Bacterial Response to Oxidative Stress and RNA Oxidation

Bacteria have to cope with oxidative stress caused by distinct Reactive Oxygen Species (ROS), derived not only from normal aerobic metabolism but also from oxidants present in their environments. The major ROS include superoxide O2−, hydrogen peroxide H2O2 and radical hydroxide HO•. To protect cells under oxidative stress, bacteria induce the expression of several genes, namely the SoxRS, OxyR and PerR regulons. Cells are able to tolerate a certain number of free radicals, but high levels of ROS result in the oxidation of several biomolecules. Strikingly, RNA is particularly susceptible to this common chemical damage. Oxidation of RNA causes the formation of strand breaks, elimination of bases or insertion of mutagenic lesions in the nucleobases. The most common modification is 8-hydroxyguanosine (8-oxo-G), an oxidized form of guanosine. The structure and function of virtually all RNA species (mRNA, rRNA, tRNA, sRNA) can be affected by RNA oxidation, leading to translational defects with harmful consequences for cell survival. However, bacteria have evolved RNA quality control pathways to eliminate oxidized RNA, involving RNA-binding proteins like the members of the MutT/Nudix family and the ribonuclease PNPase. Here we summarize the current knowledge on the bacterial stress response to RNA oxidation, namely we present the different ROS responsible for this chemical damage and describe the main strategies employed by bacteria to fight oxidative stress and control RNA damage.

RNA Quality Control Non-Denaturing Agarose "Bleach" Gel v1

To evaluate the quality of RNA without the use of toxic chemicals, samples are run in a non-denaturing agarose "bleach" gel and product segregation is used to estimate RNA integrity. Be aware that in a non-denaturing gel, the RNA will not segregate strictly on particle size due to secondary structures of the molecules. This method is for an estimation of quality only and the location of the banding in relation to a base pair ladder does not allow confident determination of the size of the RNA fragment. If true fragment size determinations are required, RNA quality should be evaluated on a denaturing gel electrophoresis system.

RNA Granules in the Mitochondria and Their Organization under Mitochondrial Stresses

The human mitochondrial genome (mtDNA) regulates its transcription products in specialised and distinct ways as compared to nuclear transcription. Thanks to its mtDNA mitochondria possess their own set of tRNAs, rRNAs and mRNAs that encode a subset of the protein subunits of the electron transport chain complexes. The RNA regulation within mitochondria is organised within specialised, membraneless, compartments of RNA-protein complexes, called the Mitochondrial RNA Granules (MRGs). MRGs were first identified to contain nascent mRNA, complexed with many proteins involved in RNA processing and maturation and ribosome assembly. Most recently, double-stranded RNA (dsRNA) species, a hybrid of the two complementary mRNA strands, were found to form granules in the matrix of mitochondria. These RNA granules are therefore components of the mitochondrial post-transcriptional pathway and as such play an essential role in mitochondrial gene expression. Mitochondrial dysfunctions in the form of, for example, RNA processing or RNA quality control defects, or inhibition of mitochondrial fission, can cause the loss or the aberrant accumulation of these RNA granules. These findings underline the important link between mitochondrial maintenance and the efficient expression of its genome.

Innate, translation-dependent silencing of an invasive transposon in Arabidopsis

Co-evolution between hosts’ and parasites’ genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence-degeneration and, ultimately, loss-of-autonomy of most transposable elements (TEs). Recognition of newly-invasive plant TEs, by contrast, involves an innate antiviral-like silencing response. To investigate this response’s activation, we studied the single-copy element EVADÉ (EVD), one of few representatives of the large Ty1/Copia family able to proliferate in Arabidopsis when epigenetically-reactivated. In Ty1/Copia-elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full-length genomic flGAG-POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly-nuclear flGAG-POL, during which an unusually intense ribosomal stalling event coincides precisely with the starting-point of sRNA production exclusively on shGAG. mRNA breakage occurring at this starting-point yields unconventional 5’OH RNA fragments that evade RNA-quality-control and concomitantly likely stimulate RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6) to initiate sRNA production. This hitherto-unrecognized “translation-dependent silencing” (TdS) is independent of codon-usage or GC-content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against de novo invasive TEs that underlies their associated sRNA patterns.

Cauliflower Mosaic Virus Utilizes Processing Bodies to Escape Translational Repression in Arabidopsis

Viral infections impose extraordinary RNA stress on a cell, triggering cellular RNA surveillance pathways like RNA decapping, nonsense-mediated decay and RNA silencing. Viruses need to maneuver between these pathways to establish infection and succeed in producing high amounts of viral proteins. Processing bodies (PBs) are integral to RNA triage in eukaryotic cells with several distinct RNA quality control pathways converging for selective RNA regulation. In this study, we investigate the role of Arabidopsis thaliana PBs during Cauliflower Mosaic Virus (CaMV) infection. We find that several PB components are co-opted into viral replication factories and support virus multiplication. This pro-viral role was not associated with RNA decay pathways but instead, we could establish PB components as essential helpers in viral RNA translation. While CaMV is normally resilient to RNA silencing, PB dysfunctions expose the virus to this pathway, similar to previous observations on transgenes. Transgenes, however, undergo RNA Quality Control dependent RNA degradation, whereas CaMV RNA remains stable but becomes translationally repressed through decreased ribosome association, revealing a unique dependence between PBs, RNA silencing and translational repression. Together, our study shows that PB components are co-opted by the virus to maintain efficient translation, a mechanism not associated with canonical PB functions.

The role of RST1 and RIPR proteins in plant RNA quality control systems

To keep mRNA homeostasis, the RNA degradation, quality control and silencing systems should act in balance in plants. Degradation of normal mRNA starts with deadenylation, then deadenylated transcripts are degraded by the SKI-exosome 3′-5′ and/or XRN4 5′-3′ exonucleases. RNA quality control systems identify and decay different aberrant transcripts. RNA silencing degrades double-stranded transcripts and homologous mRNAs. It also targets aberrant and silencing prone transcripts. The SKI-exosome is essential for mRNA homeostasis, it functions in normal mRNA degradation and different RNA quality control systems, and in its absence silencing targets normal transcripts. It is highly conserved in eukaryotes, thus recent reports that the plant SKI-exosome is associated with RST1 and RIPR proteins and that, they are required for SKI-exosome–mediated decay of silencing prone transcripts were unexpected. To clarify whether RST1 and RIPR are essential for all SKI-exosome functions or only for the elimination of silencing prone transcripts, degradation of different reporter transcripts was studied in RST1 and RIPR inactivated Nicotiana benthamiana plants. As RST1 and RIPR, like the SKI-exosome, were essential for Non-stop and No-go decay quality control systems, and for RNA silencing- and minimum ORF-mediated decay, we propose that RST1 and RIPR are essential components of plant SKI-exosome supercomplex.

Substrate discrimination and quality control require each catalytic activity of TRAMP and the nuclear RNA exosome

Quality control requires discrimination between functional and aberrant species to selectively target aberrant substrates for destruction. Nuclear RNA quality control in Saccharomyces cerevisiae includes the TRAMP complex that marks RNA for decay via polyadenylation followed by helicase-dependent 3′ to 5′ degradation by the RNA exosome. Using reconstitution biochemistry, we show that polyadenylation and helicase activities of TRAMP cooperate with processive and distributive exoribonuclease activities of the nuclear RNA exosome to protect stable RNA from degradation while selectively targeting and degrading less stable RNA. Substrate discrimination is lost when the distributive exoribonuclease activity of Rrp6 is inactivated, leading to degradation of stable and unstable RNA species. These data support a proofreading mechanism in which deadenylation by Rrp6 competes with Mtr4-dependent degradation to protect stable RNA while selectively targeting and degrading unstable RNA.

A UPF1 variant drives conditional remodeling of nonsense-mediated mRNA decay

The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination to degrade transcripts with premature stop codons and regulate thousands of human genes. Due to the major role of NMD in RNA quality control and gene expression regulation, it is important to understand how the pathway responds to changing cellular conditions. Here we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL, enables condition-dependent remodeling of NMD specificity. UPF1LL associates more stably with potential NMD target mRNAs than the major UPF1SL isoform, expanding the scope of NMD to include many transcripts normally immune to the pathway. Unexpectedly, the enhanced persistence of UPF1LL on mRNAs supports induction of NMD in response to rare translation termination events. Thus, while canonical NMD is abolished by translational repression, UPF1LL activity is enhanced, providing a mechanism to rapidly rewire NMD specificity in response to cellular stress.