The RFN element is a highly conserved domain that is found frequently in the 5 -untranslated regions of prokaryotic mRNAs that encode for flavin mononucleotide (FMN) biosynthesis and transport proteins. We report that this domain serves as the receptor for a metabolite-dependent riboswitch that directly binds FMN in the absence of proteins. Our results also indicate that in Bacillus subtilis, the riboswitch most likely controls gene expression by causing premature transcription termination of the rib-DEAHT operon and precluding access to the ribosome-binding site of ypaA mRNA. Sequence and structural analyses indicate that the RFN element is a natural FMN-binding aptamer, the allosteric character of which is harnessed to control gene expression.T he expression of certain genes is controlled by mRNA elements that form receptors for target metabolites. Selective binding of a metabolite by such an mRNA ''riboswitch'' permits a shift in the conformation to an alternative structure that results ultimately in the modulation of protein synthesis. For example, the btuB gene of Escherichia coli carries a highly conserved sequence element termed the B 12 box (1) in the 5Ј UTR of the mRNA that directly binds coenzyme B 12 with high selectivity (2). This binding event seems to operate via an allosteric mechanism that represses expression of a reporter gene to Ϸ1% of that observed in cells grown in the absence of added coenzyme B 12 . Similarly, many organisms carry a highly conserved ''thi box'' sequence (3) in mRNAs that are required for the biosynthesis of the coenzyme thiamine pyrophosphate. An expanded region of RNA that encompasses the thi box also has been shown (4) to function as a riboswitch by directly binding thiamine pyrophosphate, resulting in reduced translation of thiM and thiC mRNAs in E. coli. It is conceivable that riboswitches also could modulate transcription termination, although only proteins (5, 6) or RNAs are currently known to regulate transcription termination in trans (7).Another highly conserved but distinct RNA domain, termed the RFN element, has been identified in mRNAs of prokaryotic genes required for the biosynthesis of riboflavin and FMN (8, 9). It is known (10, 11) that FMN is required for down-regulation of the ribDEAHT operon (hereafter termed ribD) of Bacillus subtilis, which encodes several FMN biosynthetic enzymes. Furthermore, mutations within the RFN element of ribD eliminate FMN-mediated regulation (12, 13). Sequence comparisons of RFN elements from various riboflavin biosynthesis genes and ypaA (a putative riboflavin transport protein) (8, 14) have been used to generate a secondary structure model (Fig. 1A) and to establish the conserved nucleotides of this domain (8, 9). The structural model is composed of a six-stem junction wherein extensive sequence conservation exists at the bases of the stem elements and among the intervening nucleotides that form the core of the junction (Fig. 1 A).It has been proposed that either an unidentified FMNdependent protein effector (15) or perhaps...

Thiamine metabolism genes are regulated in numerous bacteria by a riboswitch class that binds the coenzyme thiamine pyrophosphate (TPP). We demonstrate that the antimicrobial action of the thiamine analog pyrithiamine (PT) is mediated by interaction with TPP riboswitches in bacteria and fungi. For example, pyrithiamine pyrophosphate (PTPP) binds the TPP riboswitch controlling the tenA operon in Bacillus subtilis. Expression of a TPP riboswitch-regulated reporter gene is reduced in transgenic B. subtilis or Escherichia coli when grown in the presence of thiamine or PT, while mutant riboswitches in these organisms are unresponsive to these ligands. Bacteria selected for PT resistance bear specific mutations that disrupt ligand binding to TPP riboswitches and derepress certain TPP metabolic genes. Our findings demonstrate that riboswitches can serve as antimicrobial drug targets and expand our understanding of thiamine metabolism in bacteria.

Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite Trypanosoma brucei, exosome secretion is induced by stress that affects trans-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites.

Interferon-gamma mRNA activates the RNA-dependent protein kinase PKR, which in turn strongly attenuates translation of interferon-gamma mRNA. Unlike riboswitches restricted to noncoding regions, the interferon-gamma RNA domain that activates PKR comprises the 5' UTR and 26 translated codons. Extensive interferon-gamma coding sequence is thus dedicated to activating PKR and blocking interferon-gamma synthesis. This implies that the PKR activator is disrupted by ribosomes during translation initiation and must refold promptly to restore PKR activation. The activator structure harbors an essential kink-turn, probably to allow formation of a pseudoknot that is critical for PKR activation. Three indispensable short helices, bordered by orientation-sensitive base pairs, align with the pseudoknot stem, generating RNA helix of sufficient length to activate PKR. Through gain-of-function mutations, we show that the RNA activator can adopt alternative conformations that activate PKR. This flexibility promotes efficient refolding of interferon-gamma mRNA, which is necessary for its dual function as translation template and activator of PKR, and which thus prevents overexpression of this inflammatory cytokine.

The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an insect and a mammalian host. Here, we investigated the presence of pseudouridines (Ψs) on the spliceosomal small nuclear RNAs (snRNAs), which may enable growth at the very different temperatures characterizing the two hosts. To this end, we performed the first high-throughput mapping of spliceosomal snRNA Ψs by small RNA Ψ-seq. The analysis revealed 42 Ψs on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding Ψ on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA Ψs are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth at elevated temperatures. We further demonstrate that Ψ strengthens U4/U6 RNA–RNA and U2B"/U2A’ proteins-U2 snRNA interaction at elevated temperatures. The existence of single hairpin RNAs that modify both the spliceosome and ribosome RNAs is unique for these parasites, and may be related to their ability to cycle between their two hosts that differ in temperature.