Pigeons and certain other avian species produce a milklike secretion in their crop sacs to nourish offspring,yet the detailed processes involved are not fully elucidated.This study investigated the crop sacs of 225-da...Pigeons and certain other avian species produce a milklike secretion in their crop sacs to nourish offspring,yet the detailed processes involved are not fully elucidated.This study investigated the crop sacs of 225-day-old unpaired non-lactating male pigeons(MN)and males initiating lactation on the first day after incubation(ML).Using RNA sequencing,ribosomeprofiling,andsingle-cell transcriptome sequencing(scRNA-seq),we identified a significant up-regulation of genes associated with ribosome assembly and protein synthesis in ML compared to MN.Results from scRNA-seq analysis identified 12distinct cell types and 22 clusters,with secretory epithelial cells(SECs)exhibiting marked expression of plasma cell markers,including IGLL1 and MZB1.RNA fluorescence in situ hybridization(RNA FISH)and IgY quantification confirmed the critical role of SECs in producing endogenous IgY during lactation.We propose that fibroblast-derived BAFF signals activate SECs,mimicking B cell transformation and enhancing protein production through the unfolded protein response(UPR).These findings shed light on the cellular dynamics of pigeon milk production and contribute to a broader understanding of avian biology.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
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
Selenium is a crucial trace element that contributes to physiological processes in the body as selenoproteins.Selenoproteins serve as an integral role in the body in controlling the redox state of cells and protecting...Selenium is a crucial trace element that contributes to physiological processes in the body as selenoproteins.Selenoproteins serve as an integral role in the body in controlling the redox state of cells and protecting against damage induced by oxidative stress.This study aimed to investigate the effects and possible mechanism of selenium on selenoproteins expression in EA.hy926 cells induced by oxidized low density lipoprotein(oxLDL).The impact of selenium on the viability of EA.hy926 cells was detected by the methylthiazolyldiphenyltetrazolium bromide(MTT)method,and intracellular reactive oxygen species(ROS)level and mitochondrial membrane potential were assessed by fluorescent probe DCFH-DA and JC-1,respectively.RNA-seq,quantitative real-time polymerase chain reaction(qPCR),and Western blot were used to investigate the selenoprotein expression.Selenoprotein mRNA translation efficiency was analyzed by ribosome profiling(Ribo-Seq)coupled with transcriptomics.Our data showed that selenium supplementation(0.5μmol/L)significantly decreased ROS production,increased mitochondrial inner membrane potential and increased the proliferative activity of EA.hy926 cells induced by oxLDL.Moreover,The protective effects of selenium against oxLDL-induced EA.hy926 cell injury were associated with the upregulation of the expressions of selenoproteins glutathione peroxidase 1(GPX1),glutathione peroxidase 4(GPX4),and thioredoxin reductase 1(TXNRD1).Furthermore,the expressions of selenoproteins GPX1 and GPX4 were hierarchically controlled,but the expressions of selenoproteins TXNRD1 were mainly regulated by oxLDL.Finally,Ribo-Seq coupled with transcriptomics results demonstrated that the expressions of selenoproteins GPX1,GPX4,and TXNRD1 were regulated at the translation process level.These findings suggested that selenium could have preventive effects in oxLDL induced EA.hy926 cell injury by regulating the selenoprotein expression,and the selenoproteins expressions at the translation level in vascular endothelial cells need further study.展开更多
Natural products play a crucial role in new drug development,but their druggability is often limited by uncertain molecular targets and insufficient research on mechanisms of action.In this study,we developed a new RP...Natural products play a crucial role in new drug development,but their druggability is often limited by uncertain molecular targets and insufficient research on mechanisms of action.In this study,we developed a new RPL19-TRAP^(KI)-seq method,combining CRISPR/Cas9 and TRAP technologies,to investigate these mechanisms.We identified and validated seven ribosomal large subunit surface proteins suitable for TRAP,selecting RPL19 for its high enrichment.We successfully established a stable cell line expressing EGFP-RPL19 using CRISPR knock-in and verified its efficiency and specificity in enriching ribosomes and translating mRNA.Integrated with next-generation sequencing,this method allows precise detection of translating mRNA.We validated RPL19-TRAP^(KI)-seq by investigating rapamycin,an mTOR inhibitor,yielding results consistent with previous reports.This optimized TRAP technology provides an accurate representation of translating mRNA,closely reflecting protein expression levels.Furthermore,we investigated SBF-1,a 23-oxa-analog of natural saponin OSW-1 with significant anti-tumor activity but an unclear mechanism.Using RPL19-TRAP^(KI)-seq,we found that SBF-1 exerts its cytotoxic effects on tumor cells by disturbing cellular oxidative phosphorylation.In conclusion,our method has been proven to be a promising tool that can reveal the mechanisms of small molecules with greater accuracy,setting the stage for future exploration of small molecules and advancing the fields of pharmacology and therapeutic development.展开更多
Conventional peptides(CPs)and non-conventional peptides(NCPs)are generated from small open reading frames,but most CPs are derived from large precursors.NCPs,which are derived from sequences other than conventional op...Conventional peptides(CPs)and non-conventional peptides(NCPs)are generated from small open reading frames,but most CPs are derived from large precursors.NCPs,which are derived from sequences other than conventional open reading frames or annotated coding sequences regions,function in plant development and adaptation to stresses.Ribosome profiling,a technique for studying translational regulation,can be used to identify NCPs.Another new technique,peptidogenomics,which integrates mass spectrometry and genomics,is becoming more widely used for identifying plant NCPs.In recent years,numerous studies have investigated the roles in monocots and dicots of miRNA-derived peptides and upstream open reading frames,which have potential for improving agronomic traits.Investigating the biological functions of NCPs will advance molecular plant breeding by identifying regulators of plant growth and development.We present an overview of NCP identification methods and recent findings about NCP biological functions.展开更多
Background:A key step in gene expression is the recognition of the stop codon to terminate translation at the correct position.However,it has been observed that ribosomes can misinterpret the stop codon and continue t...Background:A key step in gene expression is the recognition of the stop codon to terminate translation at the correct position.However,it has been observed that ribosomes can misinterpret the stop codon and continue the translation in the 3′UTR region.This phenomenon is called stop codon read-through(SCR).It has been suggested that these events would occur on a programmed basis,but the underlying mechanisms are still not well understood.Methods:Here,we present a strategy for the comprehensive identification of SCR events in the Drosophila melanogaster transcriptome by evaluating the ribosomal density profiles.The associated ribosomal leak rate was estimated for every event identified.A statistical characterization of the frequency of nucleotide use in the proximal region to the stop codon in the sequences associated to SCR events was performed.Results:The results show that the nucleotide usage pattern in transcripts with the UGA codon is different from the pattern for those transcripts ending in the UAA codon,suggesting the existence of at least two mechanisms that could alter the translational termination process.Furthermore,a linear regression models for each of the three stop codons was developed,and we show that the models using the nucleotides at informative positions outperforms those models that consider the entire sequence context to the stop codon.Conclusions:We report that distal nucleotides can affect the SCR rate in a stop-codon dependent manner.展开更多
基金supported by the Department of Agriculture and Rural Affairs of Jiangxi Province,China (JXARS-09)Science and Technology Program of Guangdong Province,China (2020B1212060060)。
文摘Pigeons and certain other avian species produce a milklike secretion in their crop sacs to nourish offspring,yet the detailed processes involved are not fully elucidated.This study investigated the crop sacs of 225-day-old unpaired non-lactating male pigeons(MN)and males initiating lactation on the first day after incubation(ML).Using RNA sequencing,ribosomeprofiling,andsingle-cell transcriptome sequencing(scRNA-seq),we identified a significant up-regulation of genes associated with ribosome assembly and protein synthesis in ML compared to MN.Results from scRNA-seq analysis identified 12distinct cell types and 22 clusters,with secretory epithelial cells(SECs)exhibiting marked expression of plasma cell markers,including IGLL1 and MZB1.RNA fluorescence in situ hybridization(RNA FISH)and IgY quantification confirmed the critical role of SECs in producing endogenous IgY during lactation.We propose that fibroblast-derived BAFF signals activate SECs,mimicking B cell transformation and enhancing protein production through the unfolded protein response(UPR).These findings shed light on the cellular dynamics of pigeon milk production and contribute to a broader understanding of avian biology.
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 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supported by grants from the National Natural Science Foundation of China(NSFC,81960588)the Ningxia Natural Science Foundation(2020AAC03146)support from the Ningxia Medical University。
文摘Selenium is a crucial trace element that contributes to physiological processes in the body as selenoproteins.Selenoproteins serve as an integral role in the body in controlling the redox state of cells and protecting against damage induced by oxidative stress.This study aimed to investigate the effects and possible mechanism of selenium on selenoproteins expression in EA.hy926 cells induced by oxidized low density lipoprotein(oxLDL).The impact of selenium on the viability of EA.hy926 cells was detected by the methylthiazolyldiphenyltetrazolium bromide(MTT)method,and intracellular reactive oxygen species(ROS)level and mitochondrial membrane potential were assessed by fluorescent probe DCFH-DA and JC-1,respectively.RNA-seq,quantitative real-time polymerase chain reaction(qPCR),and Western blot were used to investigate the selenoprotein expression.Selenoprotein mRNA translation efficiency was analyzed by ribosome profiling(Ribo-Seq)coupled with transcriptomics.Our data showed that selenium supplementation(0.5μmol/L)significantly decreased ROS production,increased mitochondrial inner membrane potential and increased the proliferative activity of EA.hy926 cells induced by oxLDL.Moreover,The protective effects of selenium against oxLDL-induced EA.hy926 cell injury were associated with the upregulation of the expressions of selenoproteins glutathione peroxidase 1(GPX1),glutathione peroxidase 4(GPX4),and thioredoxin reductase 1(TXNRD1).Furthermore,the expressions of selenoproteins GPX1 and GPX4 were hierarchically controlled,but the expressions of selenoproteins TXNRD1 were mainly regulated by oxLDL.Finally,Ribo-Seq coupled with transcriptomics results demonstrated that the expressions of selenoproteins GPX1,GPX4,and TXNRD1 were regulated at the translation process level.These findings suggested that selenium could have preventive effects in oxLDL induced EA.hy926 cell injury by regulating the selenoprotein expression,and the selenoproteins expressions at the translation level in vascular endothelial cells need further study.
基金supported by the National Key Research and Development Program of China(No.2022YFC2804800 to W.J.)the National Natural Science Foundation of China(No.22494704.,22137002 to Y.D.,92253305 to W.J.and 31971111 to C.L.)+6 种基金the Science and Technology Commission of Shanghai Municipality(Grant 20JC1410900 to Y.D.)the University Innovation Research Group in Chongqing(No.CXQT21016 to Y.D.)the Chongqing Talent Program Project(No.CQYC20200302119 to Y.D.)High-Level Innovation Platform Cultivation Plan of Chongqing(to Y.D.)Joint Fund of the Natural Science Innovation and Development Foundation of Chongqing(to Y.D.)Program for Professor of Special Appointment(Eastern Scholar)at Shanghai Institutions of Higher Learning(to W.J.)Chongqing Doctoral Express Entry Project(No.CSTB2022BSXM-JCX0044 to J.H.).
文摘Natural products play a crucial role in new drug development,but their druggability is often limited by uncertain molecular targets and insufficient research on mechanisms of action.In this study,we developed a new RPL19-TRAP^(KI)-seq method,combining CRISPR/Cas9 and TRAP technologies,to investigate these mechanisms.We identified and validated seven ribosomal large subunit surface proteins suitable for TRAP,selecting RPL19 for its high enrichment.We successfully established a stable cell line expressing EGFP-RPL19 using CRISPR knock-in and verified its efficiency and specificity in enriching ribosomes and translating mRNA.Integrated with next-generation sequencing,this method allows precise detection of translating mRNA.We validated RPL19-TRAP^(KI)-seq by investigating rapamycin,an mTOR inhibitor,yielding results consistent with previous reports.This optimized TRAP technology provides an accurate representation of translating mRNA,closely reflecting protein expression levels.Furthermore,we investigated SBF-1,a 23-oxa-analog of natural saponin OSW-1 with significant anti-tumor activity but an unclear mechanism.Using RPL19-TRAP^(KI)-seq,we found that SBF-1 exerts its cytotoxic effects on tumor cells by disturbing cellular oxidative phosphorylation.In conclusion,our method has been proven to be a promising tool that can reveal the mechanisms of small molecules with greater accuracy,setting the stage for future exploration of small molecules and advancing the fields of pharmacology and therapeutic development.
基金supported by the National Natural Science Foundation of China(31861143004)the Agricultural Science and Technology Innovation Program of CAAS to Wen-Xue Li.
文摘Conventional peptides(CPs)and non-conventional peptides(NCPs)are generated from small open reading frames,but most CPs are derived from large precursors.NCPs,which are derived from sequences other than conventional open reading frames or annotated coding sequences regions,function in plant development and adaptation to stresses.Ribosome profiling,a technique for studying translational regulation,can be used to identify NCPs.Another new technique,peptidogenomics,which integrates mass spectrometry and genomics,is becoming more widely used for identifying plant NCPs.In recent years,numerous studies have investigated the roles in monocots and dicots of miRNA-derived peptides and upstream open reading frames,which have potential for improving agronomic traits.Investigating the biological functions of NCPs will advance molecular plant breeding by identifying regulators of plant growth and development.We present an overview of NCP identification methods and recent findings about NCP biological functions.
基金LIE is funded by CONICET Ph.D.Fellowship.AMA and LD are researchers of CONICET(Argentina).JRR is Full Professor at the UNLP(Argentina).This work was supported by CONICET,Argentina(PIP2017-00059).
文摘Background:A key step in gene expression is the recognition of the stop codon to terminate translation at the correct position.However,it has been observed that ribosomes can misinterpret the stop codon and continue the translation in the 3′UTR region.This phenomenon is called stop codon read-through(SCR).It has been suggested that these events would occur on a programmed basis,but the underlying mechanisms are still not well understood.Methods:Here,we present a strategy for the comprehensive identification of SCR events in the Drosophila melanogaster transcriptome by evaluating the ribosomal density profiles.The associated ribosomal leak rate was estimated for every event identified.A statistical characterization of the frequency of nucleotide use in the proximal region to the stop codon in the sequences associated to SCR events was performed.Results:The results show that the nucleotide usage pattern in transcripts with the UGA codon is different from the pattern for those transcripts ending in the UAA codon,suggesting the existence of at least two mechanisms that could alter the translational termination process.Furthermore,a linear regression models for each of the three stop codons was developed,and we show that the models using the nucleotides at informative positions outperforms those models that consider the entire sequence context to the stop codon.Conclusions:We report that distal nucleotides can affect the SCR rate in a stop-codon dependent manner.
