transmembrane transports and the competitive metabolic pathway were rationally modified to increase Ψ production,and the resultant strain Tran-F2 produced 2.81±0.098 g/L of Ψ in shake-flask fermentation.The final strain Tran-F2 could produce 29.5 g/L of Ψ with a productivity of 0.670 g/(L·h)and the molar conversion ratio of uracil was 90.4%in fed-batch culture.As far as we know,this is the best efficiency of Ψ production using glucose and uracil as feedstock.This study provides an efficient and cost-effective biosynthetic strategy for the Ψ production in industry.
基金supported by the National Natural Science Foundation of China(22377096,92153303,21721005,91940000,92253202 and 22177087)the Ministry of Science and Technology of China(2023YFC3402200)+2 种基金China Postdoctoral Science Foundation(2023M732692)Fundamental Research Funds for the Central Universities(2042023kf0118 and 2042023kfyq05)Translational Medicine and Interdisciplinary Research Joint Foundation of Zhongnan Hospital of Wuhan University(ZNJC20222)。
文摘Colorectal cancer(CRC)is a prevalent malignancy characterized by rising incidence and mortality rates worldwide.The involvement of RNA pseudouridine synthase enzymes in CRC development is well-documented,yet the transcriptomic profile of pseudouridine(Ψ)in CRC remains largely unexplored.This study explored the transcriptomic landscape of Ψ in CRC by analyzing 21 biopsies and their corresponding adjacent healthy tissues,along with blood samples from 10 patients using PRAISE,a quantitative pseudouridine sequencing technology at base resolution.Our findings revealed significant differences in Ψ distribution and levels between CRC samples and adjacent normal tissues,correlating with the expression levels of DKC1,PUS7L,and PUS10.Notably,distinct Ψ levels of sno RNAs could serve as potential tumor biomarkers.Furthermore,we assessed the clinical utility of Ψ in both tumor and blood samples.Differentiated Ψ levels showed promising correlations with clinical markers such as cancer antigen 125,cancer antigen 153,and cancer antigen 199 in tumors,whereas Ψ sites in blood revealed enhanced correlations with routine blood indicators like white blood cells(WBCs)and alpha-fetoprotein(AFP).These findings underscore the significant role of RNA Ψ in CRC,providing valuable insights into its potential applications for clinical diagnosis and treatment.
文摘Spliceosomal RNAs are a family of small nuclear RNAs(snRNAs)that are essential for pre-mRNA splicing.All vertebrate spliceosomal snRNAs are extensively pseudouridylated after transcription.Pseudouridines in spliceosomal snRNAs are generally clustered in regions that are functionally important during splicing.Many of these modified nucleotides are conserved across species lines.Recent studies have demonstrated that spliceosomal snRNA pseudouridylation is catalyzed by two different mechanisms:an RNA-dependent mechanism and an RNA-independent mechanism.The functions of the pseudouridines in spliceosomal snRNAs(U2 snRNA in particular)have also been extensively studied.Experimental data indicate that virtually all pseudouridines in U2 snRNA are functionally important.Besides the currently known pseudouridines(constitutive modifications),recent work has also indicated that pseudouridylation can be induced at novel positions under stress conditions,thus strongly suggesting that pseudouridylation is also a regulatory modification.
文摘Urinary and serum pseudouridine concentrations were determined by high-performance liquid chromatography in 80 patients with primary liver cancer, 32 with benign space occupying lesions of the liver, 42 with liver cirrhosis and 40 healthy subjects. Their mean urinary and serum pseudouridine levels were 39.2=11.5 nmol / /μmol creatinine and 3.4 ± 1.3 μmol / L, 24.5 = 5.4 nmol / μmol creatinine and 2.5 = 0.5 μmol / L, 22.8 ± 7.8 nmol / μmol creatinine and 2.3 = 0.4 μmol / L, 26.4 ± 4.6 nmol / μmol creatinine and 2.3 = 0.4 μmol / L, respectively. Exceeding the mean plus 2SD of pseudouridine of healthy control was considered as positive value for the diagnosis of primary liver cancer. Thus the positivity of urinary and serum pseudouridine in hepatoma was 71.3% and 70.0%, respectively. The positive rate of combined pseudouridine and alpha-fetoprotein assay was 91.3% in patients with hepatoma. Besides, pseudouridine levels could elevate before positive localization and reduce to normal levels after tumor resection. The results showed that the determination of pseudouridine is of clinical significance in the diagnosis and monitoring of primary liver cancer.
基金Supported by University of Zurich:"URPP:Translational Cancer research"the"MERIT"project supported by the FP7 European Union’s ResearchInnovation Funding program
文摘The functional impact of modifications of cellular RNAs,including m RNAs,mi RNAs and lnc RNAs,is a field of intense study.The role of such modifications in cancer has started to be elucidated.Diverse and sometimes opposite effects of RNA modifications have been reported.Some RNA modifications promote,while ot-hers decrease the growth and invasiveness of cancer.The present manuscript reviews the current knowledge on the potential impacts of N6-Methyladenosine,Pse-udouridine,Inosine,2’O-methylation or methylcytidine in cancer’s RNA.It also highlights the remaining qu-estions and provides hints on research avenues and potential therapeutic applications,whereby modulating dynamic RNA modifications may be a new method to treat cancer.
基金Supported by Grants from the National Institute of Health to YiTao Yu,No.GM104077 and AG39559by the University of Rochester CTSA award(to Yi-Tao Yu)from the National Center for Advancing Translational Sciences of the National Institute of Health,No.UL1TR000042
文摘Pseudouridines(Ψs) are the most abundant and highly conserved modified nucleotides found in various stable RNAs of all organisms. Most Ψs are clustered in regions that are functionally important for pre-m RNA splicing. Ψ has an extra hydrogen bond donor that endows RNA molecules with distinct properties that contribute significantly to RNA-mediated cellular processes. Experimental data indicate that spliceosomal sn RNA pseudouridylation can be catalyzed by both RNA-dependent and RNA-independent mechanisms. Recent work has also demonstrated that pseudouridylation can be induced at novel positions under stress conditions, suggesting a regulatory role for Ψ.
基金supported by Sichuan Province Science and Technology Support Program(grant No.2022NSFSC0761)Chengdu Medical College Research Foundation(grant No.KYPY22-02)+1 种基金Chengdu Medical College Excellent-talent Program(grant No.2024qnGzn09)Chengdu Medical College Graduate Research Innovation Fund(grant No.YCX2025-01-68).
文摘RNA modifications encompass a series of dynamic chemical changes and editing events on RNA molecules,playing a pivotal role in essential physiological processes such as embryonic development,immune response,and the maintenance of cell homeostasis.By influencing RNA stability,splicing,translation,and intermolecular interactions,RNA modifications serve as crucial mechanisms regulating gene expression at the post-transcriptional level.Dysregulation of the modification machineries or aberrant modification patterns is closely associated with the onset and progression of various diseases,including tumors,metabolic disorders,cardiovascular diseases,and neurological and immune conditions,making them potential biomarkers for disease diagnosis,prognosis,and treatment.In this review,we summarize the molecular mechanisms of major RNA modifications,emphasize their functions in health and disease,and discuss their diagnostic and therapeutic value in pathological contexts.
基金supported by ARC grants DP110103805 and FT13100525 awarded to I.S.and an APA and a GRDC PhD topup scholarship awarded to A.B.
文摘The advent of high-throughput sequencing technol- ogies coupled with new detection methods of RNA modifica- tions has enabled investigation of a new layer of gene regulation - the epitranscriptome. With over loo known RNA modifications, understanding the repertoire of RNA modifications is a huge undertaking. This review summarizes what is known about RNA modifications with an emphasis on discoveries in plants. RNA ribose modifications, base methyl- ations and pseudouridylation are required for normal develop- ment in Arabidopsis, as mutations in the enzymes modifying them have diverse effects on plant development and stress responses. These modifications can regulate RNA structure, turnover and translation. Transfer RNA and ribosomal RNA modifications have been mapped extensively and their functions investigated in many organisms, including plants. Recent work exploring the locations, functions and targeting of N6-methyladenosine （m^6A）, 5-methylcytosine （m^5C）, pseudour- idine （up）, and additional modifications in mRNAs and ncRNAs are highlighted, as well as those previously known on tRNAs and rRNAs. Many questions remain as to the exact mechanisms of targeting and functions of specific modified sites and whether these modifications have distinct functions in the different classes of RNAs.
基金the Ministry of Science and Technology of China(2019YFA0110902,2019YFA0802201)。
文摘More than 160 types of post-transcriptional RNA modifications have been reported;there is substantial variation in modification type,abundance,site,and function across species,tissues,and RNA type.The recent development of high-throughput detection technology has enabled identification of diverse dynamic and reversible RNA modifications,including N6,2′-O-dimethyladenosine(m6Am),N1-methyladenosine(m1A),5-methylcytosine(m5C),N6-methyladenosine(m6A),pseudouridine(Ψ),and inosine(I).In this review,we focus on eukaryotic mRNA modifications.We summarize their biogenesis,regulatory mechanisms,and biological functions,as well as highthroughput methods for detection of mRNA modifications.We also discuss challenges that must be addressed in mRNA modification research.
