RNA base-pairing complexity in living cells visualized by correlated chemical probing

Abstract

2 SUPPORTING DISCUSSION Prior experiments on the S4-binding elementThe original double pseudoknot (DPK) model of the S4-binding element (S4E) was developed from early experiments mapping the impact of mutations on S4 binding (Fig. S6) (1). Following these initial experiments, the same library of S4E mutants has been extensively characterized by complementary biophysical and functional experiments (2-4). These data have been interpreted through the lens of the DPK model. However, as detailed below, much of this data is ambiguous and in several cases is inconsistent with the DPK structure.The DPK structure is motivated by two pairs of mutations that disrupt and rescue S4 binding in filter-binding assays (1). Disruption of the proposed PK2 interaction by mutation 17 reduces S4 binding ~10-fold and is rescued by the compensatory mutation 21 ( Fig. S6A). Disruption of PK3 by mutation 18 reduces S4 binding ~2-fold and is rescued by the compensatory mutation 22.However, the significance of these compensatory mutations is less clear when considered in the context of other mutation data. Relative to mutations spanning the kissing loop (KL) structure that uniformly and catastrophically disrupt binding, mutations to the PK2 and PK3 regions have inconsistent and modest impact on binding (Fig. S6A). Notably, multiple mutations that should disrupt PK2 and PK3 have no impact on binding. Subsequent in vivo functional assays also are inconsistent with the DPK model (2). Significantly, what should be compensatory 18+22 mutations do not rescue S4 repressive function in vivo. Finally, biophysical characterization of different S4E mutants also reveals inconsistencies of the DPK model, with the expected compensatory mutations (17+21 and 18+22) failing to rescue S4E tertiary folding (3). Overall, the inconsistent and modest impact of mutations to the proposed PK2 and PK3 interactions and the inconsistency of compensatory rescue argue against direct PK2 and PK3 pairing. Rather, these data are more consistent with these regions interacting indirectly, as would be expected for a kissing-loop type structure where nucleotides adjacent to the KL duplex contribute to tertiary stability and S4 binding but do not directly pair ( Fig. S6D).While most of the mutations tested by prior studies are not expected to impact H3, three mutations do provide evidence supporting H3 and the kissing-loop structure. Mutation 19 (G95àA) converts a GU pair in H3 to an AU pair, and as expected, has no impact on S4 3 binding in vitro or repression function in vivo (2). Mutation 18 modestly disrupts H3 and modestly decreases S4 binding affinity, although has no impact on repression function in vivo (1). Most significantly, mutation of the AGGAG Shine-Dalgarno sequence (SD; Fig. S6A) to its sequence complement, UCCUC, completely disrupts H3 and, as expected, completely abolishes S4 binding (4). By comparison, in the context of the DPK model, the SD mutation occurs in the middle of a 25-nt single-stranded loop and thus would not be expected to have such profound impa...

Time-resolved, single-molecule, correlated chemical probing of RNA

“…RNase P has a complex secondary and tertiary structure and its high-resolution structure is known 13,14 . Sites of per-nucleotide chemical modification were identified using the MaP strategy 6,8 . Both TMO and DMS react preferentially with unpaired nucleotides, with high reactivities observed at loops and bulges ( Figure 2A and Supporting Figure 2).…”

“…The challenge of directly detecting through-space structural interactions in RNA by chemical probing has been recently addressed by development of the RNA interaction group analyzed by mutational profiling (RING-MaP) technology, in which multiple chemical adducts are visualized in the same RNA strand by a processive relaxed fidelity reverse transcription reaction [6][7][8][9] .…”

“…Through-space structural communication is detected from interdependent modification events in the same molecular strand of RNA. The key advantage of the RING-MaP chemical probing method is thus the ability to directly detect, rather than merely model or predict, RNA duplexes 7,8 , tertiary interactions, and long-range structural communication 6,9,10 (Figure 1B). To date, RING-MaP correlated chemical probing has been carried out using dimethyl sulfate (DMS).…”

RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

“…RNase P and RMRP are structurally related, but sequence divergent, non-coding RNAs that bind overlapping sets of proteins to form RNP endonucleases that cleave distinct substrates 6,19,24 .…”

“…Correlations between RNP-MaP sites were computed over 3-nucleotide windows using a previously described G-test framework (RingMapper) 19 . Windows were required to be separated by > 4 nucleotides, jointly covered by more than 10,000 sequencing reads, jointly co-mutated >50 times, and have background mutation rates below 6% (Fig.…”