Rogh, S. N. | bioRxiv Science

bioRxiv2023

The role of the 5' sensing function of ribonuclease E in cyanobacteria

RNA degradation is crucial for many processes in pro- and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y towards 5-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5 sensing. Cyanobacteria, such as Synechocystis sp. PCC 6803 (Synechocystis), encode RNase E and RNase J homologs. Here, we constructed a Synechocystis strain lacking the 5 sensing function of RNase E and mapped on a transcriptome-wide level 292 5-sensing-dependent cleavage sites. These included so far unknown targets such as the 5 untranslated region of the response regulator gene lsiR; trxA, apcE and atpI mRNAs, encoding proteins related to energy metabolism; as well as sbtB and rbcLXS encoding proteins relevant for carbon fixation. Cyanobacterial 5 sensing is important for the maturation of rRNA and several tRNAs, including tRNAGluUUC. This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and most prokaryotes. We found that increased RNase activities leads to a higher copy number of the major Synechocystis plasmids pSYSA and pSYSM. The results provide a first step towards understanding the relative importance of different target mechanisms of RNase E outside Escherichia coli.

microbiology↗

bioRxiv2021

Transcriptome-wide in vivo mapping of cleavage sites for the compact cyanobacterial ribonuclease E reveals insights into its function and substrate recognition

Ribonucleases are crucial enzymes in RNA metabolism and post-transcriptional regulatory processes in bacteria. Cyanobacteria encode the two essential ribonucleases RNase E and RNase J. Cyanobacterial RNase E is shorter than homologues in other groups of bacteria and lacks both the chloroplast-specific N-terminal extension as well as the C-terminal domain typical for RNase E of enterobacteria. In order to investigate the function of RNase E in the model cyanobacterium Synechocystis sp. PCC 6803, we engineered a temperature-sensitive RNase E mutant by introducing two site-specific mutations, I65F and spontaneously occurring V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1,472 RNase-E-dependent cleavage sites. We inferred a dominating cleavage signature consisting of an adenine at the -3 and a uridine at the +2 position within a single-stranded segment of the RNA. The data identified putative RNase-E-dependent instances of operon discoordination, mRNAs likely regulated jointly by RNase E and an sRNA, potential 3 end-derived sRNAs and a dual-acting mechanism for the glutamine riboswitch. Our findings substantiate the pivotal role of RNase E in post-transcriptional regulation and suggest the redundant or concerted action of RNase E and RNase J in cyanobacteria.

microbiology↗