Christina Waldsich

RNA folding is an essential aspect underlying RNA-mediated cellular processes. Many RNAs, including large, multi-domain ribozymes, are capable of folding to the native, functional state without assistance of a protein cofactor in vitro. In the cell, trans-acting factors, such as proteins, are however known to modulate the structure and thus the fate of an RNA. DEAD-box proteins, including Mss116p, were recently found to assist folding of group I and group II introns in vitro and in vivo. The underlying mechanism(s) have been studied extensively to explore the contribution of ATP hydrolysis and duplex unwinding in helicase-stimulated intron splicing. Here we summarize the ongoing efforts to understand the novel role of DEAD-box proteins in RNA folding.

RNA folding is the most essential process underlying RNA function. While significant progress has been made in understanding the forces driving RNA folding in vitro, exploring the rules governing i...

In yeast mitochondria the DEAD-box helicase Mss116p is essential for respiratory growth by acting as group I and group II intron splicing factor. Here we provide the first structure-based insights into how Mss116p assists RNA folding in vivo. Employing an in vivo chemical probing technique, we mapped the structure of the ai5γ group II intron in different genetic backgrounds to characterize its intracellular fold. While the intron adopts the native conformation in the wt yeast strain, we found that the intron is able to form most of its secondary structure, but lacks its tertiary fold in the absence of Mss116p. This suggests that ai5γ is largely unfolded in the mss116-knockout strain and requires the protein at an early step of folding. Notably, in this unfolded state misfolded substructures have not been observed. As most of the protein-induced conformational changes are located within domain D1, Mss116p appears to facilitate the formation of this largest domain, which is the scaffold for docking of other...

Nucleotide analog interference mapping (NAIM) is a powerful chemogenetic approach that allows RNA structure and function to be characterized at the atomic level. Random modifications of base or backbone moieties are incorporated into the RNA transcript as nucleotide analog phosphorothioates. The resulting RNA pool is then subjected to a stringent selection step, in which the RNA has to accomplish a specific task, for example, folding. RNA functional groups important for this process can be identified by physical isolation of the functional and the nonfunctional RNA molecules and subsequent mapping of the modified nucleotide positions in both RNA populations by iodine cleavage of the susceptible phosphorothioate linkage. This approach has been used to analyze a variety of aspects of RNA biochemistry, including RNA structure, catalysis and ligand interaction. Here, I describe how to set up a NAIM assay for studying RNA folding. This protocol can be readily adapted to study any RNAs and their properties. The time required to complete the experiment is dependent on the length of the RNA and the number of atomic modifications tested. In general, a single NAIM experiment can be completed in 1–2 weeks, but expect a time frame of several weeks to obtain reliable and statistically meaningful results.