Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA v...Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.展开更多
Gene therapy appears as a promising strategy to treatincurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites(IRESs), RNA elements naturally pre...Gene therapy appears as a promising strategy to treatincurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites(IRESs), RNA elements naturally present in the 5' untranslated regions of a few m RNAs, constitute a powerful tool to co-express several genes of interest. IRESs are translational enhancers allowing the translational machinery to start protein synthesis by internal initiation. This feature allowed the design of multi-cistronic vectors expressing several genes from a single m RNA. IRESs exhibit tissue specificity, and drive translation in stress conditions when the global cell translation is blocked, which renders them useful for gene transfer in hypoxic conditions occurring in ischemic diseases and cancer. IRES-based viral and non viral vectors have been used successfully in preclinical and clinical assays of combined gene therapy and resulted in therapeutic benefits for various pathologies including cancers, cardiovascular diseases and degenerative diseases.展开更多
Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for han...Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.展开更多
The decoding release factor （RF） triggers termination of protein synthesis by functionally mimicking a tRNA to span the decoding centre and the peptidyl transferase centre （PTC） of the ribosome. Structurally, it m...The decoding release factor （RF） triggers termination of protein synthesis by functionally mimicking a tRNA to span the decoding centre and the peptidyl transferase centre （PTC） of the ribosome. Structurally, it must fit into a site crafted for a tRNA and surrounded by five other RNAs, namely the adjacent peptidyl tRNA carrying the completed polypeptide, the mRNA and the three rRNAs. This is achieved by extending a structural domain from the body of the protein that results in a critical conformational change allowing it to contact the PTC. A structural model of the bacterial termination complex with the accommodated RF shows that it makes close contact with the first, second and third bases of the stop codon in the mRNA with two separate loops of structure＂ the anticodon loop and the loop at the tip of helix orS. The anticodon loop also makes contact with the base following the stop codon that is known to strongly influence termination efficiency. It confirms the close contact of domain 3 of the protein with the key RNA structures of the PTC. The mRNA signal for termination includes sequences upstream as well as downstream of the stop codon, and this may reflect structural restrictions for specific combinations of tRNA and RF to be bound onto the ribosome together. An unbiased SELEX approach has been investigated as a tool to identify potential rRNA-binding contacts of the bacterial RF in its different binding conformations within the active centre of the ribosome.展开更多
Genomic DNA for Jatropha curcas ribosome inactivating protein （JRIP） was cloned from total DNA of its leaves by polymerase chain reaction （PCR）. The no intron character was confirmed. The plant expression vector p...Genomic DNA for Jatropha curcas ribosome inactivating protein （JRIP） was cloned from total DNA of its leaves by polymerase chain reaction （PCR）. The no intron character was confirmed. The plant expression vector pBI121-JRIP was constructed by inserting the JRIP gene into pBI121 plasmid. The recombinant Agrobacterium EHA105 strain harboring pBI121-JRIP was constructed by conducting pBI121-JRIP to strain EHA 105. PCR and Southern blotting were carried out, and the results proved that the JRIP gene was integrated into tobacco genome. It might provide a new material for disease resistance tobacco species breeding.展开更多
In the present experiments the changes in levels of ribosome,polysome and 3H-leucine incorporation rate in liver post-mitochondrial supernatant (PM-supernatant) were investigated in Sedericient and Se-supplented rats....In the present experiments the changes in levels of ribosome,polysome and 3H-leucine incorporation rate in liver post-mitochondrial supernatant (PM-supernatant) were investigated in Sedericient and Se-supplented rats.The results demonstrated that the amounts of ribosome and polysome as well as the ratio of polysome to ribosome in liver PM-supernatant from the Se-deficient rats were all remarkahly decreased.In the meantime,the rate of protein synthesis expressed as radioactivity or 3H-leucine incorporated into protein in the PM-supernatant system also decreased significantly.The results suggest that the decreases of ribosomes and proportion of ribosomal aggregates in PM-supernatant may be responsible for the decrease of the protein synthesis activity in liver of the Se-deficient animals.展开更多
AIM: To investigate the subcellular localization and the function of mouse transducin β-like 3(Tbl3).METHODS: The coding sequence of mouse Tbl3 was cloned from the c DNAs of a promyelocyte cell line by reverse transc...AIM: To investigate the subcellular localization and the function of mouse transducin β-like 3(Tbl3).METHODS: The coding sequence of mouse Tbl3 was cloned from the c DNAs of a promyelocyte cell line by reverse transcription-polymerase chain reaction. Fusion constructs of Tbl3 and enhanced green fluorescent protein(EGFP) were transfected into fibroblasts and examined by fluorescence microscopy to reveal the subcellular localization of tbl3. To search for nucleolar targeting sequences, scanning deletions of Tbl3-EGFP were constructed and transfected into fibroblasts. To explore the possible function of Tbl3, small hairpin RNAs(sh RNAs) were used to knock down endogenous Tbl3 in mouse promyelocytes and fibroblasts. The effects of Tbl3 knockdown on ribosomal RNA(r RNAs) synthesis or processing were studied by labeling cells with 5,6-3H-uridine followed by a chase with fresh medium for various periods. Total RNAs were purified from treated cells and subjected to gel electrophoresis and Northern analysis. Ribosome profiling by sucrose gradient centrifugation was used to compare the amounts of 40 S and 60 S ribosome subunits as well as the 80 S monosome. The impact of Tbl3 knockdown on cell growth and proliferation was examined by growth curves and colony assays.RESULTS: The largest open reading frame of mouse Tbl3 encodes a protein of 801 amino acids(AA) with an apparent molecular weight of 89-90 kilodalton. It contains thirteen WD40 repeats(an ancient protein-protein interaction motif) and a carboxyl terminus that is highly homologous to the corresponding region of the yeast nucleolar protein, utp13. Virtually nothing is known about the biological function of Tbl3. All cell lines surveyed expressed Tbl3 and the level of expression correlated roughly with cell proliferation and/or biosynthetic activity. Using Tbl3-EGFP fusion constructs we obtained the first direct evidence that Tbl3 is targeted to the nucleoli in mammalian cells. However, no previously described nucleolar targeting sequences were found in Tbl3, suggesting that the WD40 motif and/or other topological features are responsible for nucleolar targeting. Partial knockdown(by 50%-70%) of mouse Tbl3 by shR NA had no discernable effects on the processing of the 47 S pre-ribosomal RNA(pre-r RNA) or the steady-state levels of the mature 28 S, 18 S and 5.8S r RNAs but consistently increased the expression level of the 47 S pre-rR NA by two to four folds. The results of the current study corroborated the previous finding that there was no detectable rR NA processing defects in zebra fish embryos with homozygous deletions of zebra fish Tbl3. As ribosome production consumes the bulk of cellular energy and biosynthetic precursors, dysregulation of pre-rR NA synthesis can have negative effects on cell growth, proliferation and differentiation. Indeed, partial knockdown of Tbl3 in promyelocytes severely impaired their proliferation. The inhibitory effect of Tbl3 knockdown was also observed in fibroblasts, resulting in an 80% reduction in colony formation. Taken together, these results indicate that Tbl3 is a newly recognized nucleolar protein with regulatory roles at very early stages of ribosome biogenesis, perhaps at the level of rR NA gene transcription. CONCLUSION: Tbl3 is a newly recognized nucleolar protein with important regulatory roles in ribosome biogenesis.展开更多
Pathogen effectors target diverse subcellular organelles to manipulate the plant immune system.Although the nucleolus has emerged as a stress marker and several effectors are localized in the nucleolus,the roles of nu...Pathogen effectors target diverse subcellular organelles to manipulate the plant immune system.Although the nucleolus has emerged as a stress marker and several effectors are localized in the nucleolus,the roles of nucleolar-targeted effectors remain elusive.In this study,we showed that Phytophthora infestans infection of Nicotiana benthamiana results in nucleolar inflation during the transition from the biotrophic to the necrotrophic phase.Multiple P.infestans effectors were localized in the nucleolus:Pi23226 induced cell death in N.benthamiana and nucleolar inflation similar to that observed in the necrotrophic stage of infection,whereas its homolog Pi23015 and a deletion mutant(Pi23226DC)did not induce cell death or affect nucleolar size.RNA immunoprecipitation and individual-nucleotide-resolution UV crosslinking and immunoprecipitation sequencing analysis indicated that Pi23226 bound to the 30 end of 25S rRNA precursors,resulting in accumulation of unprocessed 27S pre-rRNAs.The nucleolar stress marker NAC082 was strongly upregulated under Pi23226-expressing conditions.Pi23226 subsequently inhibited global protein translation in host cells by interacting with ribosomes.Pi23226 enhanced P.infestans pathogenicity,indicating that Pi23226-induced ribosome malfunction and cell death were beneficial for pathogenesis in the host.Our results provide evidence for the molecular mechanism underlying RNA-binding effector activity in host ribosome biogenesis and lead to new insights into the nucleolar action of effectors in pathogenesis.展开更多
Carrimycin is a synthetic macrolide antibiotic that has been shown to have anti-cancer activity;however,its exact mechanism of action and molecular target were previously unknown.It was recently elucidated that Isoval...Carrimycin is a synthetic macrolide antibiotic that has been shown to have anti-cancer activity;however,its exact mechanism of action and molecular target were previously unknown.It was recently elucidated that Isovalerylspiramycin I(ISP I),the active component of carrimycin,targets selenoprotein H(SelH),a nucleolar reactive oxygen species-scavenging enzyme in the selenoprotein family.ISP I treatment accelerates SelH degradation,resulting in oxidative stress,disrupted ribosomal biogenesis,and apoptosis in tumor cells.Specifically,ISP I disrupts the association between RNA polymerase I and ribosomal DNA in the nucleolus.This inhibits ribosomal RNA transcription and subsequent ribosomal assembly,which prevents cancer cells from sustaining elevated rates of protein synthesis and cellular proliferation that are necessary for tumor growth and malignancy.In this review,we(1)describe the historical categorization and evolution of anti-cancer agents,including macrolide antibiotics,(2)outline the discovery of SelH as a target of ISP I,and(3)summarize the ways in which carrimycin has been used both clinically and at the bench to date and propose additional potential therapeutic uses.展开更多
Ribosomopathies encompass a spectrum of disorders arising from impaired ribosome biogenesis and reduced functionality.Mutation or dysexpression of the genes that disturb any finely regulated steps of ribosome biogenes...Ribosomopathies encompass a spectrum of disorders arising from impaired ribosome biogenesis and reduced functionality.Mutation or dysexpression of the genes that disturb any finely regulated steps of ribosome biogenesis can result in different types of ribosomopathies in clinic,collectively known as ribosomopathy genes.Emerging data suggest that ribosomopathy patients exhibit a significantly heightened susceptibility to cancer.Abnormal ribosome biogenesis and dysregulation of some ribosomopathy genes have also been found to be intimately associated with cancer development.The correlation between ribosome biogenesis or ribosomopathy and the development of malignancies has been well established.This work aims to review the recent advances in the research of ribosomopathy genes among human cancers and meanwhile,to excavate the potential role of these genes,which have not or rarely been reported in cancer,in the disease development across cancers.We plan to establish a theoretical framework between the ribosomopathy gene and cancer development,to further facilitate the potential of these genes as diagnostic biomarker as well as pharmaceutical targets for cancer treatment.展开更多
The roles of concealed microproteins encoded by long noncoding RNAs(lncRNAs)are gradually being exposed,but their functions in tumorigenesis are still largely unclear.Here,we identify and characterize a conserved 99-a...The roles of concealed microproteins encoded by long noncoding RNAs(lncRNAs)are gradually being exposed,but their functions in tumorigenesis are still largely unclear.Here,we identify and characterize a conserved 99-amino acid microprotein named KRASIM that is encoded by the putative lncRNA NCBP2-AS2.KRASIM is differentially expressed in normal hepatocytes and hepatocellular carcinoma(HCC)cells and can suppress HCC cell growth and proliferation.Mechanistically,KRASIM interacts and colocalizes with the KRAS protein in the cytoplasm of human HuH-7 hepatoma cells.More importantly,the overexpression of KRASIM decreases the KRAS protein level,leading to the inhibition of ERK signaling activity in HCC cells.These results demonstrate a novel microprotein repressor of the KRAS pathway for the first time and provide new insights into the regulatory mechanisms of oncogenic signaling and HCC therapy.展开更多
Dear Editor,Streptomyces can produce a large variety of secondary metabolites as a major source of anti-infective, antitumor or immune-suppressive agents widely applied in clinical treatment. Antibiotics-resistant bac...Dear Editor,Streptomyces can produce a large variety of secondary metabolites as a major source of anti-infective, antitumor or immune-suppressive agents widely applied in clinical treatment. Antibiotics-resistant bacteria are spreading at alarming rates.展开更多
A new ribosome-inactivating protein （RIP） with a molecular weight of 31 kDa induced by Cinchonaglycoside C （1） designated CIP31, was isolated from tobacco leaves. Analysis of this protein sequence indicated that i...A new ribosome-inactivating protein （RIP） with a molecular weight of 31 kDa induced by Cinchonaglycoside C （1） designated CIP31, was isolated from tobacco leaves. Analysis of this protein sequence indicated that it belongs to the RIP family and it was distinct from the other plant RIPs reported previously at its N-terminal amino acid sequence. CIP31 can directly impair synthesis of coat protein （CP） of tobacco mosaic virus （TMV）, which resulted in inhibition of TMV long distance movement and multiplication in tobacco plants at concentrations of ng/mL. Furthermore, no toxicity was shown to the growth and fertility of the plants. CIP31 was synthesized only in the presence of Cinchonaglycoside C （1） and was independent of the salicylic acid （SA） signal pathway. We provided evidence for the SA-independent biological induction of resistance.展开更多
In eukaryotic cells, initiation of protein translation is to recruit the ribosome to a specific mRNA, which is generally dependent on the 5＇ cap structure. However, protein translation can also be initiated in a cap-...In eukaryotic cells, initiation of protein translation is to recruit the ribosome to a specific mRNA, which is generally dependent on the 5＇ cap structure. However, protein translation can also be initiated in a cap-independent manner by using a cis-regulatory element termed the internal ribosome entry site （IRES）. The first experimentally validated IRES was reported in the poliovirus （Pelletier and Sonenberg, 1988）. Then eukaryotic cellular mRNAs were also validated to contain IRES elements.展开更多
Ribosome biogenesis is essential for the cell growth and division. Disruptions in ribosome biogenesis result in developmental defects and a group of diseases, known as ribosomopathies. Here, we report a mutation in ze...Ribosome biogenesis is essential for the cell growth and division. Disruptions in ribosome biogenesis result in developmental defects and a group of diseases, known as ribosomopathies. Here, we report a mutation in zebrafish urb1, which encodes an essential ribosome biogenesis protein. The urb1 cq31 mutant exhibits hypoplastic digestive organs, which is caused by impaired cell proliferation with the differentiation of digestive organ progenitors unaffected. Knockdown of mtor or raptor leads to similar hypoplastic phenotypes and reduced expression of urb1 in the digestive organs. Overexpression of Urb1 results in overgrowth of digestive organs, and can efficiently rescue the hypoplastic liver and pancreas in the mtor and raptor morphants. Reduced syntheses of free ribosomal subunits and impaired assembly of polysomes are observed in the urb1 mutant as well as in the mtor and raptor morphants, which can be rescued by the Urb1 overexpression. These data demonstrate that Urb1 plays an important role in governing ribosome biogenesis and protein synthesis downstream of mammalian/mechanistic target of rapamycin complex 1（mTORC1）, thus regulating the development of digestive organs. Our study indicates the requirement of hyperactive protein synthesis for the digestive organ development.展开更多
Small proteins specifically refer to proteins consisting of less than 100 amino acids translated from small open reading frames(s ORFs),which were usually missed in previous genome annotation.The significance of small...Small proteins specifically refer to proteins consisting of less than 100 amino acids translated from small open reading frames(s ORFs),which were usually missed in previous genome annotation.The significance of small proteins has been revealed in current years,along with the discovery of their diverse functions.However,systematic annotation of small proteins is still insufficient.Sm Prot was specially developed to provide valuable information on small proteins for scientific community.Here we present the update of Sm Prot,which emphasizes reliability of translated s ORFs,genetic variants in translated s ORFs,disease-specific s ORF translation events or sequences,and remarkably increased data volume.More components such as non-ATG translation initiation,function,and new sources are also included.Sm Prot incorporated638,958 unique small proteins curated from 3,165,229 primary records,which were computationally predicted from 419 ribosome profiling(Ribo-seq)datasets or collected from literature and other sources from 370 cell lines or tissues in 8 species(Homo sapiens,Mus musculus,Rattus norvegicus,Drosophila melanogaster,Danio rerio,Saccharomyces cerevisiae,Caenorhabditis elegans,and Escherichia coli).In addition,small protein families identified from human microbiomes were also collected.All datasets in Sm Prot are free to access,and available for browse,search,and bulk downloads at http://bigdata.ibp.ac.cn/SmProt/.展开更多
Ribosome biogenesis in the nucleolus requires numerous nucleolar proteins and small non-coding RNAs.Among them is ribosome biogenesis factor Bmsl,which is highly conserved from yeast to human.In yeast,Bmsl initiates r...Ribosome biogenesis in the nucleolus requires numerous nucleolar proteins and small non-coding RNAs.Among them is ribosome biogenesis factor Bmsl,which is highly conserved from yeast to human.In yeast,Bmsl initiates ribosome biogenesis through recruiting Rcll to pre-ribosomes.However,little is known about the biological function of Bmsl in vertebrates.Here we report that Bmsl plays an essential role in zebrafish liver development.We identified a zebrafish bms1l^（sq163） mutant which carries a T to A mutation in the gene bmsl-like（bms1l）.This mutation results in L^（152） to Q^（152） substitution in a GTPase motif in Bmsll.Surprisingly,bmsll^（sq163） mutation confers hypoplasia specifically in the liver,exocrine pancreas and intestine after 3 days post-fertilization（dpf）.Consistent with the bmsll^（sq163） mutant phenotypes,whole-mount in situ hybridization（WISH） on wild type embryos showed that bmsll transcripts are abundant in the entire digestive tract and its accessory organs.Immunostaining for phospho-Histone 3（P-H3） and TUNEL assay revealed that impairment of hepatoblast proliferation rather than cell apoptosis is one of the consequences of bms1l（sq163） giving rise to an under-developed liver.Therefore,our findings demonstrate that Bmsll is necessary for zebrafish liver development.展开更多
Heat shock response is a classical stress-induced regulatory system in bacteria, character- ized by extensive transcriptional reprogramming. To compare the impact of heat stress on the tran- scriptome and translatome ...Heat shock response is a classical stress-induced regulatory system in bacteria, character- ized by extensive transcriptional reprogramming. To compare the impact of heat stress on the tran- scriptome and translatome in Escherich＆ coli, we conducted ribosome profiling in parallel with RNA-Seq to investigate the alterations in transcription and translation efficiency when E. coli cells were exposed to a mild heat stress （from 30 ~C to 45 ~C）. While general changes in ribosome foot- prints correlate with the changes of mRNA transcripts upon heat stress, a number of genes show differential changes at the transcription and translation levels. Translation efficiency of a few genes that are related to environment stimulus response is up-regulated, and in contrast, some genes func- tioning in mRNA translation and amino acid biosynthesis are down-regulated at the translation level in response to heat stress. Moreover, our ribosome occupancy data suggest that in generalribosomes accumulate remarkably in the starting regions of ORFs upon heat stress. This study pro- vides additional insights into bacterial gene expression in response to heat stress, and suggests the presence of stress-induced but yet-to-be characterized cellular regulatory mechanisms of gene expression at translation level.展开更多
Elucidating protein translational regulation is crucial for understanding cellular function and drug development.A key molecule in protein translation is ribosome,which is a super-molecular complex extensively studied...Elucidating protein translational regulation is crucial for understanding cellular function and drug development.A key molecule in protein translation is ribosome,which is a super-molecular complex extensively studied for more than a half century.The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography,Cryo-EM,and single molecule biophysics.Current studies of the ribosome have shown multiple functional states,each with a unique conformation.In this study,we analyzed the RNA-protein distances of ribosome(2.5 MDa)complexes and compared these changes among different ribosome complexes.We found that the RNA-protein distance is significantly correlated with the ribosomal functional state.Thus,the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions.In particular,the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.展开更多
Bacteria growth depends crucially on protein synthesis,which is limited by ribosome synthesis.Ribosomal RNA(rRNA)transcription is the rate-limiting step of ribosome synthesis.It is generally proposed that the transcri...Bacteria growth depends crucially on protein synthesis,which is limited by ribosome synthesis.Ribosomal RNA(rRNA)transcription is the rate-limiting step of ribosome synthesis.It is generally proposed that the transcriptional initiation rate of rRNA operon is the primary factor that controls the r RNA synthesis.In this study,we established a convenient GFP-based reporter approach for measuring the bacterial rRNA chain elongation rate.We showed that the rRNA chain elongation rate of Escherichia coli remains constant under nutrient limitation and chloramphenicol inhibition.In contrast,rRNA chain elongation rate decreases dramatically under low temperatures.Strikingly,we found that Vibrio natriegens,the fastest growing bacteria known,has a 50%higher rRNA chain elongation rate than E.coli,which contributes to its rapid ribosome synthesis.Our study demonstrates that r RNA chain elongation rate is another important factor that affects the bacterial ribosome synthesis capacity.展开更多
基金Indo-Swiss Joint research Program (ISJRP)#17/2011
文摘Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.
文摘Gene therapy appears as a promising strategy to treatincurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites(IRESs), RNA elements naturally present in the 5' untranslated regions of a few m RNAs, constitute a powerful tool to co-express several genes of interest. IRESs are translational enhancers allowing the translational machinery to start protein synthesis by internal initiation. This feature allowed the design of multi-cistronic vectors expressing several genes from a single m RNA. IRESs exhibit tissue specificity, and drive translation in stress conditions when the global cell translation is blocked, which renders them useful for gene transfer in hypoxic conditions occurring in ischemic diseases and cancer. IRES-based viral and non viral vectors have been used successfully in preclinical and clinical assays of combined gene therapy and resulted in therapeutic benefits for various pathologies including cancers, cardiovascular diseases and degenerative diseases.
文摘Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.
文摘The decoding release factor （RF） triggers termination of protein synthesis by functionally mimicking a tRNA to span the decoding centre and the peptidyl transferase centre （PTC） of the ribosome. Structurally, it must fit into a site crafted for a tRNA and surrounded by five other RNAs, namely the adjacent peptidyl tRNA carrying the completed polypeptide, the mRNA and the three rRNAs. This is achieved by extending a structural domain from the body of the protein that results in a critical conformational change allowing it to contact the PTC. A structural model of the bacterial termination complex with the accommodated RF shows that it makes close contact with the first, second and third bases of the stop codon in the mRNA with two separate loops of structure＂ the anticodon loop and the loop at the tip of helix orS. The anticodon loop also makes contact with the base following the stop codon that is known to strongly influence termination efficiency. It confirms the close contact of domain 3 of the protein with the key RNA structures of the PTC. The mRNA signal for termination includes sequences upstream as well as downstream of the stop codon, and this may reflect structural restrictions for specific combinations of tRNA and RF to be bound onto the ribosome together. An unbiased SELEX approach has been investigated as a tool to identify potential rRNA-binding contacts of the bacterial RF in its different binding conformations within the active centre of the ribosome.
基金Project supported by Tenth Five Years Key Program Foundation of the State Science and Technology Commission of China (GrantNo .2002BA901A15)
文摘Genomic DNA for Jatropha curcas ribosome inactivating protein （JRIP） was cloned from total DNA of its leaves by polymerase chain reaction （PCR）. The no intron character was confirmed. The plant expression vector pBI121-JRIP was constructed by inserting the JRIP gene into pBI121 plasmid. The recombinant Agrobacterium EHA105 strain harboring pBI121-JRIP was constructed by conducting pBI121-JRIP to strain EHA 105. PCR and Southern blotting were carried out, and the results proved that the JRIP gene was integrated into tobacco genome. It might provide a new material for disease resistance tobacco species breeding.
文摘In the present experiments the changes in levels of ribosome,polysome and 3H-leucine incorporation rate in liver post-mitochondrial supernatant (PM-supernatant) were investigated in Sedericient and Se-supplented rats.The results demonstrated that the amounts of ribosome and polysome as well as the ratio of polysome to ribosome in liver PM-supernatant from the Se-deficient rats were all remarkahly decreased.In the meantime,the rate of protein synthesis expressed as radioactivity or 3H-leucine incorporated into protein in the PM-supernatant system also decreased significantly.The results suggest that the decreases of ribosomes and proportion of ribosomal aggregates in PM-supernatant may be responsible for the decrease of the protein synthesis activity in liver of the Se-deficient animals.
基金Supported by In part by a grant from the St.Perres Fund,No.11-02011
文摘AIM: To investigate the subcellular localization and the function of mouse transducin β-like 3(Tbl3).METHODS: The coding sequence of mouse Tbl3 was cloned from the c DNAs of a promyelocyte cell line by reverse transcription-polymerase chain reaction. Fusion constructs of Tbl3 and enhanced green fluorescent protein(EGFP) were transfected into fibroblasts and examined by fluorescence microscopy to reveal the subcellular localization of tbl3. To search for nucleolar targeting sequences, scanning deletions of Tbl3-EGFP were constructed and transfected into fibroblasts. To explore the possible function of Tbl3, small hairpin RNAs(sh RNAs) were used to knock down endogenous Tbl3 in mouse promyelocytes and fibroblasts. The effects of Tbl3 knockdown on ribosomal RNA(r RNAs) synthesis or processing were studied by labeling cells with 5,6-3H-uridine followed by a chase with fresh medium for various periods. Total RNAs were purified from treated cells and subjected to gel electrophoresis and Northern analysis. Ribosome profiling by sucrose gradient centrifugation was used to compare the amounts of 40 S and 60 S ribosome subunits as well as the 80 S monosome. The impact of Tbl3 knockdown on cell growth and proliferation was examined by growth curves and colony assays.RESULTS: The largest open reading frame of mouse Tbl3 encodes a protein of 801 amino acids(AA) with an apparent molecular weight of 89-90 kilodalton. It contains thirteen WD40 repeats(an ancient protein-protein interaction motif) and a carboxyl terminus that is highly homologous to the corresponding region of the yeast nucleolar protein, utp13. Virtually nothing is known about the biological function of Tbl3. All cell lines surveyed expressed Tbl3 and the level of expression correlated roughly with cell proliferation and/or biosynthetic activity. Using Tbl3-EGFP fusion constructs we obtained the first direct evidence that Tbl3 is targeted to the nucleoli in mammalian cells. However, no previously described nucleolar targeting sequences were found in Tbl3, suggesting that the WD40 motif and/or other topological features are responsible for nucleolar targeting. Partial knockdown(by 50%-70%) of mouse Tbl3 by shR NA had no discernable effects on the processing of the 47 S pre-ribosomal RNA(pre-r RNA) or the steady-state levels of the mature 28 S, 18 S and 5.8S r RNAs but consistently increased the expression level of the 47 S pre-rR NA by two to four folds. The results of the current study corroborated the previous finding that there was no detectable rR NA processing defects in zebra fish embryos with homozygous deletions of zebra fish Tbl3. As ribosome production consumes the bulk of cellular energy and biosynthetic precursors, dysregulation of pre-rR NA synthesis can have negative effects on cell growth, proliferation and differentiation. Indeed, partial knockdown of Tbl3 in promyelocytes severely impaired their proliferation. The inhibitory effect of Tbl3 knockdown was also observed in fibroblasts, resulting in an 80% reduction in colony formation. Taken together, these results indicate that Tbl3 is a newly recognized nucleolar protein with regulatory roles at very early stages of ribosome biogenesis, perhaps at the level of rR NA gene transcription. CONCLUSION: Tbl3 is a newly recognized nucleolar protein with important regulatory roles in ribosome biogenesis.
基金supported by National Research Foundation of Korea(NRF)grants funded by the Korean government(MSIT)(NRF-2018R1A5A1023599[SRC],NRF-2021R1A2B5B03001613,and NRF-2019R1C1C1008698).
文摘Pathogen effectors target diverse subcellular organelles to manipulate the plant immune system.Although the nucleolus has emerged as a stress marker and several effectors are localized in the nucleolus,the roles of nucleolar-targeted effectors remain elusive.In this study,we showed that Phytophthora infestans infection of Nicotiana benthamiana results in nucleolar inflation during the transition from the biotrophic to the necrotrophic phase.Multiple P.infestans effectors were localized in the nucleolus:Pi23226 induced cell death in N.benthamiana and nucleolar inflation similar to that observed in the necrotrophic stage of infection,whereas its homolog Pi23015 and a deletion mutant(Pi23226DC)did not induce cell death or affect nucleolar size.RNA immunoprecipitation and individual-nucleotide-resolution UV crosslinking and immunoprecipitation sequencing analysis indicated that Pi23226 bound to the 30 end of 25S rRNA precursors,resulting in accumulation of unprocessed 27S pre-rRNAs.The nucleolar stress marker NAC082 was strongly upregulated under Pi23226-expressing conditions.Pi23226 subsequently inhibited global protein translation in host cells by interacting with ribosomes.Pi23226 enhanced P.infestans pathogenicity,indicating that Pi23226-induced ribosome malfunction and cell death were beneficial for pathogenesis in the host.Our results provide evidence for the molecular mechanism underlying RNA-binding effector activity in host ribosome biogenesis and lead to new insights into the nucleolar action of effectors in pathogenesis.
基金supported in part by the Intramural Program of the NCI and NINDS(National Institutes of Health)and did not receive any specific grant from funding agencies(e.g.,public,commercial,or not-forprofit sectors)outside of the authors'academic institution.
文摘Carrimycin is a synthetic macrolide antibiotic that has been shown to have anti-cancer activity;however,its exact mechanism of action and molecular target were previously unknown.It was recently elucidated that Isovalerylspiramycin I(ISP I),the active component of carrimycin,targets selenoprotein H(SelH),a nucleolar reactive oxygen species-scavenging enzyme in the selenoprotein family.ISP I treatment accelerates SelH degradation,resulting in oxidative stress,disrupted ribosomal biogenesis,and apoptosis in tumor cells.Specifically,ISP I disrupts the association between RNA polymerase I and ribosomal DNA in the nucleolus.This inhibits ribosomal RNA transcription and subsequent ribosomal assembly,which prevents cancer cells from sustaining elevated rates of protein synthesis and cellular proliferation that are necessary for tumor growth and malignancy.In this review,we(1)describe the historical categorization and evolution of anti-cancer agents,including macrolide antibiotics,(2)outline the discovery of SelH as a target of ISP I,and(3)summarize the ways in which carrimycin has been used both clinically and at the bench to date and propose additional potential therapeutic uses.
基金the National Natural Science Foundation of China(Grant No.:82360542)Jiangxi Provincial Natural Science Foundation,China(Grant Nos.:20224BAB214030 and 20224BAB216072)+2 种基金Doctoral Startup Fund of Gannan Medical University,China(Grant Nos.:QD202136 and QD202132)Science and Technology Planning Projects of Fuzhou,China(Grant No.:2021FZR0101)the Natural Science Foundation of Fujian Province,China(Grant No.:2022YZ0104).
文摘Ribosomopathies encompass a spectrum of disorders arising from impaired ribosome biogenesis and reduced functionality.Mutation or dysexpression of the genes that disturb any finely regulated steps of ribosome biogenesis can result in different types of ribosomopathies in clinic,collectively known as ribosomopathy genes.Emerging data suggest that ribosomopathy patients exhibit a significantly heightened susceptibility to cancer.Abnormal ribosome biogenesis and dysregulation of some ribosomopathy genes have also been found to be intimately associated with cancer development.The correlation between ribosome biogenesis or ribosomopathy and the development of malignancies has been well established.This work aims to review the recent advances in the research of ribosomopathy genes among human cancers and meanwhile,to excavate the potential role of these genes,which have not or rarely been reported in cancer,in the disease development across cancers.We plan to establish a theoretical framework between the ribosomopathy gene and cancer development,to further facilitate the potential of these genes as diagnostic biomarker as well as pharmaceutical targets for cancer treatment.
基金This work was supported by the National Key Research and Development Program of China(2017YFA0504400)the National Natural Science Foundation of China(31370791,31671349,31770879)+2 种基金Fundamental Research Funds for the Central Universities(14lgjc18)This research was supported in part by the Guangdong Province Key Laboratory of Computational Science(13lgjc05)the Guangdong Province Computational Science Innovative Research Team(14lgjc18).
文摘The roles of concealed microproteins encoded by long noncoding RNAs(lncRNAs)are gradually being exposed,but their functions in tumorigenesis are still largely unclear.Here,we identify and characterize a conserved 99-amino acid microprotein named KRASIM that is encoded by the putative lncRNA NCBP2-AS2.KRASIM is differentially expressed in normal hepatocytes and hepatocellular carcinoma(HCC)cells and can suppress HCC cell growth and proliferation.Mechanistically,KRASIM interacts and colocalizes with the KRAS protein in the cytoplasm of human HuH-7 hepatoma cells.More importantly,the overexpression of KRASIM decreases the KRAS protein level,leading to the inhibition of ERK signaling activity in HCC cells.These results demonstrate a novel microprotein repressor of the KRAS pathway for the first time and provide new insights into the regulatory mechanisms of oncogenic signaling and HCC therapy.
基金supported by grants from the Ministry of Science and Technology of China (2015CB150600)the National Natural Science Foundation of China (31571281 and 31771378)
文摘Dear Editor,Streptomyces can produce a large variety of secondary metabolites as a major source of anti-infective, antitumor or immune-suppressive agents widely applied in clinical treatment. Antibiotics-resistant bacteria are spreading at alarming rates.
基金the NationaI NaturaI Science Foundation of China(30370957)the NaturaI Science Foundation of Yunnan Province(2003C0061M).
文摘A new ribosome-inactivating protein （RIP） with a molecular weight of 31 kDa induced by Cinchonaglycoside C （1） designated CIP31, was isolated from tobacco leaves. Analysis of this protein sequence indicated that it belongs to the RIP family and it was distinct from the other plant RIPs reported previously at its N-terminal amino acid sequence. CIP31 can directly impair synthesis of coat protein （CP） of tobacco mosaic virus （TMV）, which resulted in inhibition of TMV long distance movement and multiplication in tobacco plants at concentrations of ng/mL. Furthermore, no toxicity was shown to the growth and fertility of the plants. CIP31 was synthesized only in the presence of Cinchonaglycoside C （1） and was independent of the salicylic acid （SA） signal pathway. We provided evidence for the SA-independent biological induction of resistance.
基金supported by the grants from National Natural Science Foundation of China (Nos. 61571223 and 61171191)
文摘In eukaryotic cells, initiation of protein translation is to recruit the ribosome to a specific mRNA, which is generally dependent on the 5＇ cap structure. However, protein translation can also be initiated in a cap-independent manner by using a cis-regulatory element termed the internal ribosome entry site （IRES）. The first experimentally validated IRES was reported in the poliovirus （Pelletier and Sonenberg, 1988）. Then eukaryotic cellular mRNAs were also validated to contain IRES elements.
基金supported by the National Key Basic Research Program of China(2015CB942800)the National Natural Science Foundation of China(Nos.31330051 and 31730060)+2 种基金the 111 Program(B14037)the Natural Science Foundation Project of Chongqing(cstc2014jcyj A10088)the Fundamental Research Funds for the Central Universities(XDJK2015B011)
文摘Ribosome biogenesis is essential for the cell growth and division. Disruptions in ribosome biogenesis result in developmental defects and a group of diseases, known as ribosomopathies. Here, we report a mutation in zebrafish urb1, which encodes an essential ribosome biogenesis protein. The urb1 cq31 mutant exhibits hypoplastic digestive organs, which is caused by impaired cell proliferation with the differentiation of digestive organ progenitors unaffected. Knockdown of mtor or raptor leads to similar hypoplastic phenotypes and reduced expression of urb1 in the digestive organs. Overexpression of Urb1 results in overgrowth of digestive organs, and can efficiently rescue the hypoplastic liver and pancreas in the mtor and raptor morphants. Reduced syntheses of free ribosomal subunits and impaired assembly of polysomes are observed in the urb1 mutant as well as in the mtor and raptor morphants, which can be rescued by the Urb1 overexpression. These data demonstrate that Urb1 plays an important role in governing ribosome biogenesis and protein synthesis downstream of mammalian/mechanistic target of rapamycin complex 1（mTORC1）, thus regulating the development of digestive organs. Our study indicates the requirement of hyperactive protein synthesis for the digestive organ development.
基金supported by the National Key R&D Program of China(Grant No.2016YFC0901702)National Natural Science Foundation of China(Grant Nos.81902519,91940306,31871294,31701117,and 31970647)+4 种基金the National Key R&D Program of China(Grant Nos.2017YFC0907503,2016YFC0901002,and 2018YFA0106901)the Strategic Priority Research Program of Chinese Academy of Sciences(Grant No.XDB38040300)the 13th Five-year Informatization Plan of Chinese Academy of Sciences(Grant No.XXH13505-05)Special Investigation on Science and Technology Basic Resources,Ministry of Science and Technology,China(Grant No.2019FY100102)the National Genomics Data Center,China。
文摘Small proteins specifically refer to proteins consisting of less than 100 amino acids translated from small open reading frames(s ORFs),which were usually missed in previous genome annotation.The significance of small proteins has been revealed in current years,along with the discovery of their diverse functions.However,systematic annotation of small proteins is still insufficient.Sm Prot was specially developed to provide valuable information on small proteins for scientific community.Here we present the update of Sm Prot,which emphasizes reliability of translated s ORFs,genetic variants in translated s ORFs,disease-specific s ORF translation events or sequences,and remarkably increased data volume.More components such as non-ATG translation initiation,function,and new sources are also included.Sm Prot incorporated638,958 unique small proteins curated from 3,165,229 primary records,which were computationally predicted from 419 ribosome profiling(Ribo-seq)datasets or collected from literature and other sources from 370 cell lines or tissues in 8 species(Homo sapiens,Mus musculus,Rattus norvegicus,Drosophila melanogaster,Danio rerio,Saccharomyces cerevisiae,Caenorhabditis elegans,and Escherichia coli).In addition,small protein families identified from human microbiomes were also collected.All datasets in Sm Prot are free to access,and available for browse,search,and bulk downloads at http://bigdata.ibp.ac.cn/SmProt/.
基金supported by the grants from the National Natural Science Foundation of China(NSFC)(No.31171391) to LJLan NSFC grant(No. 30825025) to JRP and a grant from the National Research Foundation of Singapore(R-154-000-529-281) to YHH
文摘Ribosome biogenesis in the nucleolus requires numerous nucleolar proteins and small non-coding RNAs.Among them is ribosome biogenesis factor Bmsl,which is highly conserved from yeast to human.In yeast,Bmsl initiates ribosome biogenesis through recruiting Rcll to pre-ribosomes.However,little is known about the biological function of Bmsl in vertebrates.Here we report that Bmsl plays an essential role in zebrafish liver development.We identified a zebrafish bms1l^（sq163） mutant which carries a T to A mutation in the gene bmsl-like（bms1l）.This mutation results in L^（152） to Q^（152） substitution in a GTPase motif in Bmsll.Surprisingly,bmsll^（sq163） mutation confers hypoplasia specifically in the liver,exocrine pancreas and intestine after 3 days post-fertilization（dpf）.Consistent with the bmsll^（sq163） mutant phenotypes,whole-mount in situ hybridization（WISH） on wild type embryos showed that bmsll transcripts are abundant in the entire digestive tract and its accessory organs.Immunostaining for phospho-Histone 3（P-H3） and TUNEL assay revealed that impairment of hepatoblast proliferation rather than cell apoptosis is one of the consequences of bms1l（sq163） giving rise to an under-developed liver.Therefore,our findings demonstrate that Bmsll is necessary for zebrafish liver development.
基金supported by the National Natural Science Foundation of China(Grant Nos.31630087,31422016,and 31470722 to NGGrant Nos.31671381 and 91540109 to XY)
文摘Heat shock response is a classical stress-induced regulatory system in bacteria, character- ized by extensive transcriptional reprogramming. To compare the impact of heat stress on the tran- scriptome and translatome in Escherich＆ coli, we conducted ribosome profiling in parallel with RNA-Seq to investigate the alterations in transcription and translation efficiency when E. coli cells were exposed to a mild heat stress （from 30 ~C to 45 ~C）. While general changes in ribosome foot- prints correlate with the changes of mRNA transcripts upon heat stress, a number of genes show differential changes at the transcription and translation levels. Translation efficiency of a few genes that are related to environment stimulus response is up-regulated, and in contrast, some genes func- tioning in mRNA translation and amino acid biosynthesis are down-regulated at the translation level in response to heat stress. Moreover, our ribosome occupancy data suggest that in generalribosomes accumulate remarkably in the starting regions of ORFs upon heat stress. This study pro- vides additional insights into bacterial gene expression in response to heat stress, and suggests the presence of stress-induced but yet-to-be characterized cellular regulatory mechanisms of gene expression at translation level.
基金partially supported by National Institute of Health(R21/R33-GM078601 and R01-GM100701)National Science Foundation(MCB-1151343)in the US
文摘Elucidating protein translational regulation is crucial for understanding cellular function and drug development.A key molecule in protein translation is ribosome,which is a super-molecular complex extensively studied for more than a half century.The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography,Cryo-EM,and single molecule biophysics.Current studies of the ribosome have shown multiple functional states,each with a unique conformation.In this study,we analyzed the RNA-protein distances of ribosome(2.5 MDa)complexes and compared these changes among different ribosome complexes.We found that the RNA-protein distance is significantly correlated with the ribosomal functional state.Thus,the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions.In particular,the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.
基金the National Natural Science Foundation of China(31700089,31700039,31870028 and 31970027)self-determined research funds of CCNU from the colleges’basic research and operation of MOE(CCNU18KFY01,CCNU19TS028 and CCNU20TS023)。
文摘Bacteria growth depends crucially on protein synthesis,which is limited by ribosome synthesis.Ribosomal RNA(rRNA)transcription is the rate-limiting step of ribosome synthesis.It is generally proposed that the transcriptional initiation rate of rRNA operon is the primary factor that controls the r RNA synthesis.In this study,we established a convenient GFP-based reporter approach for measuring the bacterial rRNA chain elongation rate.We showed that the rRNA chain elongation rate of Escherichia coli remains constant under nutrient limitation and chloramphenicol inhibition.In contrast,rRNA chain elongation rate decreases dramatically under low temperatures.Strikingly,we found that Vibrio natriegens,the fastest growing bacteria known,has a 50%higher rRNA chain elongation rate than E.coli,which contributes to its rapid ribosome synthesis.Our study demonstrates that r RNA chain elongation rate is another important factor that affects the bacterial ribosome synthesis capacity.
