Senecavirus A(SVA)has a positive-sense,single-stranded RNA genome.Its 5´untranslated region harbors an internal ribosome entry site(IRES),comprising 10 larger or smaller stem-loop structures(including a pseudokno...Senecavirus A(SVA)has a positive-sense,single-stranded RNA genome.Its 5´untranslated region harbors an internal ribosome entry site(IRES),comprising 10 larger or smaller stem-loop structures(including a pseudoknot)that have been demonstrated to be well conserved.However,it is still unclear whether each stem-loop subdomain,such as a single stem or loop,is also highly conserved.To clarify this issue in the present study,a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis(SDM)on the IRES.The SDM-modified scenarios included:(1)stem-formed complementary sequences exchanging with each other;(2)loop transversion;(3)loop transition;and(4)point mutations.All cDNA clones were separately transfected into cells for rescuing viable viruses,whereas only four SVAs of interest could be recovered,and were genetically stable during 20 passages.One progeny grew significantly slower than the other three did.The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity.Our previous study indicated that a single motif from any of the ten stem structures,if completely mutated,would cause the failure of virus recovery.Interestingly,our present study revealed three stem structures,whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs.Moreover,one apical loop was demonstrated to have the ability to tolerate its own full-length transition,also having no impact on the recovery of sequence-modifying SVA.The present study suggested that not every stem-loop structure was strictly conserved in its conformation,while the full-length IRES itself was well conserved.This provides a new research direction on interaction between the IRES and many factors.展开更多
MicroRNAs （miRNAs） usually contain 19-24 nucleotides and have been identified as important eukaryotic gene regulators. Applications of various computational approaches have simplified the task by predicting miRNAs f...MicroRNAs （miRNAs） usually contain 19-24 nucleotides and have been identified as important eukaryotic gene regulators. Applications of various computational approaches have simplified the task by predicting miRNAs from available sequence data sources. In this study, we identified a conserved miR414 from a computational analysis of EST sequence data available from Stevia rebaudiana. In addition, we also identified six conserved miRNAs namely miR169, miR319, miR414, miR164, miR167 and miR398 using stem-loop RT-PCR analysis. Hence, miR414 was commonly identified using both methods. The expression analysis of these miRNAs documented their roles in growth and development of Stevia. Furthermore, the detected miRNAs were found to target genes involved in plant growth, development, metabolism and signal transducfion. This is the first study reporting these conserved miRNAs and their expression in Stevia.展开更多
MicroRNAs are an important subclass of non-coding RNAs (ncRNA), and serve as main players into RNA interference (RNAi). Mature microRNA derived from stem-loop structure called precursor. Identification of precursor mi...MicroRNAs are an important subclass of non-coding RNAs (ncRNA), and serve as main players into RNA interference (RNAi). Mature microRNA derived from stem-loop structure called precursor. Identification of precursor microRNA (pre-miRNA) is essential step to target microRNA in whole genome. The present work proposed 25 novel local features for identifying stem- loop structure of pre-miRNAs, which captures characteristics on both the sequence and structure. Firstly, we pulled the stem of hairpins and aligned the bases in bulges and internal loops used ‘―’, and then counted 24 base-pairs (‘AA’, ‘AU’, …, ‘―G’, except ‘――’) in pulled stem (formalized by length of pulled stem) as features vector of Support Vector Machine (SVM). Performances of three classifiers with our features and different kernels trained on human data were all superior to Triplet-SVM-classifier’s in po- sitive and negative testing data sets. Moreover, we achieved higher prediction accuracy through combining 7 global sequence-structure. The result indicates validity of novel local features.展开更多
To study the relationship between the structures of rice and sorghum psbA 3 UTR and gene expres-sional activity, six chimeric luc genes that encode luciferase under control of rice or sorghum psbA 5 UTR and 3’UTR or ...To study the relationship between the structures of rice and sorghum psbA 3 UTR and gene expres-sional activity, six chimeric luc genes that encode luciferase under control of rice or sorghum psbA 5 UTR and 3’UTR or only 5 UTR were constructed. The levels of LUC accumulation in E . coli and the transcript stability in soluble pro-tein extracts of rice, sorghum chloroplast and E. coli were examined respectively, and a detailed analysis about the function of these two 3’UTR has been carried out. Here the regulation effect of these 3’UTR are reported: ( i ) When having the same promoter, the chimeric genes with rice 3 UTR produce LUC much more than that with sorghum 3 UTR; ( ii ) elimination of 3 UTR results in the fluctuation of LUC accumulation no matter whether it is under control of rice or sorghum psbA 5’UTR; ( Hi ) rice psbA 3’UTR exhibits a greater effect on stabilizing tran-scripts; ( IV ) psbA 3’lR-RNAs are more stable in chloroplast protein extracts than in E. coli protein extracts.展开更多
Two microRNA （miRNA） quantification methods, namely, poly（A） reverse transcription （RT）-quantitative real-time polymerase chain reaction （qPCR） and stem-loop RT-qPCR, have been developed for quantifying miRNA ...Two microRNA （miRNA） quantification methods, namely, poly（A） reverse transcription （RT）-quantitative real-time polymerase chain reaction （qPCR） and stem-loop RT-qPCR, have been developed for quantifying miRNA expression. In the present study, five miRNAs, including miR166, miR167, miR168, miR159, and miR396, with different sequence frequencies, were selected as targets to compare their expression profiles in five wheat tissues by applying the two methods and deep sequencing. The study aimed to determine a simple, reliable and high-throughput method for detecting miRNA expressions in wheat tissues. Results showed that the miRNA expression profiles determined by poly（A） RT-qPCR were more consistent with those obtained by deep sequencing. Further analysis indicated that the correlation coefficients of the data obtained by poly（A） RT-qPCR and deep sequencing （0.739, P≤0.01） were higher than those obtained by stem-loop RT-qPCR and deep sequencing （0.535, P≤0.01）. The protocol used for poly（A） RT-qPCR is simpler than that for stem-loop RT-qPCR. Thus, poly（A） RT-qPCR was a more suitable high-throughput assay for detecting miRNA expression profiles. To the best of our knowledge, this study was the first to compare these two miRNA quantification methods. We also provided useful information for quantifying miRNA in wheat or other plant species.展开更多
Long inverted repeats(LIRs) are evolutionarily and functionally important structures in genomes because of their involvement in RNA interference, DNA recombination, and gene duplication. Identification of LIRs is high...Long inverted repeats(LIRs) are evolutionarily and functionally important structures in genomes because of their involvement in RNA interference, DNA recombination, and gene duplication. Identification of LIRs is highly complicated when mismatches and indels between the repeats are permitted. Long inverted repeat explorer(Lirex) was developed and introduced in this report. Written in Java, Lirex provides a user-friendly interface and allows users to specify LIR searching criteria, such as length of the region, as well as pattern and size of the repeats. Recombinogenic LIRs can be selected on the basis of mismatch rate and internal spacer size from identified LIRs. Lirex, as a cross-platform tool to identify LIRs in a genome, may assist in designing following experiments to explore the function of LIRs. Our tool can identify more LIRs than other LIR searching tools. Lirex is publicly available at http://124.16.219.129/Lirex.展开更多
The 2.2 kb EcoRI fragment containing the chloroplast pshA gene from millet (Setaria italica) has been cloned and the nucleotide sequence of the 5’-noncoding region has been determined. The 5’-flanking region is foun...The 2.2 kb EcoRI fragment containing the chloroplast pshA gene from millet (Setaria italica) has been cloned and the nucleotide sequence of the 5’-noncoding region has been determined. The 5’-flanking region is found to contain prokaryote-like promoter structures: compared with prokaryotic promoters, the“-35”box (TTGACA) shows 100% homology while, in the ‘-10’box (TATACT), one different nucleotide is found. In addition, between these two boxes, there is a consensus sequence“TATATA”.just as in prokaryotic promoters. All these results indicate that millet psbA promoter has both prokaryotic and eukaryotic characteristies. The mRNA leader region of millet pshA gene is 87 bp, the same length as sorghum. However, an additional CTATTT sequence is found as compared with rice and an additional TTTT, as with wheat, barley and rye. So the differences between C3 and C4 plants may be universal in the family of Gramineae. Furthermore, computer analysis shows that a small stem-loop structure might be formed in pshA mRNA leader region in these six plants. The above-mentioned additional CTATTT sequence happens to be just located within the stem-loop structure, thus leading to different sizes of the stem-loops among these six species. It is likely that this small secondary structure may have some effect on the expression and regulation of the psbA gene.展开更多
基金This work was supported by the National Natural Science Found ation of China(32273000)the Qingdao Demonstration Project for People-benefit from Science and Techniques,China(23-2-8-xdny-14nsh and 24-2-8-xdny-4-nsh)+1 种基金the National Program of Undergraduate Innovation and Entrepreneurship,China(202310435039)the Open Project Fund of State Key Laboratory of Microbial Technology,China(M2023-03)。
文摘Senecavirus A(SVA)has a positive-sense,single-stranded RNA genome.Its 5´untranslated region harbors an internal ribosome entry site(IRES),comprising 10 larger or smaller stem-loop structures(including a pseudoknot)that have been demonstrated to be well conserved.However,it is still unclear whether each stem-loop subdomain,such as a single stem or loop,is also highly conserved.To clarify this issue in the present study,a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis(SDM)on the IRES.The SDM-modified scenarios included:(1)stem-formed complementary sequences exchanging with each other;(2)loop transversion;(3)loop transition;and(4)point mutations.All cDNA clones were separately transfected into cells for rescuing viable viruses,whereas only four SVAs of interest could be recovered,and were genetically stable during 20 passages.One progeny grew significantly slower than the other three did.The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity.Our previous study indicated that a single motif from any of the ten stem structures,if completely mutated,would cause the failure of virus recovery.Interestingly,our present study revealed three stem structures,whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs.Moreover,one apical loop was demonstrated to have the ability to tolerate its own full-length transition,also having no impact on the recovery of sequence-modifying SVA.The present study suggested that not every stem-loop structure was strictly conserved in its conformation,while the full-length IRES itself was well conserved.This provides a new research direction on interaction between the IRES and many factors.
文摘MicroRNAs （miRNAs） usually contain 19-24 nucleotides and have been identified as important eukaryotic gene regulators. Applications of various computational approaches have simplified the task by predicting miRNAs from available sequence data sources. In this study, we identified a conserved miR414 from a computational analysis of EST sequence data available from Stevia rebaudiana. In addition, we also identified six conserved miRNAs namely miR169, miR319, miR414, miR164, miR167 and miR398 using stem-loop RT-PCR analysis. Hence, miR414 was commonly identified using both methods. The expression analysis of these miRNAs documented their roles in growth and development of Stevia. Furthermore, the detected miRNAs were found to target genes involved in plant growth, development, metabolism and signal transducfion. This is the first study reporting these conserved miRNAs and their expression in Stevia.
文摘MicroRNAs are an important subclass of non-coding RNAs (ncRNA), and serve as main players into RNA interference (RNAi). Mature microRNA derived from stem-loop structure called precursor. Identification of precursor microRNA (pre-miRNA) is essential step to target microRNA in whole genome. The present work proposed 25 novel local features for identifying stem- loop structure of pre-miRNAs, which captures characteristics on both the sequence and structure. Firstly, we pulled the stem of hairpins and aligned the bases in bulges and internal loops used ‘―’, and then counted 24 base-pairs (‘AA’, ‘AU’, …, ‘―G’, except ‘――’) in pulled stem (formalized by length of pulled stem) as features vector of Support Vector Machine (SVM). Performances of three classifiers with our features and different kernels trained on human data were all superior to Triplet-SVM-classifier’s in po- sitive and negative testing data sets. Moreover, we achieved higher prediction accuracy through combining 7 global sequence-structure. The result indicates validity of novel local features.
基金Project supported by the Chinese National High-Tech Project.
文摘To study the relationship between the structures of rice and sorghum psbA 3 UTR and gene expres-sional activity, six chimeric luc genes that encode luciferase under control of rice or sorghum psbA 5 UTR and 3’UTR or only 5 UTR were constructed. The levels of LUC accumulation in E . coli and the transcript stability in soluble pro-tein extracts of rice, sorghum chloroplast and E. coli were examined respectively, and a detailed analysis about the function of these two 3’UTR has been carried out. Here the regulation effect of these 3’UTR are reported: ( i ) When having the same promoter, the chimeric genes with rice 3 UTR produce LUC much more than that with sorghum 3 UTR; ( ii ) elimination of 3 UTR results in the fluctuation of LUC accumulation no matter whether it is under control of rice or sorghum psbA 5’UTR; ( Hi ) rice psbA 3’UTR exhibits a greater effect on stabilizing tran-scripts; ( IV ) psbA 3’lR-RNAs are more stable in chloroplast protein extracts than in E. coli protein extracts.
基金the National Natural Science Foundation of China(30871578)
文摘Two microRNA （miRNA） quantification methods, namely, poly（A） reverse transcription （RT）-quantitative real-time polymerase chain reaction （qPCR） and stem-loop RT-qPCR, have been developed for quantifying miRNA expression. In the present study, five miRNAs, including miR166, miR167, miR168, miR159, and miR396, with different sequence frequencies, were selected as targets to compare their expression profiles in five wheat tissues by applying the two methods and deep sequencing. The study aimed to determine a simple, reliable and high-throughput method for detecting miRNA expressions in wheat tissues. Results showed that the miRNA expression profiles determined by poly（A） RT-qPCR were more consistent with those obtained by deep sequencing. Further analysis indicated that the correlation coefficients of the data obtained by poly（A） RT-qPCR and deep sequencing （0.739, P≤0.01） were higher than those obtained by stem-loop RT-qPCR and deep sequencing （0.535, P≤0.01）. The protocol used for poly（A） RT-qPCR is simpler than that for stem-loop RT-qPCR. Thus, poly（A） RT-qPCR was a more suitable high-throughput assay for detecting miRNA expression profiles. To the best of our knowledge, this study was the first to compare these two miRNA quantification methods. We also provided useful information for quantifying miRNA in wheat or other plant species.
基金supported by the National Natural Science Foundation of China (Grant Nos. 41476104 and 31460001)the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB06010201)+1 种基金the International Science and Technology Cooperation Project of Hainan Province, China (Grant No. KJHZ2015-22)the support from the Institute of Deep-Sea Science and Engineering, Chinese Academy of Sciences (Grant Nos. SIDSSE-BR-201303 and SIDSSE-201305)
文摘Long inverted repeats(LIRs) are evolutionarily and functionally important structures in genomes because of their involvement in RNA interference, DNA recombination, and gene duplication. Identification of LIRs is highly complicated when mismatches and indels between the repeats are permitted. Long inverted repeat explorer(Lirex) was developed and introduced in this report. Written in Java, Lirex provides a user-friendly interface and allows users to specify LIR searching criteria, such as length of the region, as well as pattern and size of the repeats. Recombinogenic LIRs can be selected on the basis of mismatch rate and internal spacer size from identified LIRs. Lirex, as a cross-platform tool to identify LIRs in a genome, may assist in designing following experiments to explore the function of LIRs. Our tool can identify more LIRs than other LIR searching tools. Lirex is publicly available at http://124.16.219.129/Lirex.
文摘The 2.2 kb EcoRI fragment containing the chloroplast pshA gene from millet (Setaria italica) has been cloned and the nucleotide sequence of the 5’-noncoding region has been determined. The 5’-flanking region is found to contain prokaryote-like promoter structures: compared with prokaryotic promoters, the“-35”box (TTGACA) shows 100% homology while, in the ‘-10’box (TATACT), one different nucleotide is found. In addition, between these two boxes, there is a consensus sequence“TATATA”.just as in prokaryotic promoters. All these results indicate that millet psbA promoter has both prokaryotic and eukaryotic characteristies. The mRNA leader region of millet pshA gene is 87 bp, the same length as sorghum. However, an additional CTATTT sequence is found as compared with rice and an additional TTTT, as with wheat, barley and rye. So the differences between C3 and C4 plants may be universal in the family of Gramineae. Furthermore, computer analysis shows that a small stem-loop structure might be formed in pshA mRNA leader region in these six plants. The above-mentioned additional CTATTT sequence happens to be just located within the stem-loop structure, thus leading to different sizes of the stem-loops among these six species. It is likely that this small secondary structure may have some effect on the expression and regulation of the psbA gene.
