In many Proteobacteria, the RNA helicase RhlB is a component of the RNA degradosome, a multi-protein complex involved in RNA processing and degradation. Within this complex, RhlB interacts with the scaffold endoribonuclease RNase E. In Escherichia coli, allosteric activation of RhlB by RNase E has defined the current paradigm for RhlB regulation. Here, we identify a distinct clade of RhlB helicases, exemplified by Pseudomonas aeruginosa RhlB, which we designate Type II. Unlike Type I RhlB, Type II RhlB helicases contain an N-terminal intrinsically disordered region that drives RNA-dependent liquid-liquid phase separation and enhances RhlB activity. Biochemical, structural, and functional analyses show that P. aeruginosa RNase E binds RhlB through an interface distinct from that described in the E. coli model and, rather than stimulating activity, antagonizes RhlB phase separation. Excessive RhlB condensation impairs bacterial growth at low temperature, and RNase E-mediated control of RhlB condensation maintains growth under these conditions. Together, these findings reveal that conserved RNA degradosome components can engage in distinct regulatory interactions across species and identify condensate dissolution as a novel mechanism regulating RNA helicase activity.

The RNA degradosome is a bacterial multi-protein complex mediating mRNA processing and degradation. In Pseudomonadota, this complex assembles on the C-terminal domain (CTD) of RNase E through short linear motifs (SLiMs) that determine its composition and functionality. In the human pathogen Pseudomonas aeruginosa, the RNase E CTD exhibits limited similarity to that of model organisms, impeding our understanding of RNA metabolic processes in this bacterium. Our study systematically maps the interactions mediated by the P. aeruginosa RNase E CTD and highlights its critical role in transcript regulation and cellular functions. We identified the SLiMs crucial for membrane attachment, RNA binding and complex clustering, as well as for direct binding to the core components PNPase and RhlB. Transcriptome analyses of RNase E CTD mutants revealed altered expression of genes involved in quorum sensing, type III secretion, and amino acid metabolism. Additionally, we show that the mutants are impaired in cold adaptation, pH response, and virulence in an infection model. Overall, this work establishes the essential role of the RNA degradosome in driving bacterial adaptability and pathogenicity.

RNA helicases-central enzymes in RNA metabolism- often feature intrinsically disordered regions (IDRs) that enable phase separation and complex molecular interactions. In the bacterial pathogen Pseudomonas aeruginosa , the non-redundant RhlE1 and RhlE2 RNA helicases share a conserved REC catalytic core but differ in C-terminal IDRs. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both IDRs facilitate RNA binding and phase separation, localizing proteins in cytoplasmic clusters. However, RhlE2 IDR is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping IDRs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The REC RhlE1 -IDR RhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The REC RhlE2 -IDR RhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases.

Background Because of COVID-19, the 2020 written medical examinations were replaced by mandatory formative online assessments. This study aimed to determine students’ performance, self-assessment of performance, and perception about the switch from a summative to a formative approach. Methods Medical students from year 2 to 5 (n=648) were included. They could repeat each test once or twice. They rated their performance after each attempt and were then given their score. Detailed feedback was given at the end of the session. An online survey determined medical students’ perception about the reorganization of education. Two items concerned the switch from summative to formative assessments Results Formative assessments involved 2385 examinees totaling 3197 attempts. Among examinees, 30.8% made at least 2 attempts. Scores increased significantly at the second attempt (median 9.4, IQR 10.8), and duration decreased (median -31.0, IQR 48.0). More than half of examinees (54.6%) underestimated their score, female students more often than male. Low performers overestimated, while high performers underestimated their scores. Students approved of the switch to formative assessments. Stress was lessened but motivation for learning decreased. Conclusions Medical students’ better scores at a second attempt support a benefit of detailed feedback, learning time and re-test opportunity on performance. Decreased learning motivation and a minority of students repeating the formative assessments point to the positive influence of summative assessment on learning.

Background: Because of COVID-19, the 2020 written medical examinations were replaced by mandatory formative online assessments. This study aimed to determine students’ performance, self-assessment of performance, and perception about the switch from a summative to a formative approach. Methods: Medical students from year 2 to 5 (n=648) were included. They could repeat each test once or twice. They rated their performance after each attempt and were then given their score. Detailed feedback was given at the end of the session. An online survey determined medical students’ perception about the reorganization of education. Two items concerned the switch from summative to formative assessments Results: Formative assessments involved 2385 examinees totaling 3197 attempts. Among examinees, 30.8% made at least 2 attempts. Scores increased significantly at the second attempt (median 9.4, IQR 10.8), and duration decreased (median -31.0, IQR 48.0). More than half of examinees (54.6%) underestimated their score, female students more often than male. Low performers overestimated, while high performers underestimated their scores. Students approved of the switch to formative assessments. Stress was lessened but motivation for learning decreased. Conclusions: Medical students’ better scores at a second attempt support a benefit of detailed feedback, learning time and re-test opportunity on performance. Decreased learning motivation and a minority of students repeating the formative assessments point to the positive influence of summative assessment on learning.

Background: Because of COVID-19, the 2020 written medical examinations were replaced by mandatory formative online assessments. This study aimed to determine students’ performance, self-assessment of performance, and perception about the switch from a summative to a formative approach. Methods: Medical students from year 2 to 5 (n=648) were included. They could repeat each test once or twice. They rated their performance after each attempt and were then given their score. Detailed feedback was given at the end of the session. An online survey determined medical students’ perception about the reorganization of education. Two items concerned the switch from summative to formative assessments Results: Formative assessments involved 2385 examinees totaling 3197 attempts. Among examinees, 30.8% made at least 2 attempts. Scores increased significantly at the second attempt (median 9.4, IQR 10.8), and duration decreased (median -31.0, IQR 48.0). More than half of examinees (54.6%) underestimated their score, female students more often than male. Low performers overestimated, while high performers underestimated their scores. Students approved of the switch to formative assessments. Stress was lessened but motivation for learning decreased. Conclusions: Medical students’ better scores at a second attempt support a benefit of detailed feedback, learning time and re-test opportunity on performance. Decreased learning motivation and a minority of students repeating the formative assessments point to the positive influence of summative assessment on learning.

RNA helicases represent attractive drug targets as their activity is linked to several human diseases and impacts microbial infectious processes. While some inhibitors of human RNA helicases demonstrated therapeutic potential as anticancer and antiviral drugs in preclinical trials, chemical inhibition of microbial RNA helicases is less investigated. Here, we address this matter by focusing on the RhlE proteobacterial group of RNA helicases. Having previously shown that the RhlE2 RNA helicase is important for the virulence of the opportunistic pathogen Pseudomonas aeruginosa, we screened a library of 1280 molecules for inhibitors of RhlE2 RNA-dependent ATP hydrolytic activity. The most potent inhibitor is the diazo compound Chicago Sky Blue (CSB). Using hydrogen-deuterium exchange mass spectrometry and biochemical assays, we mapped CSB binding to RhlE2 catalytic core and defined its inhibitory mechanism. Targeting microbial RNA helicases as therapeutic strategy is challenging due to potential side-effects linked to protein conservation across life kingdoms. Interestingly, our structure-activity relationship analysis delineates other diazo dyes closely related to CSB differentially affecting RhlE homologs. Our work could thus be exploited for future drug development studies, which are extremely timely considering the increasing spread of antibiotic resistance among bacterial pathogens.

Plasmids need to ensure their transmission to both daughter-cells when their host divides, but should at the same time avoid overtaxing their hosts by directing excessive host-resources toward production of plasmid factors. Naturally occurring plasmids have therefore evolved regulatory mechanisms to restrict their copy-number in response to the volume of the cytoplasm. In many plasmid families, copy-number control is mediated by a small plasmid-specified RNA, which is continuously produced and rapidly degraded, to ensure that its concentration is proportional to the current plasmid copy-number. We show here that pSA564 from the RepA_N-family is regulated by a small antisense RNA (RNA1), which, when over-expressed in trans , blocks plasmid replication and cures the bacterial host. The 5′ untranslated region (5′UTR) of the plasmid replication initiation gene ( repA ) potentially forms two mutually exclusive secondary structures, ON and OFF, where the latter both sequesters the repA ribosome binding site and acts as a rho -independent transcriptional terminator. Duplex formation between RNA1 and the 5′UTR shifts the equilibrium to favor the putative OFF-structure, enabling a single small RNA to down-regulate repA expression at both transcriptional and translational levels. We further examine which sequence elements on the antisense RNA and on its 5′UTR target are needed for this regulation. Finally, we identify the host-encoded exoribonucleases RNase J1 and J2 as the enzymes responsible for rapidly degrading the replication-inhibiting section of RNA1. This region accumulates and blocks RepA expression in the absence of either RNase J1 or J2, which are therefore essential host factors for pSA564 replication in Staphylococcus aureus .

The RhlE DEAD-box RNA helicase protein family is widespread among Proteobacteria, but it is the least understood due to the lack of a clear biological function. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. RhlE1 and RhlE2 diverged during P. aeruginosa evolution; our data indicate that this resulted in a non-redundant biological role, a distinct molecular action and an enzymatic activity differentially stimulated by RNA. Whereas RhlE1 is specifically necessary for bacteria growth in cold, we show that RhlE2 acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts and multiple functionalities indispensable not only for P. aeruginosa environmental adaptation, but also for its virulence. The global action of RhlE2 relies on a unique C-terminal extension, which establishes an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery.

RNA helicases are fundamental players in RNA metabolism: they remodel RNA secondary structures and arrange ribonucleoprotein complexes. While DExH-box RNA helicases function in ribosome biogenesis and splicing in eukaryotes, information is scarce about bacterial homologs. HrpB is the only bacterial DExH-box protein whose structure is solved. Besides the catalytic core, HrpB possesses three accessory domains, conserved in all DExH-box helicases, plus a unique C-terminal extension (CTE). The function of these auxiliary domains remains unknown. Here, we characterize genetically and biochemically Pseudomonas aeruginosa HrpB homolog. We reveal that the auxiliary domains shape HrpB RNA preferences, affecting RNA species recognition and catalytic activity. We show that, among several types of RNAs, the single-stranded poly(A) and the highly structured MS2 RNA strongly stimulate HrpB ATPase activity. In addition, deleting the CTE affects only stimulation by structured RNAs like MS2 and rRNAs, while deletion of accessory domains results in gain of poly(U)-dependent activity. Finally, using hydrogen-deuterium exchange, we dissect the molecular details of HrpB interaction with poly(A) and MS2 RNAs. The catalytic core interacts with both RNAs, triggering a conformational change that reorients HrpB. Regions within the accessory domains and CTE are, instead, specifically responsive to MS2. Altogether, we demonstrate that in bacteria, like in eukaryotes, DExH-box helicase auxiliary domains are indispensable for RNA handling.

RNA decay and RNA maturation are important steps in the regulation of bacterial gene expression. RNase J, which is present in about half of bacterial species, has been shown to possess both endo- and 5 to 3 exo-ribonuclease activities. The exonucleolytic activity is clearly involved in the degradation of mRNA and in the maturation of at least the 5 end of 16S rRNA in the 2 Firmicutes Staphylococcus aureus and Bacillus subtilis. The endoribonuclease activity of RNase J from several species has been shown to be weak in vitro and 3-D structural data of different RNase J orthologs have not provided a clear explanation for the molecular basis of this activity. Here, we show that S. aureus RNase J1 is a manganese dependent homodimeric enzyme with strong 5 to 3 exo-ribonuclease as well as endo-ribonuclease activity. In addition, we demonstrated that SauJ1 can efficiently degrade 5 triphosphorylated RNA. Our results highlight RNase J1 as an important player in RNA turnover in S. aureus.

Staphylococcus aureus is a versatile opportunistic pathogen that adapts readily to a variety of different growth conditions. This adaptation requires a rapid regulation of gene expression including the control of mRNA abundance. The CshA DEAD-box RNA helicase was previously shown to be required for efficient turnover of the agr quorum sensing mRNA. Here we show by transcriptome-wide RNA sequencing and microarray analyses that CshA is required for the degradation of bulk mRNA. Moreover a subset of mRNAs is significantly stabilised in absence of CshA. Deletion of the C-terminal extension affects RNA turnover similar to the full deletion of the cshA gene. In accordance with RNA decay data, the C-terminal region of CshA is required for an RNA-independent interaction with components of the RNA degradation machinery. The C-terminal truncation of CshA reduces its ATPase activity and this reduction cannot be compensated at high RNA concentrations. Finally, the deletion of the C-terminal extension does affect growth at low temperatures, but to a significantly lesser degree than the full deletion, indicating that the core of the helicase can assume a partial function and opening the possibility that CshA is involved in different cellular processes.

RNA helicases of the DEAD-box and DEAH-box families are important players in many processes involving RNA molecules. These proteins can modify RNA secondary structures or intermolecular RNA interactions and modulate RNA-protein complexes. In bacteria, they are known to be involved in ribosome biogenesis, RNA turnover and translation initiation. They thereby play an important role in the adaptation of bacteria to changing environments and to respond to stress conditions.

Background The TRIM5 proteins are cellular restriction factors that prevent retroviral infection in a species-specific manner. Multiple experiments indicate that restriction activity requires accessory host factors, including E2-enzymes. To better understand the mechanism of restriction, we conducted yeast-two hybrid screens to identify proteins that bind to two TRIM5 orthologues. Results The only cDNAs that scored on repeat testing with both TRIM5 orthologues were the proteasome subunit PSMC2 and ubiquitin. Using co-immunoprecipitation assays, we demonstrated an interaction between TRIM5α and PSMC2, as well as numerous other proteasome subunits. Fluorescence microscopy revealed co-localization of proteasomes and TRIM5α cytoplasmic bodies. Forster resonance energy transfer (FRET) analysis indicated that the interaction between TRIM5 and PSMC2 was direct. Previous imaging experiments demonstrated that, when cells are challenged with fluorescently-labeled HIV-1 virions, restrictive TRIM5α orthologues assemble cytoplasmic bodies around incoming virion particles. Following virus challenge, we observed localization of proteasome subunits to rhTRIM5α cytoplasmic bodies that contained fluorescently labeled HIV-1 virions. Conclusions Taken together, the results presented here suggest that localization of the proteasome to TRIM5α cytoplasmic bodies makes an important contribution to TRIM5α-mediated restriction.

Copyright information: Taken from Biochemical and genetic analysis of RNA cap guanine-N2 methyltransferases from and Nucleic Acids Research 2007;35(5):1411-1420.Published online 6 Feb 2007PMCID:PMC1865056.© 2007 The Author(s). A p( ) haploid strain of was transformed with plasmids bearing wild-type , mutants and or wild-type . A control transformation was performed with the empty pREP81x vector. Leu transformants were tested for growth on medium containing FOA. The plates were photographed after 3 days of incubation at 30°C.

TRIM5 is an E3 ubiquitin ligase with known antiretroviral restriction factor activity, although the mechanisms involved are poorly understood. Luban and colleagues now demonstrate that TRIM5 activates innate immune signalling pathways and acts as a pattern recognition receptor specific for the retrovirus capsid lattice. TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm1,2. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice3,4 and several reports indicate that TRIM5 has a role in signal transduction5,6,7, but the precise mechanism of restriction is unknown8. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13–UEV1A (also known as UBE2N–UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13–UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13–UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.