Attachment of the RNA degradosome to the bacterial inner cytoplasmic membrane prevents wasteful degradation of rRNA in ribosome assembly intermediates.

RNA processing and degradation shape the transcriptome by generating stable molecules that are necessary for translation (rRNA and tRNA) and by facilitating the turnover of mRNA, which is necessary for the posttranscriptional control of gene expression. In bacteria and the plant chloroplast, RNA degradosomes are multienzyme complexes that process and degrade RNA. In many bacterial species, the endoribonuclease RNase E is the central component of the RNA degradosome. RNase E-based RNA degradosomes are inner membrane proteins in a large family of gram-negative bacteria (ß- and γ-Proteobacteria). Until now, the reason for membrane localization was not understood. Here, we show that a mutant strain of Escherichia coli, in which the RNA degradosome is localized to the interior of the cell, has high levels of 20S and 40S particles that are defective intermediates in ribosome assembly. These particles have aberrant protein composition and contain rRNA precursors that have been cleaved by RNase E. After RNase E cleavage, rRNA fragments are degraded to nucleotides by exoribonucleases. In vitro, rRNA in intact ribosomes is resistant to RNase E cleavage, whereas protein-free rRNA is readily degraded. We conclude that RNA degradosomes in the nucleoid of the mutant strain interfere with cotranscriptional ribosome assembly. We propose that membrane-attached RNA degradosomes in wild-type cells control the quality of ribosome assembly after intermediates are released from the nucleoid. That is, the compact structure of mature ribosomes protects rRNA against cleavage by RNase E. Turnover of a proportion of intermediates in ribosome assembly explains slow growth of the mutant strain. Competition between mRNA and rRNA degradation could be the cause of slower mRNA degradation in the mutant strain. We conclude that attachment of the RNA degradosome to the bacterial inner cytoplasmic membrane prevents wasteful degradation of rRNA precursors, thus explaining the reason for conservation of membrane-attached RNA degradosomes throughout the ß- and γ-Proteobacteria.

Sex-Specific Effects of Long-Term Antipsychotic Drug Treatment on Adipocyte Tissue and the Crosstalk to Liver and Brain in Rats.

Antipsychotic drug (APD) medication can lead to metabolic dysfunctions and weight gain, which together increase morbidity and mortality. Metabolically active visceral adipose tissue (VAT) in particular plays a crucial role in the etiopathology of these metabolic dysregulations. Here, we studied the effect of 12 weeks of drug medication by daily oral feeding of clozapine and haloperidol on the perirenal fat tissue as part of VAT of male and female Sprague Dawley rats in the context of complex former investigations on brain, liver, and blood. Adipocyte area values were determined, as well as triglycerides, non-esterified fatty acids (NEFAs), glucose, glycogen, lactate, malondialdehyde equivalents, ferric iron and protein levels of Perilipin-A, hormone-sensitive-lipase (HSL), hepcidin, glucose transporter-4 (Glut-4) and insulin receptor-ß (IR-ß). We found increased adipocyte mass in males, with slightly higher adipocyte area values in both males and females under clozapine treatment. Triglycerides, NEFAs, glucose and oxidative stress in the medicated groups were unchanged or slightly decreased. In contrast to controls and haloperidol-medicated rats, perirenal adipocyte mass and serum leptin levels were not correlated under clozapine. Protein expressions of perilipin-A, Glut-4 and HSL were decreased under clozapine treatment. IR-ß expression changed sex-specifically in the clozapine-medicated groups associated with higher hepcidin levels in the perirenal adipose tissue of clozapine-treated females. Taken together, clozapine and haloperidol had a smaller effect than expected on perirenal adipose tissue. The perirenal adipose tissue shows only weak changes in lipid and glucose metabolism. The main changes can be seen in the proteins examined, and probably in their effect on liver metabolism.

RNA processing machineries in Archaea: the 5'-3' exoribonuclease aRNase J of the ß-CASP family is engaged specifically with the helicase ASH-Ski2 and the 3'-5' exoribonucleolytic RNA exosome machinery.

A network of RNA helicases, endoribonucleases and exoribonucleases regulates the quantity and quality of cellular RNAs. To date, mechanistic studies focussed on bacterial and eukaryal systems due to the challenge of identifying the main drivers of RNA decay and processing in Archaea. Here, our data support that aRNase J, a 5'-3' exoribonuclease of the ß-CASP family conserved in Euryarchaeota, engages specifically with a Ski2-like helicase and the RNA exosome to potentially exert control over RNA surveillance, at the vicinity of the ribosome. Proteomic landscapes and direct protein-protein interaction analyses, strengthened by comprehensive phylogenomic studies demonstrated that aRNase J interplay with ASH-Ski2 and a cap exosome subunit. Finally, Thermococcus barophilus whole-cell extract fractionation experiments provide evidences that an aRNase J/ASH-Ski2 complex might exist in vivo and hint at an association of aRNase J with the ribosome that is emphasised in absence of ASH-Ski2. Whilst aRNase J homologues are found among bacteria, the RNA exosome and the Ski2-like RNA helicase have eukaryotic homologues, underlining the mosaic aspect of archaeal RNA machines. Altogether, these results suggest a fundamental role of ß-CASP RNase/helicase complex in archaeal RNA metabolism.

Detachment of the RNA degradosome from the inner membrane of Escherichia coli results in a global slowdown of mRNA degradation, proteolysis of RNase E and increased turnover of ribosome-free transcripts.

The reason for RNase E attachment to the inner membrane is largely unknown. To understand the cell biology of RNA degradation, we have characterized a strain expressing RNase E lacking the membrane attachment site (cytoplasmic RNase E). Genome-wide data show a global slowdown in mRNA degradation. There is no correlation between mRNA stabilization and the function or cellular location of encoded proteins. The activity of cRNase E is comparable to the wild-type enzyme in vitro, but the mutant protein is unstable in vivo. Autoregulation of cRNase E synthesis compensates for protein instability. cRNase E associates with other proteins to assemble a cytoplasmic RNA degradosome. CsrB/C sRNAs, whose stability is regulated by membrane-associated CsrD, are stabilized. Membrane attachment of RNase E is thus necessary for CsrB/C turnover. In contrast to mRNA stability, ribosome-free transcripts are sensitive to inactivation by cRNase E. Our results show that effects on RNA degradation are not due to the differences in the activity or level of cRNase E, or failure to assemble the RNA degradosome. We propose that membrane attachment is necessary for RNase E stability, functional interactions with membrane-associated regulatory factors and protection of ribosome-free transcripts from premature interactions with RNase E in the nucleoid.

Effects of haloperidol and clozapine on synapse-related gene expression in specific brain regions of male rats.

We investigated the effects of clozapine and haloperidol, drugs that are widely used in the treatment of schizophrenia, on gene expression in six cortical and subcortical brain regions of adult rats. Drug treatments started at postnatal day 85 and continued over a 12-week period. Ten animals received haloperidol (1 mg/kg bodyweight) and ten received clozapine (20 mg/kg bodyweight) orally each day. Ten control rats received no drugs. The ten genes selected for this study did not belong to the dopaminergic or serotoninergic systems, which are typically targeted by the two substances, but coded for proteins of the cytoskeleton and proteins belonging to the synaptic transmitter release machinery. Quantitative real-time PCR was performed in the prelimbic cortex, cingulate gyrus (CG1) and caudate putamen and in the hippocampal cornu ammonis 1 (CA1), cornu ammonis 3 (CA3) and dentate gyrus. Results show distinct patterns of gene expression under the influence of the two drugs, but also distinct gene regulations dependent on the brain regions. Haloperidol-medicated animals showed statistically significant downregulation of SNAP-25 in CA3 (p = 0.0134) and upregulation of STX1A in CA1 (p = 0.0133) compared to controls. Clozapine-treated animals showed significant downregulation of SNAP-25 in CG1 (p = 0.0013). Our results clearly reveal that the drugs' effects are different between brain regions. These effects are possibly indirectly mediated through feedback mechanisms by proteins targeted by the drugs, but direct effects of haloperidol or clozapine on mechanisms of gene expression cannot be excluded.

Membrane recognition and dynamics of the RNA degradosome.

RNase E, which is the central component of the multienzyme RNA degradosome, serves as a scaffold for interaction with other enzymes involved in mRNA degradation including the DEAD-box RNA helicase RhlB. Epifluorescence microscopy under live cell conditions shows that RNase E and RhlB are membrane associated, but neither protein forms cytoskeletal-like structures as reported earlier by Taghbalout and Rothfield. We show that association of RhlB with the membrane depends on a direct protein interaction with RNase E, which is anchored to the inner cytoplasmic membrane through an MTS (Membrane Targeting Sequence). Molecular dynamics simulations show that the MTS interacts with the phospholipid bilayer by forming a stabilized amphipathic α-helix with the helical axis oriented parallel to the plane of the bilayer and hydrophobic side chains buried deep in the acyl core of the membrane. Based on the molecular dynamics simulations, we propose that the MTS freely diffuses in the membrane by a novel mechanism in which a large number of weak contacts are rapidly broken and reformed. TIRFm (Total Internal Reflection microscopy) shows that RNase E in live cells rapidly diffuses over the entire inner membrane forming short-lived foci. Diffusion could be part of a scanning mechanism facilitating substrate recognition and cooperativity. Remarkably, RNase E foci disappear and the rate of RNase E diffusion increases with rifampicin treatment. Control experiments show that the effect of rifampicin is specific to RNase E and that the effect is not a secondary consequence of the shut off of E. coli transcription. We therefore interpret the effect of rifampicin as being due to the depletion of RNA substrates for degradation. We propose a model in which formation of foci and constraints on diffusion arise from the transient clustering of RNase E into cooperative degradation bodies.

Small RNA binding to 5' mRNA coding region inhibits translational initiation.

Small noncoding RNAs (sRNAs) have predominantly been shown to repress bacterial mRNAs by masking the Shine-Dalgarno (SD) or AUG start codon sequence, thereby preventing 30S ribosome entry and, consequently, translation initiation. However, many recently identified sRNAs lack obvious SD and AUG complementarity, indicating that sRNA-mediated translational control could also take place at other mRNA sites. We report that Salmonella RybB sRNA represses ompN mRNA translation by pairing with the 5' coding region. Results of systematic antisense interference with 30S binding to ompN and unrelated mRNAs suggest that sRNAs can act as translational repressors by sequestering sequences within the mRNA down to the fifth codon, even without SD and AUG start codon pairing. This "five codon window" for translational control in the 5' coding region of mRNA not only has implications for sRNA target predictions but might also apply to cis-regulatory systems such as RNA thermosensors and riboswitches.

The social fabric of the RNA degradosome.

Bacterial transcripts each have a characteristic half-life, suggesting that the processes of RNA degradation work in an active and selective manner. Moreover, the processes are well controlled, thereby ensuring that degradation is orderly and coordinated. Throughout much of the bacterial kingdom, RNA degradation processes originate through the actions of assemblies of key RNA enzymes, known as RNA degradosomes. Neither conserved in composition, nor unified by common evolutionary ancestry, RNA degradosomes nonetheless can be found in divergent bacterial lineages, implicating a common requirement for the co-localisation of RNA metabolic activities. We describe how the cooperation of components in the representative degradosome of Escherichia coli may enable controlled access to transcripts, so that they have defined and programmable lifetimes. We also discuss how this cooperation contributes to precursor processing and to the riboregulation of intricate post-transcriptional networks in the control of gene expression. The E. coli degradosome interacts with the cytoplasmic membrane, and we discuss how this interaction may spatially organise the assembly and contribute to subunit cooperation and substrate capture. This article is part of a Special Issue entitled: RNA Decay mechanisms.

Replicative resolution of integron cassette insertion.

Site-specific recombination catalyzed by tyrosine recombinases follows a common pathway consisting of two consecutive strand exchanges. The first strand exchange generates a Holliday junction (HJ), which is resolved by a second strand exchange. In integrons, attC sites recombine as folded single-stranded substrates. Only one of the two attC site strands, the bottom one, is efficiently bound and cleaved by the integrase during the insertion of gene cassettes at the double-stranded attI site. Due to the asymmetry of this complex, a second strand exchange on the attC bottom strand (bs) would form linearized abortive recombination products. We had proposed that HJ resolution would rely on an uncharacterized mechanism, probably replication. Using an attC site carried on a plasmid with each strand specifically tagged, we followed the destiny of each strand after recombination. We demonstrated that only one strand, the one carrying the attC bs, is exchanged. Furthermore, we show that the recombination products contain the attC site bs and its entire de novo synthesized complementary strand. Therefore, we demonstrate the replicative resolution of single-strand recombination in integrons and rule out the involvement of a second strand exchange of any kind in the attC×attI reaction.

Neuroreceptor Inhibition by Clozapine Triggers Mitohormesis and Metabolic Reprogramming in Human Blood Cells.

The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood. Here, we analysed the effects of clozapine (2.5-20 µM) on metabolic fluxes, cell respiration, and intracellular ATP in human HL60 cells. Some results were confirmed in leukocytes of clozapine-treated patients. Neuroreceptor inhibition under clozapine reduced Akt activation with decreased glucose uptake, thereby inducing ER stress and the unfolded protein response (UPR). Metabolic profiling by liquid-chromatography/mass-spectrometry revealed downregulation of glycolysis and the pentose phosphate pathway, thereby saving glucose to keep the electron transport chain working. Mitochondrial respiration was dampened by upregulation of the F0F1-ATPase inhibitory factor 1 (IF1) leading to 30-40% lower oxygen consumption in HL60 cells. Blocking IF1 expression by cotreatment with epigallocatechin-3-gallate (EGCG) increased apoptosis of HL60 cells. Upregulation of the mitochondrial citrate carrier shifted excess citrate to the cytosol for use in lipogenesis and for storage as triacylglycerol in lipid droplets (LDs). Accordingly, clozapine-treated HL60 cells and leukocytes from clozapine-treated patients contain more LDs than untreated cells. Since mitochondrial disturbances are described in the pathophysiology of schizophrenia, clozapine-induced mitohormesis is an excellent way to escape energy deficits and improve cell survival.

Evolutionary and functional insights into the Ski2-like helicase family in Archaea: a comparison of Thermococcales ASH-Ski2 and Hel308 activities.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The Ski2-like proteins are primordial helicases that play an active role in eukaryotic RNA homeostasis pathways, with multiple homologs having specialized functions. The significance of the expansion and diversity of Ski2-like proteins in Archaea, the third domain of life, has not yet been established. Here, by studying the phylogenetic diversity of Ski2-like helicases among archaeal genomes and the enzymatic activities of those in Thermococcales, we provide further evidence of the function of this protein family in archaeal metabolism of nucleic acids. We show that, in the course of evolution, ASH-Ski2 and Hel308-Ski2, the two main groups of Ski2-like proteins, have diverged in their biological functions. Whereas Hel308 has been shown to mainly act on DNA, we show that ASH-Ski2, previously described to be associated with the 5'-3' aRNase J exonuclease, acts on RNA by supporting an efficient annealing activity, but also an RNA unwinding with a 3'-5' polarity. To gain insights into the function of Ski2, we also analyse the transcriptome of Thermococcus barophilus ΔASH-Ski2 mutant strain and provide evidence of the importance of ASH-Ski2 in cellular metabolism pathways related to translation.

Evidence for an autonomous 5' target recognition domain in an Hfq-associated small RNA.

The abundant class of bacterial Hfq-associated small regulatory RNAs (sRNAs) parallels animal microRNAs in their ability to control multiple genes at the posttranscriptional level by short and imperfect base pairing. In contrast to the universal length and seed pairing mechanism of microRNAs, the sRNAs are heterogeneous in size and structure, and how they regulate multiple targets is not well understood. This paper provides evidence that a 5' located sRNA domain is a critical element for the control of a large posttranscriptional regulon. We show that the conserved 5' end of RybB sRNA recognizes multiple mRNAs of Salmonella outer membrane proteins by ≥7-bp Watson-Crick pairing. When fused to an unrelated sRNA, the 5' domain is sufficient to guide target mRNA degradation and maintain σ(E)-dependent envelope homeostasis. RybB sites in mRNAs are often conserved and flanked by 3' adenosine. They are found in a wide sequence window ranging from the upstream untranslated region to the deep coding sequence, indicating that some targets might be repressed at the level of translation, whereas others are repressed primarily by mRNA destabilization. Autonomous 5' domains seem more common in sRNAs than appreciated and might improve the design of synthetic RNA regulators.

A tale of two mRNA degradation pathways mediated by RNase E.

RNase E is an essential endoribonuclease with a preference for RNA substrates with 5'-monophosphate ends. Primary transcripts, which have 5' triphosphate ends, are thus protected from RNase E. Their conversion to 5'-monophosphate transcripts by RppH is a prerequisite for RNase E-mediated processing and degradation. 5'-monophosphate recognition involves binding to a subdomain in the catalytic core of RNase E known as the 5' sensor. There are, however, transcripts that can be attacked directly by RNase E in a 5'-end-independent pathway. Direct entry involves elements outside of the catalytic domain that are located in the carboxyl terminal half (CTH) of RNase E. Strains harbouring rne alleles that express variants of RNase E in which 5' sensing (rneR169Q) or direct entry (rneΔCTH) are inactivated, are viable. However, the rneR169Q/rneΔCTH and ΔrppH/rneΔCTH combinations are synthetic lethal suggesting that the essential function(s) of RNase E requires at least one of these pathways to be active. A striking result is the demonstration that mutations affecting Rho-dependent transcription termination can overcome synthetic lethality by a pathway that requires RNase H. It is hypothesized that R-loop formation and RNase H cleavage substitute for RNase E-dependent RNA processing and mRNA degradation.

Structural basis for broad DNA-specificity in integron recombination.

Lateral DNA transfer--the movement of genetic traits between bacteria--has a profound impact on genomic evolution and speciation. The efficiency with which bacteria incorporate genetic information reflects their capacity to adapt to changing environmental conditions. Integron integrases are proteins that mediate site-specific DNA recombination between a proximal primary site (attI) and a secondary target site (attC) found within mobile gene cassettes encoding resistance or virulence factors. The lack of sequence conservation among attC sites has led to the hypothesis that a sequence-independent structural recognition determinant must exist within attC. Here we report the crystal structure of an integron integrase bound to an attC substrate. The structure shows that DNA target site recognition and high-order synaptic assembly are not dependent on canonical DNA but on the position of two flipped-out bases that interact in cis and in trans with the integrase. These extrahelical bases, one of which is required for recombination in vivo, originate from folding of the bottom strand of attC owing to its imperfect internal dyad symmetry. The mechanism reported here supports a new paradigm for how sequence-degenerate single-stranded genetic material is recognized and exchanged between bacteria.

Structural features of single-stranded integron cassette attC sites and their role in strand selection.

We recently showed that cassette integration and deletion in integron platforms were occurring through unconventional site-specific recombination reactions involving only the bottom strand of attC sites. The lack of sequence conservation among attC sites led us to hypothesize that sequence-independent structural recognition determinants must exist within attC sites. The structural data obtained from a synaptic complex of the Vibrio cholerae integrase with the bottom strand of an attC site has shown the importance of extra helical bases (EHB) inside the stem-loop structure formed from the bottom strand. Here, we systematically determined the contribution of three structural elements common to all known single-stranded attC site recombination substrates (the EHBs, the unpaired central spacer (UCS), and the variable terminal structure (VTS)) to strand choice and recombination. Their roles have been evaluated in vivo in the attIxattC reaction context using the suicide conjugation assay we previously developed, but also in an attCxattC reaction using a deletion assay. Conjugation was used to deliver the attC sites in single-stranded form. Our results show that strand choice is primarily directed by the first EHB, but the presence of the two other EHBs also serves to increase this strand selection. We found that the structure of the central spacer is essential to achieve high level recombination of the bottom strand, suggesting a dual role for this structure in active site exclusion and for hindering the reverse reaction after the first strand exchange. Moreover, we have shown that the VTS has apparently no role in strand selectivity.

Sex-dependent effects of long-term clozapine or haloperidol medication on red blood cells and liver iron metabolism in Sprague Dawley rats as a model of metabolic syndrome.

BACKGROUND: Patients with liver diseases often have some form of anemia. Hematological dyscrasias are known side effects of antipsychotic drug medication and the occurrence of agranulocytosis under clozapine is well described. However, the sex-dependent impact of clozapine and haloperidol on erythrocytes and symptoms like anemia, and its association with hepatic iron metabolism has not yet been completely clarified. Therefore, in the present study, we investigated the effect of both antipsychotic drugs on blood parameters and iron metabolism in the liver of male and female Sprague Dawley rats. METHODS: After puberty, rats were treated orally with haloperidol or clozapine for 12 weeks. Blood count parameters, serum ferritin, and liver transferrin bound iron were determined by automated counter. Hemosiderin (Fe3+) was detected in liver sections by Perl's Prussian blue staining. Liver hemoxygenase (HO-1), 5'aminolevulinate synthase (ALAS1), hepcidin, heme-regulated inhibitor (HRI), cytochrome P4501A1 (CYP1A1) and 1A2 (CYP1A2) were determined by Western blotting. RESULTS: We found anemia with decreased erythrocyte counts, associated with lower hemoglobin and hematocrit, in females with haloperidol treatment. Males with clozapine medication showed reduced hemoglobin and increased red cell distribution width (RDW) without changes in erythrocyte numbers. High levels of hepatic hemosiderin were found in the female clozapine and haloperidol medicated groups. Liver HRI was significantly elevated in male clozapine medicated rats. CYP1A2 was significantly reduced in clozapine medicated females. CONCLUSIONS: The characteristics of anemia under haloperidol and clozapine medication depend on the administered antipsychotic drug and on sex. We suggest that anemia in rats under antipsychotic drug medication is a sign of an underlying liver injury induced by the drugs. Changing hepatic iron metabolism under clozapine and haloperidol may help to reduce these effects of liver diseases.

Phylogenetic Diversity of Lhr Proteins and Biochemical Activities of the Thermococcales aLhr2 DNA/RNA Helicase.

Helicase proteins are known to use the energy of ATP to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of DNA and RNA metabolisms and participate in numerous repair mechanisms that maintain cellular integrity. The archaeal Lhr-type proteins are SF2 helicases that are mostly uncharacterized. They have been proposed to be DNA helicases that act in DNA recombination and repair processes in Sulfolobales and Methanothermobacter. In Thermococcales, a protein annotated as an Lhr2 protein was found in the network of proteins involved in RNA metabolism. To investigate this, we performed in-depth phylogenomic analyses to report the classification and taxonomic distribution of Lhr-type proteins in Archaea, and to better understand their relationship with bacterial Lhr. Furthermore, with the goal of envisioning the role(s) of aLhr2 in Thermococcales cells, we deciphered the enzymatic activities of aLhr2 from Thermococcus barophilus (Tbar). We showed that Tbar-aLhr2 is a DNA/RNA helicase with a significant annealing activity that is involved in processes dependent on DNA and RNA transactions.

Sex-dependent alterations of dopamine receptor and glucose transporter density in rat hypothalamus under long-term clozapine and haloperidol medication.

OBJECTIVE: Sex-dependent disturbances of peripheral glucose metabolism are known complications of antipsychotic drug treatment. The influence of long-term clozapine and haloperidol medication on hypothalamus, maintaining aspects of internal body homeostasis, has not yet been completely clarified. METHODS: After puberty, male and female Sprague Dawley rats were fed orally with ground pellets containing haloperidol (1 mg/kgBW/day) or clozapine (20 mg/kgBW/day) for 12 weeks. The hypothalamic protein expression of dopamine receptors D2R and D4R, melanocortin receptor MC4R, and glucose transporters Glut1 and Glut3 was examined. Glucose, glycogen, lactate, and pyruvate levels were determined, also malondialdehyde equivalents as markers of oxidative stress. RESULTS: D2R expression was increased in the male haloperidol and clozapine group but decreased in females medicated with clozapine. D4R expression was upregulated under clozapine medication. While females showed increased Glut1, Glut3 was elevated in both male and female clozapine-medicated animals. We found no changes of hypothalamic malondialdehyde, glycogen, and MC4R. Hypothalamic lactate was elevated in the female clozapine group. CONCLUSION: Clozapine sex-dependently affects the expression of D2R, Glut1, and Glut3. The upregulation of the glucose transporters indicates glucose deprivation in the endothelial cells and consequently in astrocytes and neurons. Increased hypothalamic lactate in females under clozapine points to enhanced glycolysis with a higher glucose demand to produce the required energy. Haloperidol did not change the expression of the glucose transporters and upregulated D2R only in males.

Insights into RNA-processing pathways and associated RNA-degrading enzymes in Archaea.

RNA-processing pathways are at the centre of regulation of gene expression. All RNA transcripts undergo multiple maturation steps in addition to covalent chemical modifications to become functional in the cell. This includes destroying unnecessary or defective cellular RNAs. In Archaea, information on mechanisms by which RNA species reach their mature forms and associated RNA-modifying enzymes are still fragmentary. To date, most archaeal actors and pathways have been proposed in light of information gathered from Bacteria and Eukarya. In this context, this review provides a state of the art overview of archaeal endoribonucleases and exoribonucleases that cleave and trim RNA species and also of the key small archaeal proteins that bind RNAs. Furthermore, synthetic up-to-date views of processing and biogenesis pathways of archaeal transfer and ribosomal RNAs as well as of maturation of stable small non-coding RNAs such as CRISPR RNAs, small C/D and H/ACA box guide RNAs, and other emerging classes of small RNAs are described. Finally, prospective post-transcriptional mechanisms to control archaeal messenger RNA quality and quantity are discussed.

Large-Scale Measurement of mRNA Degradation in Escherichia coli: To Delay or Not to Delay.

In this study, we compared different computational methods used for genome-wide determination of mRNA half-lives in Escherichia coli with a special focus on the impact on considering a delay before the onset of mRNA decay after transcription arrest. A wide variety of datasets were analyzed coming from different technical methods for mRNA quantification (microarrays, RNA-seq, and RT-qPCR) and different bacterial growth conditions. The exponential decay of mRNA levels was fitted using both linear and exponential models and with or without a delay. We showed that for all the models, independently of mRNA quantification methods and growth conditions, ignoring the delay resulted in only a modest overestimation of the half-life. For approximately 80% of the mRNAs, differences in mRNA half-life values were less than 34s. The correlation between half-lives estimated with and without a delay was extremely high. However, the slope of the linear regression between the half-lives with and without a delay tended to decrease with the delay. For the few mRNAs for which taking into account the delay influenced the estimated half-life, the impact was dependent on the model and the growth condition. The smallest impact was obtained for the linear model.