Search

CRISPR type III systems, which are abundantly found in archaea, recognize and degrade RNA in their specific response to invading nucleic acids. Therefore, these systems can be harnessed for gene knockdown technologies even in hyperthermophilic archaea to study essential genes. We show here the broader usability of this posttranscriptional silencing technology by expanding the application to further essential genes and systematically analysing and comparing silencing thresholds and escape mutants. Synthetic guide RNAs expressed from miniCRISPR cassettes were used to silence genes involved in cell division ( cdvA ), transcription ( rpo8 ), and RNA metabolism ( smAP2 ) of the two crenarchaeal model organisms Saccharolobus solfataricus and Sulfolobus acidocaldarius . Results were systematically analysed together with those obtained from earlier experiments of cell wall biogenesis ( slaB ) and translation ( aif5A ). Comparison of over 100 individual transformants revealed gene-specific silencing maxima ranging between 40 and 75%, which induced specific knockdown phenotypes leading to growth retardation. Exceedance of this threshold by strong miniCRISPR constructs was not tolerated and led to specific mutation of the silencing miniCRISPR array and phenotypical reversion of cultures. In two thirds of sequenced reverted cultures, the targeting spacers were found to be precisely excised from the miniCRISPR array, indicating a still hypothetical, but highly active recombination system acting on the dynamics of CRISPR spacer arrays. Our results indicate that CRISPR type III - based silencing is a broadly applicable tool to study in vivo functions of essential genes in Sulfolobales which underlies a specific mechanism to avoid malignant silencing overdose.

Translation factor a/eIF5A is highly conserved in Eukarya and Archaea. The eukaryal eIF5A protein is required for transit of ribosomes across consecutive proline codons, whereas the function of the archaeal orthologue remains unknown. Here, we provide a first hint for an involvement of Sulfolobus solfataricus (Sso) aIF5A in translation. CRISPR-mediated knock down of the aif5A gene resulted in strong growth retardation, underlining a pivotal function. Moreover, in vitro studies revealed that Sso aIF5A is endowed with endoribonucleolytic activity. Thus, aIF5A appears to be a moonlighting protein that might be involved in protein synthesis as well as in RNA metabolism.

At low temperatures the Escherichia coli rpoS mRNA, encoding the stationary phase sigma factor RpoS, forms an intramolecular secondary structure (iss) that impedes translation initiation. Under these conditions the small RNA DsrA, which is stabilzed by Hfq, forms a duplex with rpoS mRNA sequences opposite of the ribosome-binding site (rbs). Both the DEAD box helicase CsdA and Hfq have been implicated in DsrA·rpoS duplex formation. Hfq binding to A-rich sequences in the rpoS leader has been suggested to restructure the mRNA, and thereby to accelerate DsrA·rpoS duplex formation, which, in turn, was deemed to free the rpoS rbs and to permit ribosome loading on the mRNA. Several experiments designed to elucidate the role of Hfq in DsrA-mediated translational activation of rpoS mRNA have been conducted in vitro. Here, we assessed RpoS synthesis in vivo to further study the role of Hfq in rpoS regulation. We show that RpoS synthesis was reduced when DsrA was ectopically overexpressed at 24 °C in the absence of Hfq despite of DsrA·rpoS duplex formation. This observation indicated that DsrA·rpoS annealing may not be sufficient for efficient ribosome loading on rpoS mRNA. In addition, a HfqG29A mutant protein was employed, which is deficient in binding to A-rich sequences present in the rpoS leader but proficient in DsrA binding. We show that DsrA·rpoS duplex formation occurs in the presence of the HfqG29A mutant protein at low temperature, whereas synthesis of RpoS was greatly diminished. RNase T1 footprinting studies of DsrA·rpoS duplexes in the absence and presence of Hfq or HfqG29A indicated that Hfq is required to resolve a stem-loop structure in the immediate coding region of rpoS mRNA. These in vivo studies corroborate the importance of the A-rich sequences in the rpoS leader and strongly suggest that Hfq, besides stabilizing DsrA and accelerating DsrA·rpoS duplex formation, is also required to convert the rpoS mRNA into a translationally competent form.

A His-tag is often added by means of recombinant DNA technology to a heterologous protein of interest, which is then over-produced in Escherchia coli and purified by one-step immobilized metal-affinity chromatography (IMAC). Owing to the presence of 24 histidines at the C-termini of the hexameric E. coli RNA chaperone Hfq, the protein co-purifies with His-tagged proteins of interest. As Hfq can bind to distinct RNA substrates with high affinity, its presence can obscure studies performed with (putative) RNA binding activities purified by IMAC. Here, we present results for a seemingly positive RNA-binding activity, exemplifying that false-positive results can be avoided if the protein of interest is either subjected to further purification step(s) or produced in an E. coli hfq- strain.

The opportunistic human pathogen Pseudomonas aeruginosa is frequently associated with nosocomial infections, and can be life threatening in immunosuppressed, cancer and cystic fibrosis patients. Virulence in P. aeruginosa is combinatorial, and results from the activation of several genetic programs that regulate motility, attachment to the host epithelium as well as the synthesis of exotoxins. The pathogen has a high survival capacity in the host owing to its metabolic versatility, nutrient scavenging and resistance against both, antibiotics and immune defenses. Adaptive responses to various environmental stresses and stimuli are often regulated by small regulatory RNAs (sRNA). In this review, we summarize the current knowledge on the regulation and function of P. aeruginosa sRNAs that titrate regulatory proteins, base-pair with target mRNAs, and which are derived from CRISPR elements.

The ribosome binding site of Escherichia coli rpoS mRNA, encoding the stationary sigma-factor RpoS, is sequestered by an inhibitory stem-loop structure (iss). Translational activation of rpoS mRNA at low temperature and during exponential growth includes Hfq-facilitated duplex formation between rpoS and the small regulatory RNA DsrA as well as a concomitant re-direction of RNAse III cleavage in the 5´-untranslated region of rpoS upon DsrA·rpoS annealing. In this way, DsrA-mediated regulation does not only activate rpoS translation by disrupting the inhibitory secondary structure but also stabilizes the rpoS transcript. Although minor structural changes by Hfq have been observed in rpoS mRNA, a prevailing question concerns unfolding of the iss in rpoS at low growth temperature. Here, we have identified the DEAD-box helicase CsdA as an ancillary factor required for low temperature activation of RpoS synthesis by DsrA. The lack of RpoS synthesis observed in the csdA mutant strain at low growth temperature could be attributed to a lack of duplex formation between rpoS and DsrA, showing that at low temperature the sole action of Hfq is not sufficient to permit DsrA·rpoS annealing. An interactome study has previously indicated an association between Hfq and CsdA. However, immunological assays did not reveal a physical interaction between Hfq and CsdA. These findings add to a model, wherein Hfq binds upstream of the rpoS iss and presents DsrA in a conformation receptive to annealing. Melting of the iss by CsdA may then permit DsrA·rpoS duplex formation, and consequently rpoS translation.

RNA molecules face difficulties when folding into their native structures. In the cell, proteins can assist RNAs in reaching their functionally active states by binding and stabilizing a specific structure or, in a quite opposite way, by interacting in a non-specific manner. These proteins can either facilitate RNA-RNA interactions in a reaction termed RNA annealing, or they can resolve non-functional inhibitory structures. The latter is defined as "RNA chaperone activity" and is the main topic of this review. Here we define RNA chaperone activity in a stringent way and we review those proteins for which RNA chaperone activity has been clearly demonstrated. These proteins belong to quite diverse families such as hnRNPs, histone-like proteins, ribosomal proteins, cold shock domain proteins and viral nucleocapsid proteins. DExD/H-box containing RNA helicases are discussed as a special family of enzymes that restructure RNA or RNPs in an ATP-dependent manner. We further address the different mechanisms RNA chaperones might use to promote folding including the recently proposed theory of protein disorder as a key element in triggering RNA-protein interactions. Finally, we present a new website for proteins with RNA chaperone activity which compiles all the information on these proteins with the perspective to promote the understanding of their activity.