RNA-helicases are proteins required for the unwinding of occluding secondary RNA structures, especially at low temperatures. In this work, we have deleted all 4 DExD-box RNA helicases in various combinations in the Gram-positive pathogen Listeria monocytogenes. Our results show that 3 out of 4 RNA-helicases were important for growth at low temperatures, whereas the effect was less prominent at 37°C. Over-expression of one RNA-helicase, Lmo1450, was able to overcome the reduced growth of the quadruple mutant strain at temperatures above 26°C, but not at lower temperatures. The maturation of ribosomes was affected in different degrees in the various strains at 20°C, whereas the effect was marginal at 37°C. This was accompanied by an increased level of immature 23S rRNA precursors in some of the RNA-helicase mutants at low temperatures. Although the expression of the PrfA regulated virulence factors ActA and LLO decreased in the quadruple mutant strain, this strain showed a slightly increased infection ability. Interestingly, even though the level of the virulence factor LLO was decreased in the quadruple mutant strain as compared with the wild-type strain, the hly-transcript (encoding LLO) was increased. Hence, our results could suggest a role for the RNA-helicases during translation. In this work, we show that DExD-box RNA-helicases are involved in bacterial virulence gene-expression and infection of eukaryotic cells.

Double-stranded RNAs are an important class of functional macromolecules in living systems. They are usually found as part of highly specialized intracellular machines that control diverse cellular events, ranging from virus replication, antiviral defense, RNA interference, to regulation of gene activities and genomic integrity. Within different intracellular machines, the RNA duplex is often found in association with specific RNA-dependent ATPases, including Dicer, RIG-I and DRH-3 proteins. These duplex RNA-activated ATPases represent an emerging group of motor proteins within the large and diverse super family 2 nucleic acid-dependent ATPases (which are historically defined as SF2 helicases). The duplex RNA-activated ATPases share characteristic molecular features for duplex RNA recognition, including motifs (e.g., motifs IIa and Vc) and an insertion domain (HEL2i), and they require double-strand RNA binding for their enzymatic activities. Proteins in this family undergo large conformational changes concomitant with RNA binding, ATP binding and ATP hydrolysis in order to achieve their functions, which include the release of signaling domains and the recruitment of partner proteins. The duplex RNA-activated ATPases represent a distinct and fascinating group of nanomechanical molecular motors that are essential for duplex RNA sensing and processing in diverse cellular pathways.

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.

A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase. Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis. Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43. Changes in the expression levels of Prp43-interacting G-patch proteins modulate the cellular localization of Prp43 and G-patch protein overexpression causes accumulation of the helicase in the cytoplasm or nucleoplasm. Overexpression of several G-patch proteins also leads to defects in ribosome biogenesis that are consistent with withdrawal of the helicase from this pathway. Together, these findings suggest that the availability of cofactors and the sequestering of the helicase are means to regulate the activity of multifunctional RNA helicases and their distribution between different cellular processes.

DEAD-box RNA helicases are present in almost all living organisms and participate in various processes of RNA metabolism. Bacterial proteins of this large family were shown to be required for translation initiation, ribosome biogenesis and RNA decay. The latter is primordial for rapid adaptation to changing environmental conditions. In particular, the RhlB RNA helicase from E. coli was shown to assist the bacterial degradosome machinery. Recently, the CshA DEAD-box proteins from Bacillus subtilis and Staphylococcus aureus were shown to interact with proteins that are believed to form the degradosome. S. aureus can cause life-threatening disease, with particular concern focusing on biofilm formation on catheters and prosthetic devices, since in this form the bacteria are almost impossible to eradicate both by the immune system and antibiotic treatment. This persistent state relies on the expression of surface encoded proteins that allow attachment to various surfaces, and contrasts with the dispersal mode of growth that relies on the secretion of proteins such as hemolysins and proteases. The switch between these two states is mainly mediated by the Staphylococcal cell density sensing system encoded by agr. We show that inactivation of the cshA DEAD-box gene results in dysregulation of biofilm formation and hemolysis through modulation of agr mRNA stability. Importantly, inactivation of the agrA gene in the cshA mutant background reverses the defect, indicating that cshA is genetically upstream of agr and that a delicate balance of agr mRNA abundance mediated through stability control by CshA is critical for proper expression of virulence factors.

Ribosome synthesis is an essential cellular process, and perturbation of human ribosome production is linked to cancer and genetic diseases termed ribosomopathies. During their assembly, pre-ribosomal particles undergo numerous structural rearrangements, which establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome biogenesis. RNA helicases are essential mediators of such remodelling events and here, we demonstrate that the DEAH-box RNA helicase DHX37 is required for maturation of the small ribosomal subunit in human cells. Our data reveal that the presence of DHX37 in early pre-ribosomal particles is monitored by a quality control pathway and that failure to recruit DHX37 leads to pre-rRNA degradation. Using an in vivo crosslinking approach, we show that DHX37 binds directly to the U3 small nucleolar RNA (snoRNA) and demonstrate that the catalytic activity of the helicase is required for dissociation of the U3 snoRNA from pre-ribosomal complexes. This is an important event during ribosome assembly as it enables formation of the central pseudoknot structure of the small ribosomal subunit. We identify UTP14A as a direct interaction partner of DHX37 and our data suggest that UTP14A can act as a cofactor that stimulates the activity of the helicase in the context of U3 snoRNA release.

CsdA is one of five E. coli DEAD-box helicases and as a cold-shock protein assists RNA structural remodeling at low temperatures. The helicase has been shown to catalyze duplex unwinding in an ATP-dependent way and accelerate annealing of complementary RNAs, but detailed kinetic analyses are missing. Therefore, we performed kinetic measurements using a coupled annealing and strand displacement assay with high temporal resolution to analyze how CsdA balances the two converse activities. We furthermore tested the hypothesis that the unwinding activity of DEAD-box helicases is largely determined by the substrate's thermodynamic stability using full-length CsdA and a set of RNAs with constant length, but increasing GC content. The rate constants for strand displacement did indeed decrease with increasing duplex stability, with a calculated free energy between -31.3 and -40 kcal/mol being the limit for helix unwinding. Thus, our data generally support the above hypothesis, showing that for CsdA substrate thermal stability is an important rate limiting factor.

RNA helicases contribute to diverse aspects of RNA metabolism through their functions in re-arranging RNA structures. Identification of the remodelling targets of RNA helicases is a critical step in elucidating their cellular functions. Here, we show that, in contrast to many other ribosome biogenesis factors, the DExD box ATPase DDX55 predominantly localizes to the nucleoplasm and we identify a nuclear localization signal within the C-terminal region of the protein. DDX55 associates with pre-ribosomal subunits in human cells and is required for maturation of large subunit pre-rRNAs. Interestingly, in vitro analyses show that DDX55 selectively associates with double-stranded RNA substrates, which also stimulate its ATPase activity, and our data suggest that the C-terminal region of DDX55 contributes to this substrate specificity. The C-terminal region of DDX55 is also necessary for recruitment of the helicase to pre-ribosomes and, using in vivo crosslinking, we reveal a binding site for DDX55 in helix H62 of the 28S ribosomal RNA. Taken together, these data highlight the importance of the C-terminal region of DDX55 in substrate specificity and recruitment, and identify domain IV as a potential remodelling target of DDX55 during LSU biogenesis.

DEAD-box RNA-helicases catalyze the reorganization of structured RNAs and the formation of RNP complexes. The cyanobacterium Synechocystis sp. PCC 6803 encodes a single DEAD-box RNA helicase, CrhR (Slr0083), whose expression is regulated by abiotic stresses that alter the redox potential of the photosynthetic electron transport chain, including temperature downshift. Despite its proposed effect on RNA metabolism and its known relevance in cold-stress adaptation, the reported impact of a CrhR knockout on the cold adaption of the transcriptome only identified eight affected genes. Here, we utilized a custom designed microarray to assess the impact of the absence of CrhR RNA helicase activity on the transcriptome, independent of cold stress. CrhR truncation impacts an RNA subset comprising ~10% of the ncRNA and also ~10% of the mRNA transcripts. While equal numbers of mRNAs showed increased as well as decreased abundance, more than 90% of the ncRNAs showed enhanced expression in the absence of CrhR, indicative of a negative effect on ncRNA transcription or stability. We further tested the effect of CrhR on the stability of strongly responding RNAs that identify examples of post-transcriptional and transcriptional regulation. The data suggest that CrhR impacts multiple aspects of RNA metabolism in Synechocystis.

Assembly of eukaryotic ribosomal subunits is a complex and dynamic process involving the action of more than 200 trans-acting assembly factors. Although recent cryo-electron microscopy structures have provided information on architecture of several pre-ribosomal particles and the binding sites of many AFs, the RNA and protein interactions of many other AFs not captured in these snapshots still remain elusive. RNA helicases are key regulators of structural rearrangements within pre-ribosomal complexes and here we have analysed the eIF4A-like RNA helicase Fal1 and its putative cofactor Sgd1. Our data show that these proteins interact directly via the MIF4G domain of Sgd1 and that the MIF4G domain of Sgd1 stimulates the catalytic activity of Fal1 in vitro. The catalytic activity of Fal1, and the interaction between Fal1 and Sgd1, are required for efficient pre-rRNA processing at the A0, A1 and A2 sites. Furthermore, Sgd1 co-purifies the early small subunit biogenesis factors Lcp5 and Rok1, suggesting that the Fal1-Sgd1 complex likely functions within the SSU processome. In vivo crosslinking data reveal that Sgd1 binds to helix H12 of the 18S rRNA sequence and we further demonstrate that this interaction is formed by the C-terminal region of the protein, which is essential for its function in ribosome biogenesis.

Ribosome production is an essential cellular process involving a plethora of trans-acting factors, such as nucleases, methyltransferases, RNA helicases and kinases that catalyse key maturation steps. Precise temporal and spatial regulation of such enzymes is essential to ensure accurate and efficient subunit assembly. Here, we focus on the maturation of the 3' end of the 18S rRNA in human cells. We reveal that human RIO2 is an active kinase that phosphorylates both itself and the rRNA methyltransferase DIM1 in vitro. In contrast to yeast, our data confirm that human DIM1 predominantly acts in the nucleus and we further demonstrate that the 21S pre-rRNA is the main target for DIM1-catalysed methylation. We show that the PIN domain of the endonuclease NOB1 is required for site 3 cleavage, while the zinc ribbon domain is essential for pre-40S recruitment. Furthermore, we also demonstrate that NOB1, PNO1 and DIM1 bind to a region of the pre-rRNA encompassing the 3' end of 18S and the start of ITS1, in vitro. Interestingly, NOB1 is present in the cell at higher levels than other pre-40S factors. We provide evidence that NOB1 is multimeric within the cell and show that NOB1 multimerisation is lost when ribosome biogenesis is blocked. Taken together, our data indicate a dynamic interplay of key factors associated with the 3' end of the 18S rRNA during human pre-40S biogenesis and highlight potential mechanisms by which this process can be regulated.