| The processing of mRNA at post-transcriptional is crucial for bacteria to coordinate gene expression in vivo.Compared to transcriptional regulation,which involves RNA synthesis,post-transcriptional processing provides a manner of regulation that saves time and energy relative to de novo RNA synthesis.The rapid turnover of mRNA contributes to the ability of bacteria to adapt very quickly to changing nutritional or environmental.To resolve the potential conflict between the equimolar stoichiometry of transcripts within an operon and the non-equimolar stoichiometry of subunits frequently necessitated,one strategy employed by the bacteria is selective RNA processing and stabilization(SRPS),where the primary mRNA transcribed as an operon is processed by nucleases into segments first and then variation in stability among the segments contribute to differential gene expression,and finally maintains the normal function of protein complex in vivo.Ruminiclostridium cellulolyticum(previously Clostridium cellulolyticum)produces cellulosomes,which are high-molecular-mass protein complexes assembled by dockerin modules in the cellulosomal catalytic components and cohesin modules on the scaffoldins.The cellulase and hemicellulase from R.cellulolyticum are encoded a large operon called cip-cel and xyl-doc,respectively.Our previous study reveal that the SRPS employed by R.cellulolyticum to regulate the transcription of genes from cip-cel operon,In which the radio of each subunit of cluster and the different fates of each processed RNA portion have been regulated,In this study,the cleavage sites and key elements involved in cip-cel mRNA processing at post-transcriptional level have been analysised in R.cellulolyticum.The main results as follows.Firstly,eleven intergenic regions(IRs)from the cip-cel operon were respectively inserted into the dual-fluorescent-proteins reporter system which constructed by our lab to determine the RNA processing events.The results showed that there are six cleavage sites in cip-cel operon,which located in the first,second,fourth,fifth,seven and tenth IRs(named IR1,IR2,IR4,IR5,IR7 and IR10).Secondly,a non-radioactive method of primer extension by employing near-infrared fluorescent cyanine dye Cy5.5 and a high-throughput sequencing method have been developed,which employed to identify the processing sites of cip-cel operon.Six processing sites of cip-cel mRNA were identified,which located at the upstream or downstream regions of the stem-loop.The methods we developed in this study not only detected the RNA cleavage sites of cip-cel mRNA precisely,but also avoids the use of hazardous radioactive isotope reagents for labeling and shortens the processing time,which will provide a safer and more reliable identification method for exploring transcription initiation sites and RNA processing sites in post-transcription level of bacteria.Thirdly,To gain further insight into cip-cel mRNA processing,secondary structures from six IRs were analyzed and compared using Mfold,which were located at the upstream/downstream of the processing sites in cip-cel mRNA.Different mutants from IRs were constructed in which their stem-loops have been mutant.Interestingly,the RNA cleavage no longer occurred when the stem-loop deleted or destroyed,moreover,the abundance of monocistronic transcripts was significantly decreased.Therefore,our findings reveal that the stem-loop of IRs from cip-cel operon acting as processing signals which induce ribonuclease to cleavage mRNA at specific sites in the post-transcriptional level.These discovery will provides a new idea for identing the endonuclease involved in cip-cel mRNA processing.Finaly,The stem-loops involved in cip-cel mRNA processing can be classified into three groups according to the characteristics of their structures.Such as,Type I of IR1-SL1,IR5-SL1,and IR10-SL,which have a perfect stem;Type II of IR2-SL and IR7-SL1,which have many unpaired regions;and Type III of IR4-SL2,which has an AT-pair region at its bottom.Our results further demonstrated that the 2-GC-pair-regions located at stem of Type I and Type II stem-loop were crucial for RNA cleavage.In addition,the IR4-SL1 and the AT-pair region of IR4-SL2 which belong to Type III stem-loop contributed together to the location of the cleavage site.In this study,all processing sites in the cip-cel cluster were accurately identified,the role of the stem-loop in RNA processing and the stability of transcripts was analyzed,and the the sequence/structure requirements for processing of the cip-cel mRNA haven been unveiled,which elucidate the molecular mechanism of R.cellulolyticum regulating the differential expression of genes in the cip-cel cluster through SRPS at the posttranscriptional level.Therefore,this study lays a theoretical foundation for research on the regulatory mechanism of cellulosome expression and its assembly,and also provides a new idea for elucidate the regulatory mechanism of RNA at the post-transcriptional level of bacteria,which has important biological significance for the design of synthetic biological tools for regulating gene expression in the future. |
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