Identification of nonspecific RNA-binding proteins leads to characterization of Lhp1 as a robust ATP-independent RNA chaperone




Middleton, Aaron Travis

Journal Title

Journal ISSN

Volume Title



RNAs with biological functions that depend on higher-order structures are vulnerable to forming inactive alternative structures. These alternative, misfolded structures can be stable enough to require intervention by proteins that promote conformational transitions. These RNA chaperone proteins are biologically critical, and they have long been hypothesized to possess nonspecific RNA binding as a general property – empowering a small collection of RNA chaperones to facilitate the folding of a large and diverse transcriptome. To identify new candidates for this nonspecific RNA chaperone activity, we performed affinity purifications of budding yeast RNA-binding proteins, using an exogenous and non-orthologous RNA as bait: the highly structured ribozyme derived from the Tetrahymena thermophila Group I intron. Proteins that co-purified with the RNA were identified by mass spectrometry, and analysis of multiple replicates revealed a sizable consistent population that was highly enriched in RNA-binding proteins. From this pool of nonspecific RBPs, we selected the yeast La protein Lhp1 for further investigation. In vitro kinetic assays with purified Lhp1 showed that it robustly accelerates refolding of the Tetrahymena ribozyme from a stable misfolded state to its catalytically active conformation, suggesting that Lhp1 indeed possesses broad RNA chaperone activity. Comparisons between this ATP-independent RNA chaperone and the ATP-dependent DEAD-box helicase CYT-19 highlighted the surprising efficiency of Lhp1 chaperone activity in single-turnover refolding assays, as well as some significant obstacles attributed to its lack of ATP utilization. Our findings suggest that Lhp1 may rely on other proteins in vivo to rescue activity after cycles of duplex unwinding. Overall, the validation of this candidate from our proteomic survey as a general RNA chaperone encourages us to pursue additional candidates in the future.


LCSH Subject Headings