Dissecting RNA chaperone activity

Abstract

Many RNA-binding proteins help RNAs to fold via their RNA chaperone activity. This term has been used widely without accounting for the diversity of the observed reactions, which include complex events like restructuring of misfolded catalytic RNAs, promoting the assembly of RNA-protein complexes, and mediating RNA-RNA interactions. Proteins display very diverse activities depending on the assays used to measure RNA chaperone activity. To classify proteins with this activity, we compared three exemplary proteins from E. coli, host factor Hfq, ribosomal protein S1, and the histone-like protein StpA for their abilities to promote two simple reactions, RNA annealing and strand displacement. The results of a FRET-based assay show that S1 promotes only RNA strand displacement while Hfq solely enhances RNA annealing. StpA, in contrast, is active in both reactions. To test whether the two activities can be assigned to different domains of the bipartite-structured StpA, we assayed the purified N-and C-terminal domains separately. While both domains are unable to promote RNA annealing, we can attribute the RNA strand displacement activity of StpA to the C-terminal domain. Correlating with their RNA annealing activities, only Hfq and full-length StpA display simultaneous binding of two RNAs, suggesting a matchmaker-like model for this activity. For StpA, this ''RNA crowding'' requires protein-protein interactions, since a dimerization-deficient StpA mutant lost the ability to bind and anneal two RNAs. These results underline the difference between the two reaction types, making it necessary to distinguish and classify proteins according to their specific RNA chaperone activities.

Phosphorylation of ORF1p is required for L1 retrotransposition

“…RNA binding and nucleic acid chaperone activity are important biochemical properties of ORF1p, and facilitated annealing of RNA is an essential component of chaperone activity (64); therefore, we compared this activity for WT and mutant ORF1p using FRET (65). In this assay, RNA annealing allows transfer of emission energy from an excited donor fluorophore on one RNA strand to an acceptor fluorophore on its complementary strand (Fig.…”

RNAs actively cycle on the Sm-like protein Hfq

“…Association and dissociation rates of RNA-Hfq pairs vary. Some RNAs have strong distal or proximal face preferences, which may result in simultaneous binding of two RNAs, rather than competition (de Haseth and Uhlenbeck 1980a; Mikulecky et al 2004;Sun and Wartell 2006;Rajkowitsch and Schroeder 2007;Vecerek et al 2008;Link et al 2009). In the model (Fig.…”

“…Hfq-RNA interactions show a preference of U-rich for proximal and A-rich RNA sequences for distal face binding (de Haseth and Uhlenbeck 1980a;Mikulecky et al 2004). Simultaneous binding may occur on both sides as well, which could facilitate intermolecular base-pairing and regulation (Rajkowitsch and Schroeder 2007).…”

HCV NS3 protein helicase domain assists RNA structure conversion

“…The assay was performed with C2 and C 0 3 RNAs (0.5 nM each), C2 RNA alone (0.5 nM), or C 0 3 RNA alone (0.5 nM), the indicated concentration of NS3H (upper panels) or mNS3H (lower panels), 2.5 mM ATP, and 1.5 mM MnCl 2 at 37°C for 1 h. The reaction mixtures were treated with proteinase K before resolving by gel electrophoresis for detecting dsRNA formation (lanes 1-5). Alternatively, the reaction mixtures were directly resolved by gel electrophoresis for detecting RNA binding (lanes [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. Notably, a large fraction of the RNA-protein complex did not migrate into the gel well at 100 nM and 400 nM NS3H or mNS3H.…”