Impact of RNase E and RNase J on Global mRNA Metabolism in the Cyanobacterium Synechocystis PCC6803

Abstract

mRNA levels result from an equilibrium between transcription and degradation. Ribonucleases (RNases) facilitate the turnover of mRNA, which is an important way of controlling gene expression, allowing the cells to adjust transcript levels to a changing environment. In contrast to the heterotrophic model bacteria Escherichia coli and Bacillus subtilis, RNA decay has not been studied in detail in cyanobacteria. Synechocystis sp. PCC6803 encodes orthologs of both E. coli and B. subtilis RNases, including RNase E and RNase J, respectively. We show that in vitro Sy RNases E and J have an endonucleolytic cleavage specificity that is very similar between them and also compared to orthologous enzymes from E. coli, B. subtilis, and Chlamydomonas. Moreover, Sy RNase J displays a robust 5 -exoribonuclease activity similar to B. subtilis RNase J1, but unlike the evolutionarily related RNase J in chloroplasts. Both nucleases are essential and gene deletions could not be fully segregated in Synechocystis. We generated partially disrupted strains of Sy RNase E and J that were stable enough to allow for their growth and characterization. A transcriptome analysis of these strains partially depleted for RNases E and J, respectively, allowed to observe effects on specific transcripts. RNase E altered the expression of a larger number of chromosomal genes and antisense RNAs compared to RNase J, which rather affects endogenous plasmid encoded transcripts. Our results provide the first description of the main transcriptomic changes induced by the partial depletion of two essential ribonucleases in cyanobacteria.

Escherichia coliRNase E can efficiently replace RNase Y inBacillus subtilis

“…As shown in Figure 4 , all three enzymes can cleave at the same site, in a single stranded sequence 6–9 nucleotides upstream of the leader terminator structure (Figure 4B ). This corroborates previous observations that suggested a similar cleavage specificity for RNase Y and RNase E on the one hand ( 12 ) and RNase E and RNase J on the other hand ( 35 , 43 ).…”

“…This allowed to show that all three ribonucleases can indeed cleave at the same site in a single substrate (Figure 4 ). We previously found that cyanobacterial orthologs of RNase E and RNase J have an endonucleolytic cleavage specificity that is very similar between them and also compared to the orthologous enzymes in E. coli and B. subtilis ( 35 ). Together, our in vitro data support the view that the three totally unrelated ribonucleases Y, E and J have evolved independently towards a common activity and been maintained in bacteria throughout evolution.…”

“…168 (NCBI accession number for chromosome: NC_000964.3) and its annotation were retrieved from the NCBI: ( https://www.ncbi.nlm.nih.gov/genome/?term=Bacillus±subtilis ) and from Subtiwiki ( 34 ). Reads processing, mapping and differential gene expression analysis were performed according to ( 35 ). Briefly, single-end 100-nt reads were mapped to the reference genome using bwa and allowing soft-clipping in the alignment parameters (bwa mem) ( 36 ).…”

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

“…PCC 6803 (in the following Synechocystis), a unicellular model cyanobacterium. Both RNase E as well as RNase J are essential in Synechocystis (20)(21)(22). Besides cyanobacteria, only alphaproteobacteria, several actinobacteria and fibrobacteres contain both RNase E and RNase J, but lack RNase Y (2).…”

The temperature-regulated DEAD-box RNA helicase CrhR interactome: Autoregulation and photosynthesis-related transcripts

“…SigG is associated with the oxidative stress response caused by high light (Huckauf et al, 2000) and is ectopically upregulated in a ∆ rpaA mutant in the dark (Köbler et al, 2018). Furthermore, interacting transcripts encoding enzymes associated with RNA turnover and metabolism were identified, including rnj/slr0551 encoding ribonuclease J, an enzyme possessing both endonuclease and a robust 5'-exoribonuclease activity (Cavaiuolo et al, 2020), slr0053 encoding the rRNA maturation RNase YbeY and, as an enzyme of RNA metabolism, crhR itself.…”