Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria.

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.

Formative assessments during COVID-19 pandemic: an observational study on performance and experiences of medical students.

: 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. : Medical students from year 2 to 5 (n=648) were included.

Mechanism of inhibition of bacterial RNA helicases by diazo dyes and implications for antimicrobial drug development.

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.

The DEAD-box RNA helicase RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs.

RNase J1 and J2 Are Host-Encoded Factors for Plasmid Replication.

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.

Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences.

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.

Both exo- and endo-nucleolytic activities of RNase J1 from Staphylococcus aureus are manganese dependent and active on triphosphorylated 5'-ends.

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 C-terminal region of the RNA helicase CshA is required for the interaction with the degradosome and turnover of bulk RNA in the opportunistic pathogen Staphylococcus 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.

Bacterial versatility requires DEAD-box RNA helicases.

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.

TRIM5α associates with proteasomal subunits in cells while in complex with HIV-1 virions.

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.

TRIM5 is an innate immune sensor 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 cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice.

Short double-stranded RNAs with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as RIG-I decoys.

Arenavirus RNA genomes are initiated by a "prime and realign" mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to blunt-ended (5' ppp)dsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes.

The double-stranded RNA binding domain of the vaccinia virus E3L protein inhibits both RNA- and DNA-induced activation of interferon beta.

Vaccinia virus, a large DNA virus that replicates in the cytoplasm, expresses its E3L protein to inhibit the cellular innate immune response and apoptosis. E3L is a bifunctional protein that contains an N-terminal DNA binding domain (BD) and a C-terminal double-stranded RNA (dsRNA)-BD (residues 100-190), both of which contribute to viral pathogenesis by blocking the activation of cellular genes that respond to the viral infection. We report that expression of the dsRNA-BD alone inhibits not only the dsRNA-induced activation of interferon beta (IFNbeta) but also that of 5'-triphosphate single-stranded RNA and DNA-induced IFNbeta activation even though E3L(100-190) does not bind the latter two pathogen-associated molecular patterns.

RIG-I and dsRNA-induced IFNbeta activation.

Except for viruses that initiate RNA synthesis with a protein primer (e.g., picornaviruses), most RNA viruses initiate RNA synthesis with an NTP, and at least some of their viral (ppp)RNAs remain unblocked during the infection.

Genetic and biochemical analysis of yeast and human cap trimethylguanosine synthase: functional overlap of 2,2,7-trimethylguanosine caps, small nuclear ribonucleoprotein components, pre-mRNA splicing factors, and RNA decay pathways.

Trimethylguanosine synthase (Tgs1) is the enzyme that converts standard m(7)G caps to the 2,2,7-trimethylguanosine (TMG) caps characteristic of spliceosomal small nuclear RNAs. Fungi and mammalian somatic cells are able to grow in the absence of Tgs1 and TMG caps, suggesting that an essential function of the TMG cap might be obscured by functional redundancy. A systematic screen in budding yeast identified nonessential genes that, when deleted, caused synthetic growth defects with tgs1Delta.

Activation of the beta interferon promoter by unnatural Sendai virus infection requires RIG-I and is inhibited by viral C proteins.

As infection with wild-type (wt) Sendai virus (SeV) normally activates beta interferon (IFN-beta) very poorly, two unnatural SeV infections were used to study virus-induced IFN-beta activation in mouse embryonic fibroblasts: (i) SeV-DI-H4, which is composed mostly of small, copyback defective interfering (DI) genomes and whose infection overproduces short 5'-triphosphorylated trailer RNAs (pppRNAs) and underproduces viral V and C proteins, and (ii) SeV-GFP(+/-), a coinfection that produces wt amounts of viral gene products but that also produces both green fluorescent protein (GFP) mRNA and its complement, which can form double-stranded RNA (dsRNA) with capped 5' ends. We found that (i) virus-induced signaling to IFN-beta depended predominantly on RIG-I (as opposed to mda-5) for both SeV infections, i.e.

Sendai virus RNA polymerase scanning for mRNA start sites at gene junctions.

Mini-genomes expressing two reporter genes and a variable gene junction were used to study Sendai virus RNA polymerase (RdRp) scanning for the mRNA start signal of the downstream gene (gs2). We found that RdRp could scan the template efficiently as long as the initiating uridylate of gs2 (3' UCCCnnUUUC) was preceded by the conserved intergenic region (3' GAA) and the last 3 uridylates of the upstream gene end signal (ge1; 3' AUUCUUUUU). The end of the leader sequence (3' CUAAAA, which precedes gs1) could also be used for gene2 expression, but this sequence was considerably less efficient.

Biochemical and genetic analysis of RNA cap guanine-N2 methyltransferases from Giardia lamblia and Schizosaccharomyces pombe.

RNA cap guanine-N2 methyltransferases such as Schizosaccharomyces pombe Tgs1 and Giardia lamblia Tgs2 catalyze methylation of the exocyclic N2 amine of 7-methylguanosine. Here we performed a mutational analysis of Giardia Tgs2, entailing an alanine scan of 17 residues within the minimal active domain. Alanine substitutions at Phe18, Thr40, Asp76, Asn103 and Asp140 reduced methyltransferase specific activity to <3% of wild-type Tgs2, thereby defining these residues as essential.

Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin's antifungal activity.

Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guanine-binding pocket, alone and in combination.

Poxvirus mRNA cap methyltransferase. Bypass of the requirement for the stimulatory subunit by mutations in the catalytic subunit and evidence for intersubunit allostery.

The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit vD1-(540-844) and a stimulatory subunit vD12. The poxvirus enzyme can function in vivo in Saccharomyces cerevisiae in lieu of the essential cellular cap methyltransferase Abd1. Coexpression of both poxvirus subunits is required to complement the growth of abd1delta cells.

Different strategies for carboxyl-terminal domain (CTD) recognition by serine 5-specific CTD phosphatases.

The phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II, consisting of ((1)YSPTSPS(7))(n) heptad repeats, encodes information about the state of the transcriptional apparatus that can be conveyed to factors that regulate mRNA synthesis and processing. Here we describe how the CTD code is read by two classes of protein phosphatases, plant CPLs and yeast Ssu72, that specifically dephosphorylate Ser(5) in vitro. The CPLs and Ssu72 recognize entirely different positional cues in the CTD primary structure.

Giardia lamblia RNA cap guanine-N2 methyltransferase (Tgs2).

Tgs1 is the enzyme responsible for converting 7-methylguanosine RNA caps to the 2,2,7-trimethylguanosine cap structures of small nuclear and small nucleolar RNAs. Whereas budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe encode a single Tgs1 protein, the primitive eukaryote Giardia lamblia encodes two paralogs, Tgs1 and Tgs2. Here we show that purified Tgs2 is a monomeric enzyme that catalyzes methyl transfer from AdoMet (K(m) of 6 microm) to m(7)GDP (K(m) of 65 microm; k(cat) of 14 min(-1)) to form m(2,7)GDP.

Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis.

The Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase Ecm1 has been characterized structurally but not biochemically. Here we show that purified Ecm1 is a monomeric protein that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to GTP. The reaction is cofactor-independent and optimal at pH 7.

Specificity and mechanism of RNA cap guanine-N2 methyltransferase (Tgs1).

The 2,2,7-trimethylguanosine (TMG) cap structure is characteristic of certain eukaryotic small nuclear and small nucleolar RNAs. Prior studies have suggested that cap trimethylation might be contingent on cis-acting elements in the RNA substrate, protein components of a ribonucleoprotein complex, or intracellular localization of the RNA substrate. However, the enzymatic requirements for TMG cap formation remain obscure because TMG synthesis has not been reconstituted in vitro from defined components.