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

Hoffmann, Ute; Lichtenberg, Elisabeth; Rogh, Said N.; Bilger, R.; Reimann, Viktoria; Heyl, Florian; Backofen, Rolf; Steglich, Claudia; Hess, Wolfgang R.; Wilde, Annegret

doi:10.1101/2023.01.13.523895

Cited by 3 publications

(8 citation statements)

References 102 publications

(213 reference statements)

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“…This result supports our idea that the ratio between the ssr7036 and the asRNA1 transcript amount and their RNase E-mediated cleavage and degradation is important for copy number control of the plasmid pSYSA. In a parallel study, a 3.8-fold increase in the pSYSA copy number was observed in strains overexpressing RNase E and RNase HII ( Hoffmann et al, 2023 ), which matches the results of this work. Hence, we conclude that an elevated ssr7036 transcript level leads to a higher pSYSA copy number.…”

Section: Resultssupporting

confidence: 89%

“…Positions which were classified as TSS are indicated by “TSS” next to the respective peaks. Data was analyzed as described previously ( Hoffmann et al, 2021 , 2023 ) using DESeq2 ( Love et al, 2014 ). DESeq2 assigns adjusted p values based on the Wald test and uses the Benjamini-Hochberg procedure to correct for multiple testing.…”

Section: Resultsmentioning

confidence: 99%

“…In the second analyzed dataset, a strain harbouring a 5′-sensing-deficient RNase E variant, rne (5p), was compared to rne (WT) and WT. 5′ sensing describes a central mechanism by which RNase E can recognize its targets ( Hoffmann et al, 2023 ). In this analysis, processing sites with higher read counts in rne (WT) or rne (5p) compared to Synechocystis WT indicated cleavage sites of the RNase E native enzyme or the 5′-sensing-deficient enzyme, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…All vectors and constructs used in this study are listed in Table 1 . Construction of strains rne (WT), rne (Ts) and rne (5p) were previously described ( Hoffmann et al, 2021 , 2023 ). For the construction of plasmids VIII.22 and VIII.23, the primers P1 and P2 were used to inversely amplify the pUC19 backbone (for primer details see Table 2 ).…”

Section: Methodsmentioning

confidence: 99%

“…Previous analyses after the transient inactivation of RNase E by temperature shift (TIER-seq) revealed that transcripts derived from the four major plasmids accumulated differentially between cells expressing an unmodified form of this riboendonuclease, rne (WT) and a temperature-sensitive form, rne (Ts) ( Hoffmann et al, 2021 ). Moreover, the joint overexpression of RNase E and RNase HII led up to 3.8- and 2.4-fold increased copy numbers of plasmids pSYSA and pSYSM, respectively ( Hoffmann et al, 2023 ). These results were interpreted as pointing toward a possible role of RNase E in copy number regulation of these replicons.…”

Section: Introductionmentioning

confidence: 99%

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Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA

Kaltenbrunner

Reimann²,

Hoffmann³

et al. 2023

Front. Microbiol.

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Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The ~100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encoded ssr7036 transcript. Together with a cis-encoded abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion of slr7037 led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence of slr7037 was required for successful replication of a pSYSA-derived vector in another model cyanobacterium, Synechococcus elongatus PCC 7942. Therefore, we annotated the protein encoded by slr7037 as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus in Synechocystis sp. PCC 6803.

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA

Kaltenbrunner

Reimann²,

Hoffmann³

et al. 2023

Front. Microbiol.

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A conserved protein inhibitor brings under check the activity of RNase E in cyanobacteria

Liu,

Lin,

Yuan

et al. 2023

Nucleic Acids Research

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The bacterial ribonuclease RNase E plays a key role in RNA metabolism. Yet, with a large substrate spectrum and poor substrate specificity, its activity must be well controlled under different conditions. Only a few regulators of RNase E are known, limiting our understanding on posttranscriptional regulatory mechanisms in bacteria. Here we show that, RebA, a protein universally present in cyanobacteria, interacts with RNase E in the cyanobacterium Anabaena PCC 7120. Distinct from those known regulators of RNase E, RebA interacts with the catalytic region of RNase E, and suppresses the cleavage activities of RNase E for all tested substrates. Consistent with the inhibitory function of RebA on RNase E, depletion of RNase E and overproduction of RebA caused formation of elongated cells, whereas the absence of RebA and overproduction of RNase E resulted in a shorter-cell phenotype. We further showed that the morphological changes caused by altered levels of RNase E or RebA are dependent on their physical interaction. The action of RebA represents a new mechanism, potentially conserved in cyanobacteria, for RNase E regulation. Our findings provide insights into the regulation and the function of RNase E, and demonstrate the importance of balanced RNA metabolism in bacteria.

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Interactors and effects of overexpressing YlxR/RpnM, a conserved RNA binding protein in cyanobacteria

Hemm,

Miucci,

Riediger

et al. 2024

Preprint

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Throughout the tree of life RNA-binding proteins play important roles in the regulation of gene expression and RNA metabolism, but they are only poorly characterized in cyanobacteria. Here, we analyzed the predicted RNA-binding protein Ssr1238 from the model cyanobacteriumSynechocystissp. PCC 6803. Ssr1238 is encoded in a syntenic region within thenusA-ssr1238-infBoperon, similar to genes encoding such proteins in gram-positive bacteria. Overexpression of Ssr1238 for 24 h led to slightly higher levels of RNase P RNA, 44 tRNAs, and several stress-related mRNAs. Co-immunoprecipitation of proteins followed by MS analysis and sequencing of UV crosslinked, co-immunoprecipitated RNA samples identified potential interaction partners of Ssr1238. The most highly enriched transcript was RNase P RNA, and RnpA, the protein component of RNase P, was among the most highly enriched proteins, consistent with recent findings that YlxR proteins can influence the enzymatic activity of RNase P. A second highly enriched transcript derived from the 3’ region of genessl3177, which encodes a rare lipoprotein homolog, a central enzyme in cell wall remodeling during cell division. The data also showed a strong connection to the RNA maturation and modification system indicated by co-precipitation of RNA methyltransferase Sll1967, the A-adding tRNA nucleotidyltransferase Sll1253, and queuine tRNA-ribosyltransferase Slr0713, as well as riboendonuclease E and enolase. Surprisingly, cyanophycin synthetase and urease were highly enriched as well. Therefore, Ssr1238 specifically binds to two different transcripts and appears to participate in the coordination of RNA maturation, aspects of nitrogen metabolism and translation, and cell division. Our results are consistent with recent findings that theB. subtilisYlxR protein functions as an RNase P modulator (RnpM), extend its proposed role to the phylum cyanobacteria, and suggest additional functionalities.

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

Cited by 3 publications

References 102 publications

Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA

Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA

A conserved protein inhibitor brings under check the activity of RNase E in cyanobacteria

Interactors and effects of overexpressing YlxR/RpnM, a conserved RNA binding protein in cyanobacteria

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