iCLIP analysis of RNA substrates of the archaeal exosome.

BACKGROUND: The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3' to 5' direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3'-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. RESULTS: To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17-19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5' parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3'-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5'-ends of RNAs was detected. CONCLUSIONS: In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5'-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3'-5' direction.

A response regulator of the OmpR family is part of the regulatory network controlling the oxidative stress response of Rhodobacter sphaeroides.

As a free-living bacterium Rhodobacter sphaeroides needs to respond to many environmental stresses. Oxidative stress, membrane stress or heat stress induce the ompR-1 gene encoding a protein of the OmpR family. Overexpression of OmpR-1 results in increased resistance to organic peroxides and diamide. Our data demonstrate that OmpR-1 positively affects expression of several sRNAs with an established role in R. sphaeroides stress defences and negatively affects the promoter of the rpoHI gene. The RpoHI sigma factor has a main role in the activation of many stress responses. Thus OmpR-1 has a balancing effect on the activation of the RpoHI regulon. We present a model with OmpR-1 as part of a regulatory network controlling stress defences in R. sphaeroides.

RNase E cleavage shapes the transcriptome of Rhodobacter sphaeroides and strongly impacts phototrophic growth.

Bacteria adapt to changing environmental conditions by rapid changes in their transcriptome. This is achieved not only by adjusting rates of transcription but also by processing and degradation of RNAs. We applied TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) for the transcriptome-wide identification of RNase E cleavage sites and of 5' RNA ends, which are enriched when RNase E activity is reduced in Rhodobacter sphaeroides. These results reveal the importance of RNase E for the maturation and turnover of mRNAs, rRNAs, and sRNAs in this guanine-cytosine-rich α-proteobacterium, some of the latter have well-described functions in the oxidative stress response. In agreement with this, a role of RNase E in the oxidative stress response is demonstrated. A remarkably strong phenotype of a mutant with reduced RNase E activity was observed regarding the formation of photosynthetic complexes and phototrophic growth, whereas there was no effect on chemotrophic growth.

6S RNA in Rhodobacter sphaeroides: 6S RNA and pRNA transcript levels peak in late exponential phase and gene deletion causes a high salt stress phenotype.

The function of 6S RNA, a global regulator of transcription, was studied in the photosynthetic α-proteobacterium Rhodobacter sphaeroides. The cellular levels of R. sphaeroides 6S RNA peak toward the transition to stationary phase and strongly decrease during extended stationary phase. The synthesis of so-called product RNA transcripts (mainly 12-16-mers) on 6S RNA as template by RNA polymerase was found to be highest in late exponential phase. Product RNA ≥ 13-mers are expected to trigger the dissociation of 6S RNA:RNA polymerase complexes. A 6S RNA deletion in R. sphaeroides had no impact on growth under various metabolic and oxidative stress conditions (with the possible exception of tert-butyl hydroperoxide stress). However, the 6S RNA knockout resulted in a robust growth defect under high salt stress (0.25 M NaCl). Remarkably, the sspA gene encoding the putative salt stress-induced membrane protein SspA and located immediately downstream of the 6S RNA (ssrS) gene on the antisense strand was expressed at elevated levels in the ΔssrS strain when grown in the presence of 250 mM NaCl.

The Conserved Dcw Gene Cluster of R. sphaeroides Is Preceded by an Uncommonly Extended 5' Leader Featuring the sRNA UpsM.

Cell division and cell wall synthesis mechanisms are similarly conserved among bacteria. Consequently some bacterial species have comparable sets of genes organized in the dcw (division and cell wall) gene cluster. Dcw genes, their regulation and their relative order within the cluster are outstandingly conserved among rod shaped and gram negative bacteria to ensure an efficient coordination of growth and division. A well studied representative is the dcw gene cluster of E. coli. The first promoter of the gene cluster (mraZ1p) gives rise to polycistronic transcripts containing a 38 nt long 5' UTR followed by the first gene mraZ. Despite reported conservation we present evidence for a much longer 5' UTR in the gram negative and rod shaped bacterium Rhodobacter sphaeroides and in the family of Rhodobacteraceae. This extended 268 nt long 5' UTR comprises a Rho independent terminator, which in case of termination gives rise to a non-coding RNA (UpsM). This sRNA is conditionally cleaved by RNase E under stress conditions in an Hfq- and very likely target mRNA-dependent manner, implying its function in trans. These results raise the question for the regulatory function of this extended 5' UTR. It might represent the rarely described case of a trans acting sRNA derived from a riboswitch with exclusive presence in the family of Rhodobacteraceae.

An RNAi-Based Control of Fusarium graminearum Infections Through Spraying of Long dsRNAs Involves a Plant Passage and Is Controlled by the Fungal Silencing Machinery.

Meeting the increasing food and energy demands of a growing population will require the development of ground-breaking strategies that promote sustainable plant production. Host-induced gene silencing has shown great potential for controlling pest and diseases in crop plants. However, while delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a promising concept, it requires the generation of transgenic crop plants which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species. Using the agronomically important barley-Fusarium graminearum pathosystem, we alternatively demonstrate that a spray application of a long noncoding dsRNA (791 nt CYP3-dsRNA), which targets the three fungal cytochrome P450 lanosterol C-14α-demethylases, required for biosynthesis of fungal ergosterol, inhibits fungal growth in the directly sprayed (local) as well as the non-sprayed (distal) parts of detached leaves. Unexpectedly, efficient spray-induced control of fungal infections in the distal tissue involved passage of CYP3-dsRNA via the plant vascular system and processing into small interfering (si)RNAs by fungal DICER-LIKE 1 (FgDCL-1) after uptake by the pathogen. We discuss important consequences of this new finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential as a new plant protection strategy.

Regulation of a polyamine transporter by the conserved 3' UTR-derived sRNA SorX confers resistance to singlet oxygen and organic hydroperoxides in Rhodobacter sphaeroides.

Singlet oxygen is generated by bacteriochlorophylls when light and oxygen are simultaneously present in Rhodobacter sphaeroides. Singlet oxygen triggers a specific response that is partly regulated by the alternative sigma factor RpoHI/HII. The sRNA RSs2461 has previously been identified as an RpoHI/HII-dependent sRNA and is derived from the 3' UTR of the mRNA for an OmpR-type transcriptional regulator. Similar to the RpoHI/HII-dependent CcsR and SorY sRNAs, RSs2461 affects the resistance of R. sphaeroides against singlet oxygen and was therefore renamed here SorX. Furthermore, SorX has a strong impact on resistance against organic hydroperoxides that usually occur as secondary damages downstream of singlet oxygen. The 75-nt SorX 3' fragment, which is generated by RNase E cleavage and highly conserved among related species, represents the functional entity. A target search identified potA mRNA, which encodes a subunit of a polyamine transporter, as a direct SorX target and stress resistance via SorX could be linked to potA. The PotABCD transporter is an uptake system for spermidine in E. coli. While spermidine is generally described as beneficial during oxidative stress, we observed significantly increased sensitivity of R. sphaeroides to organic hydroperoxides in the presence of spermidine. We therefore propose that the diminished import of spermidine, due to down-regulation of potA by SorX, counteracts oxidative stress. Together with results from other studies this underlines the importance of regulated transport to bacterial stress defense.

The sRNA SorY confers resistance during photooxidative stress by affecting a metabolite transporter in Rhodobacter sphaeroides.

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen ((1)O2) in the facultative photosynthetic bacterium Rhodobacter sphaeroides. We have identified SorY as an sRNA, which is induced under several stress conditions and confers increased resistance against (1)O2. SorY by direct interaction affects the takP mRNA, encoding a TRAP-T transporter. We present a model in which SorY reduces the metabolite flux into the tricarboxylic acid cycle (TCA cycle) by reducing malate import through TakP. It was previously shown that oxidative stress in bacteria leads to switch from glycolysis to the pentose phosphate pathway and to reduced activity of the TCA cycle. As a consequence the production of the prooxidant NADH is reduced and production of the protective NADPH is enhanced. In R. sphaeroides enzymes for glycolysis, pentose phosphate pathway, Entner-Doudoroff pathway and gluconeogenesis are induced in response to (1)O2 by the alternative sigma factor RpoHII. The same is true for the sRNA SorY. By limiting malate import SorY thus contributes to the balance of the metabolic fluxes under photooxidative stress conditions. This assigns a so far unknown function to an sRNA in oxidative stress response.

RNase J is required for processing of a small number of RNAs in Rhodobacter sphaeroides.

All bacteria contain multiple exoribonucleases to ensure a fast breakdown of different RNA molecules, either for maturation or for complete degradation to the level of mononucleotides. This efficient RNA degradation plays pivotal roles in the post-transcriptional gene regulation, in RNA processing and maturation as well as in RNA quality control mechanisms and global adaption to stress conditions. Besides different 3'-to-5' exoribonucleases mostly with overlapping functions in vivo many bacteria additionally possess the 5'-to-3' exoribonuclease, RNase J, to date the only known bacterial ribonuclease with this activity. An RNA-seq approach was applied to identify specific targets of RNase J in the α-proteobacterium Rhodobacter sphaeroides. Only few transcripts were strongly affected by the lack of RNase J implying that its function is mostly required for specific processing/degradation steps in this bacterium. The accumulation of diverse RNA fragments in the RNase J deletion mutant points to RNA features that apparently cannot be targeted by the conventional 3'-exoribonucleases in Gram-negative bacteria.

DegS and RseP homologous proteases are involved in singlet oxygen dependent activation of RpoE in Rhodobacter sphaeroides.

Singlet oxygen ((1)O2) is the main agent of photooxidative stress and is generated by photosensitizers as (bacterio)chlorophylls. It leads to the damage of cellular macromolecules and therefore photosynthetic organisms have to mount an adaptive response to (1)O2 formation. A major player of the photooxidative stress response in Rhodobacter sphaeroides is the alternative sigma factor RpoE, which is inactivated under non-stress conditions by its cognate anti-sigma factor ChrR. By using random mutagenesis we identified RSP_1090 to be required for full activation of the RpoE response under (1)O2 stress, but not under organic peroxide stress. In this study we show that both RSP_1090 and RSP_1091 are required for full resistance towards (1)O2. Moreover, we revealed that the DegS and RseP homologs RSP_3242 and RSP_2710 contribute to (1)O2 resistance and promote ChrR proteolysis. The RpoE signaling pathway in R. sphaeroides is therefore highly similar to that of Escherichia coli, although very different anti-sigma factors control RpoE activity. Based on the acquired results, the current model for RpoE activation in response to (1)O2 exposure in R. sphaeroides was extended.

Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase-encoding genes confers strong resistance to Fusarium species.

Head blight, which is caused by mycotoxin-producing fungi of the genus Fusarium, is an economically important crop disease. We assessed the potential of host-induced gene silencing targeting the fungal cytochrome P450 lanosterol C-14α-demethylase (CYP51) genes, which are essential for ergosterol biosynthesis, to restrict fungal infection. In axenic cultures of Fusarium graminearum, in vitro feeding of CYP3RNA, a 791-nt double-stranded (ds)RNA complementary to CYP51A, CYP51B, and CYP51C, resulted in growth inhibition [half-maximum growth inhibition (IC50) = 1.2 nM] as well as altered fungal morphology, similar to that observed after treatment with the azole fungicide tebuconazole, for which the CYP51 enzyme is a target. Expression of the same dsRNA in Arabidopsis and barley rendered susceptible plants highly resistant to fungal infection. Microscopic analysis revealed that mycelium formation on CYP3RNA-expressing leaves was restricted to the inoculation sites, and that inoculated barley caryopses were virtually free of fungal hyphae. This inhibition of fungal growth correlated with in planta production of siRNAs corresponding to the targeted CYP51 sequences, as well as highly efficient silencing of the fungal CYP51 genes. The high efficiency of fungal inhibition suggests that host-induced gene-silencing targeting of the CYP51 genes is an alternative to chemical treatments for the control of devastating fungal diseases.