Genome-Wide Search for Intracellular Factors Affecting RNA Folding Via in Vivo Oligonucleotide Hybridization

Specific intracellular factors (e.g. proteins, metal ions, etc.) help RNAs reach their functionally active structures. However, detecting these factors and unveiling their influence on folding is challenging, particularly in vivo. Hereby, we present a novel approach where a transposon library is screened to detect cellular factors influencing the model Tetrahymena group I (gI) intron folding in vivo in Escherichia coli by utilizing a fluorescence-based oligonucleotide hybridization assay. Our in vivo structural probing assay utilizes a riboregulator that expresses green fluorescence protein (GFP) when oligonucleotide hybridization occurs between the probe and a structurally accessible local region within a target RNA, providing valuable information on RNA folding. We have profiled the gI intron in vivo in both wild type E. coli cells and in an StpA mutant strain; the suppressor of td phenotype A (stpA) protein acts as an RNA folding chaperone for the gI intron. Specifically, when probing a central region (P3-P4) that is critical for folding of the intron, we have confirmed that this factor influences gI intron folding, as previously reported in vitro.  This region undergoes transient reconfigurations giving rise to low abundance folding intermediates, one of which is termed alt-P3. We will discuss these findings as well as the application of this intracellular fluorescence-based system for a genome-wide search of other factors that affect gI intron folding in vivo. In particular, we probe the dynamic P3-P4 region of the gI intron in screening a transposon library. Significant differences in fluorescence relative to the wild type cell’s fluorescence pattern can be accredited to inhibited or enhanced RNA folding, affected by specific cellular factors.