Location of the RNA-processing enzymes RNase III, RNase E and RNase P in the Eschenchia coli cell

AbstractRibonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5′ tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5′ leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.

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RNA processing in Aquifex aeolicus involves RNase E/G and an RNase P-like activity

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2007.11.165 ◽

2008 ◽

Vol 366 (2) ◽

pp. 457-463 ◽

Cited By ~ 12

Author(s):

Tania B. Lombo ◽

Vladimir R. Kaberdin

Keyword(s):

Rna Processing ◽

Rnase P ◽

Rnase E ◽

Aquifex Aeolicus

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Impact of PNPase on the transcriptome of Rhodobacter sphaeroides and its cooperation with RNase III and RNase E

BMC Genomics ◽

10.1186/s12864-021-07409-4 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Daniel-Timon Spanka ◽

Carina Maria Reuscher ◽

Gabriele Klug

Keyword(s):

Rhodobacter Sphaeroides ◽

Rna Processing ◽

Regulatory Networks ◽

Organic Peroxide ◽

Rna Degradation ◽

Major Influence ◽

Rnase E ◽

Sequencing Data ◽

Rnase Iii ◽

Gram Negative

Abstract Background The polynucleotide phosphorylase (PNPase) is conserved among both Gram-positive and Gram-negative bacteria. As a core part of the Escherichia coli degradosome, PNPase is involved in maintaining proper RNA levels within the bacterial cell. It plays a major role in RNA homeostasis and decay by acting as a 3′-to-5′ exoribonuclease. Furthermore, PNPase can catalyze the reverse reaction by elongating RNA molecules in 5′-to-3′ end direction which has a destabilizing effect on the prolonged RNA molecule. RNA degradation is often initiated by an endonucleolytic cleavage, followed by exoribonucleolytic decay from the new 3′ end. Results The PNPase mutant from the facultative phototrophic Rhodobacter sphaeroides exhibits several phenotypical characteristics, including diminished adaption to low temperature, reduced resistance to organic peroxide induced stress and altered growth behavior. The transcriptome composition differs in the pnp mutant strain, resulting in a decreased abundance of most tRNAs and rRNAs. In addition, PNPase has a major influence on the half-lives of several regulatory sRNAs and can have both a stabilizing or a destabilizing effect. Moreover, we globally identified and compared differential RNA 3′ ends in RNA NGS sequencing data obtained from PNPase, RNase E and RNase III mutants for the first time in a Gram-negative organism. The genome wide RNA 3′ end analysis revealed that 885 3′ ends are degraded by PNPase. A fair percentage of these RNA 3′ ends was also identified at the same genomic position in RNase E or RNase III mutant strains. Conclusion The PNPase has a major influence on RNA processing and maturation and thus modulates the transcriptome of R. sphaeroides. This includes sRNAs, emphasizing the role of PNPase in cellular homeostasis and its importance in regulatory networks. The global 3′ end analysis indicates a sequential RNA processing: 5.9% of all RNase E-dependent and 9.7% of all RNase III-dependent RNA 3′ ends are subsequently degraded by PNPase. Moreover, we provide a modular pipeline which greatly facilitates the identification of RNA 5′/3′ ends. It is publicly available on GitHub and is distributed under ICS license.

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Knockout of the plastid RNase E leads to defective RNA processing and chloroplast ribosome deficiency

The Plant Journal ◽

10.1111/j.1365-313x.2010.04377.x ◽

2010 ◽

Vol 64 (5) ◽

pp. 851-863 ◽

Cited By ~ 37

Author(s):

Michael Walter ◽

Katrin Piepenburg ◽

Mark Aurel Schöttler ◽

Kerstin Petersen ◽

Sabine Kahlau ◽

...

Keyword(s):

Rna Processing ◽

Rnase E ◽

Defective Rna

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RNA Processing and Degradation by RNase III

Control of Messenger RNA Stability ◽

10.1016/b978-0-08-091652-1.50009-8 ◽

1993 ◽

pp. 71-116 ◽

Cited By ~ 52

Author(s):

Donald Court

Keyword(s):

Rna Processing ◽

Rnase Iii

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Endoribonuclease-mediated control of hns mRNA stability constitutes a key regulatory pathway for Salmonella Typhimurium pathogenicity island 1 expression

PLoS Pathogens ◽

10.1371/journal.ppat.1009263 ◽

2021 ◽

Vol 17 (2) ◽

pp. e1009263 ◽

Cited By ~ 1

Author(s):

Minho Lee ◽

Minkyung Ryu ◽

Minju Joo ◽

Young-Jin Seo ◽

Jaejin Lee ◽

...

Keyword(s):

Rna Processing ◽

Mrna Stability ◽

Type Iii Secretion ◽

Transcriptional Repression ◽

Environmental Changes ◽

Pathogenicity Island ◽

Cleavage Sites ◽

Regulatory Pathway ◽

Rnase Iii ◽

Rnase G

Bacteria utilize endoribonuclease-mediated RNA processing and decay to rapidly adapt to environmental changes. Here, we report that the modulation of hns mRNA stability by the endoribonuclease RNase G plays a key role in Salmonella Typhimurium pathogenicity. We found that RNase G determines the half-life of hns mRNA by cleaving its 5′ untranslated region and that altering its cleavage sites by genome editing stabilizes hns mRNA, thus decreasing S. Typhimurium virulence in mice. Under anaerobic conditions, the FNR-mediated transcriptional repression of rnc encoding RNase III, which degrades rng mRNA, and simultaneous induction of rng transcription resulted in rapid hns mRNA degradation, leading to the derepression of genes involved in the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS). Together, our findings show that RNase III and RNase G levels-mediated control of hns mRNA abundance acts as a regulatory pathway upstream of a complex feed-forward loop for SPI-1 expression.

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RNA Processing and Degradation by RNase K and RNase E

Control of Messenger RNA Stability ◽

10.1016/b978-0-08-091652-1.50008-6 ◽

1993 ◽

pp. 53-70 ◽

Cited By ~ 17

Author(s):

Öjar Melefors ◽

Urban Lundberg ◽

Alexander Von Gabain

Keyword(s):

Rna Processing ◽

Rnase E

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Geneticin Stabilizes the Open Conformation of the 5′ Region of Hepatitis C Virus RNA and Inhibits Viral Replication

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02511-15 ◽

2015 ◽

Vol 60 (2) ◽

pp. 925-935 ◽

Cited By ~ 3

Author(s):

Ascensión Ariza-Mateos ◽

Rosa Díaz-Toledano ◽

Timothy M. Block ◽

Samuel Prieto-Vega ◽

Alex Birk ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Therapeutic Potential ◽

Kinetic Studies ◽

Rnase P ◽

Start Codon ◽

Hcv Rna ◽

Rnase Iii ◽

Entry Site ◽

Content Type

ABSTRACTThe aminoglycoside Geneticin (G418) is known to inhibit cell culture proliferation, via virus-specific mechanisms, of two different virus genera from the familyFlaviviridae. Here, we tried to determine whether Geneticin can selectively alter the switching of the nucleotide 1 to 570 RNA region of hepatitis C virus (HCV) and, if so, whether this inhibits viral growth. Two structure-dependent RNases known to specifically cleave HCV RNA were tested in the presence or absence of the drug. One was theSynechocystissp. RNase P ribozyme, which cleaves the tRNA-like domain around the AUG start codon under high-salt buffer conditions; the second wasEscherichia coliRNase III, which recognizes a double-helical RNA switch element that changes the internal ribosome entry site (IRES) from a closed (C) conformation to an open (O) one. While the drug did not affect RNase P activity, it did inhibit RNase III in the micromolar range. Kinetic studies indicated that the drug favors the switch from the C to the O conformation of the IRES by stabilizing the distal double-stranded element and inhibiting further processing of the O form. We demonstrate that, because the RNA in this region is highly conserved and essential for virus survival, Geneticin inhibits HCV Jc1 NS3 expression, the release of the viral genomic RNA, and the propagation of HCV in Huh 7.5 cells. Our study highlights the crucial role of riboswitches in HCV replication and suggests the therapeutic potential of viral-RNA-targeted antivirals.

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