Inactivation of RNase P in Escherichia coli significantly changes post-transcriptional RNA metabolism

Ribonuclease P (RNase P) is the only ribonuclease responsible for 5'-end maturation of tRNAs in all kingdoms of life. In Escherichia coli, it is also essential for separation of pre-tRNAs from multiple polycistronic transcripts. Using RNA sequencing (RNA-seq), we show here that RNase P affects the abundance of ~44% of the expressed mRNAs demonstrating a much more widespread role in mRNA decay than previously thought. Furthermore, our data also demonstrate for the first time that the polyadenylation of transcripts by PAP I modulates RNase P-mediated mRNA decay as well as tRNA processing. Overall design: RNA-seq experiments were conducted on RNAs isolated from wild type, DpcnB, rnpA49 and DpcnB rnpA49 strains of E. coli. For each library (12 libraries comprising three biological replicates for each strain), ~42 million cDNAs were sequenced out of which the number of reads that mapped uniquely to MG1655 genome was between 76-86%. Furthermore, ERCC (External RNA Control Consortium) RNAs were spiked into the RNA-seq libraries in known amounts and then aligned to ERCC references. These alignments were then used to calculate a Pearson correlation coefficient for each sample. The ERCC control correlation coefficients were 0.98, 0.95 and 0.95 for three replicates of the wild type strain; 0.97, 0.95, 0.94 for three replicates of the DpcnB mutant; 0.98, 0.98, 0.98 for the three replicates of the rnpA49 mutant and 0.98, 0.97, 0,97 for the three replicates of the rnpA49 DpcnB double mutant (data not shown). Based on the nearly identical correlation coefficients of the three replicates for each strain, the RNA-seq reads from the three replicates of each strain were merged.