Lukas Rajkowitsch

RNA chaperones, RNA annealers and RNA helicases.

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.

Structural insights into the dynamics and function of the C-terminus of the E. coli RNA chaperone Hfq

The hexameric Escherichia coli RNA chaperone Hfq (HfqEc) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of HfqEc. These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of HfqEc.

Assays for the RNA chaperone activity of proteins

Proteins with RNA chaperone activity promote RNA folding by loosening the structure of misfolded RNAs or by preventing their formation. How these proteins achieve this activity is still unknown, the mechanism is not understood and it is unclear whether this activity is always based on the same mechanism or whether different RNA chaperones use different mechanisms. To address this question, we compare and discuss in this paper a set of assays that have been used to measure RNA chaperone activity. In some assays, this activity is related to the acceleration of monomolecular reactions such as group I intron cis-splicing or anti-termination of transcription. Hereby, it is proposed that the proteins release the RNAs from folding traps, which represent the kinetic barriers during the folding process and involve the loosening of structural elements. In most assays, however, bimolecular reactions are monitored, which include the simple acceleration of annealing of two complementary RNAs, the turnover stimulation of ribozyme cleavage and group I intron trans-splicing. The acceleration of these reactions most probably involves the unfolding of structures that interfere with annealing or folding and may in addition provoke annealing by crowding. Most assays are performed in vitro, where conditions might differ substantially from intracellular conditions, and two assays have been reported that detect RNA chaperone activity in vivo.

coupling RNA annealing and strand displacement: a FRET-based microplate reader assay for RNA chaperone activity

Proteins with RNA chaperone activity help RNAs to obtain their native conformations, and many of them are active in the two basic reactions-RNA annealing and strand displacement. Therefore, we developed a time-saving in vitro assay that detects protein-facilitated annealing and strand displacement of fluorophore-labeled oligoribonucleotides in a microplate reader The two reactions are followed byfluorescence resonance energy transfer (FRET) in real-time, and the effect of the proteins on the reaction constants can be quantified. The high-throughput property of the fluorescence microplate reader the kinetic characterization, and the material-saving aspect of this assay enables a fast and convenient classification of proteins according to their RNA chaperone activity in annealing and strand displacement.

Structural and Biochemical Studies on ATP Binding and Hydrolysis by the Escherichia coli RNA Chaperone Hfq

In Escherichia coli the RNA chaperone Hfq is involved in riboregulation by assisting base-pairing between small regulatory RNAs (sRNAs) and mRNA targets. Several structural and biochemical studies revealed RNA binding sites on either surface of the donut shaped Hfq-hexamer. Whereas sRNAs are believed to contact preferentially the YKH motifs present on the proximal site, poly(A)15 and ADP were shown to bind to tripartite binding motifs (ARE) circularly positioned on the distal site. Hfq has been reported to bind and to hydrolyze ATP. Here, we present the crystal structure of a C-terminally truncated variant of E. coli Hfq (Hfq65) in complex with ATP, showing that it binds to the distal R-sites. In addition, we revisited the reported ATPase activity of full length Hfq purified to homogeneity. At variance with previous reports, no ATPase activity was observed for Hfq. In addition, FRET assays neither indicated an impact of ATP on annealing of two model oligoribonucleotides nor did the presence of ATP induce strand displacement. Moreover, ATP did not lead to destabilization of binary and ternary Hfq-RNA complexes, unless a vast stoichiometric excess of ATP was used. Taken together, these studies strongly suggest that ATP is dispensable for and does not interfere with Hfq-mediated RNA transactions.