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

Nucleic Acids Res. 2021 Dec 16;49(22):13075-13091. doi: 10.1093/nar/gkab1161.

Abstract

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 the spontaneously occurred V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1472 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 mRNAs likely regulated jointly by RNase E and an sRNA and potential 3' end-derived sRNAs. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Binding Sites / genetics
  • Cyanobacteria / enzymology
  • Cyanobacteria / genetics*
  • Endoribonucleases / genetics*
  • Endoribonucleases / metabolism
  • Gene Expression Profiling / methods*
  • Hydrolysis
  • Point Mutation
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism
  • RNA-Seq / methods
  • Sequence Homology, Amino Acid
  • Spectrophotometry / methods
  • Substrate Specificity
  • Synechocystis / enzymology
  • Synechocystis / genetics
  • Transcriptome*

Substances

  • Bacterial Proteins
  • RNA, Bacterial
  • Endoribonucleases
  • ribonuclease E