E-MTAB-6993
RNA-seq for identifying toxin MazF and MqsR cleavage sites in toxin expression and subsequent recovery cultures of Escherichia coli (mRNA and ncRNA excluding rRNA)
Bacterial toxin-antitoxin systems are thought to help bacteria to reduce their metabolism in various stressful conditions. Many toxins of the bacterial toxin-antitoxin systems are ribonucleases. Such toxins have been mostly viewed as degraders of mRNA, however recently it was demonstrated that they are also capable of cleaving non-coding RNA. MazF toxin is also hypothesized to reprogram E. colis translational machinery. Current cDNA libraries are part of a project which aims to identify the major RNA cleavage sites (in mRNA, rRNA and regulatory non-coding RNA) of MazF and MqsR toxins in E. coli. We wanted to elaborate on the roles of MazF and MqsR in E. coli: is MazF really involved in reprogramming the translational machinery or is MazF, along with MqsR, just a robust cleaver of RNA?   We extracted total RNA from cultures where the expression of MazF or MqsR was induced for 2h and from cultures that had been recovering from toxin production for 30 minutes; RNA from log phase culture was used as the control.  We used strand specific random primed paired end RNA sequencing data to locate the major cleavage sites. To map the cleavage sites, we counted 5' end stacks of forward reads in each genomic position and compared them with total coverage. MazF and MqsR cleave at specific recognition sequences, ?ACA and G?C respectively, which allowed us to eliminate the false positives. One of our initial aims was also to look for irregular RNA ligation events following toxin expression, thus we used long, 300 base reads in sequencing.
Bacterial toxin-antitoxin systems are thought to help bacteria to reduce their metabolism in various stressful conditions. Many toxins of the bacterial toxin-antitoxin systems are ribonucleases. Such toxins have been mostly viewed as degraders of mRNA, however recently it was demonstrated that they are also capable of cleaving non-coding RNA. MazF toxin is also hypothesized to reprogram E. colis translational machinery. Current cDNA libraries are part of a project which aims to identify the major RNA cleavage sites (in mRNA, rRNA and regulatory non-coding RNA) of MazF and MqsR toxins in E. coli. We wanted to elaborate on the roles of MazF and MqsR in E. coli: is MazF really involved in reprogramming the translational machinery or is MazF, along with MqsR, just a robust cleaver of RNA?   We extracted total RNA from cultures where the expression of MazF or MqsR was induced for 2h and from cultures that had been recovering from toxin production for 30 minutes; RNA from log phase culture was used as the control.  We used strand specific random primed paired end RNA sequencing data to locate the major cleavage sites. To map the cleavage sites, we counted 5' end stacks of forward reads in each genomic position and compared them with total coverage. MazF and MqsR cleave at specific recognition sequences, ?ACA and G?C respectively, which allowed us to eliminate the false positives. One of our initial aims was also to look for irregular RNA ligation events following toxin expression, thus we used long, 300 base reads in sequencing.