Messenger RNA Structure Regulates Translation Initiation: A Mechanism Exploited from Bacteria to Humans

Mustoe, Anthony M.; Corley, Meredith; Laederach, Alain; Weeks, Kevin M.

doi:10.1021/acs.biochem.8b00395

Cited by 56 publications

(64 citation statements)

References 5 publications

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“…Thus, the kissing-loop structure does not preclude formation of an entrapped S4•S4E•mRNA ternary complex. Nevertheless, we suggest a modest revision of the mechanism of S4 translation inhibition that builds on the renewed appreciation of RNA unfolding kinetics as a major ratelimiting step of translation initiation (9). In the absence of S4, the H3 helix (which overlaps the SD sequence and start codon of rpsM) is very unstable, allowing efficient unfolding and accommodation of the rpsM mRNA into the 30S mRNA cleft.…”

Section: Implications For Mechanism S4 Translation Inhibitionmentioning

confidence: 86%

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

Mustoe

Lama

Irving

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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171

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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...

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Section: Implications For Mechanism S4 Translation Inhibitionmentioning

confidence: 86%

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

Mustoe

Lama

Irving

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

171

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“…RNA molecules are strongly driven to fold back on themselves into base-paired secondary structures. These structures play central roles in RNA biology, from mediating complex functions such as RNA catalysis and specific ligand recognition, to more broadly tuning RNA sequence accessibility to regulate processes such as translation initiation (1,2). Furthermore, many RNAs fold into multiple structures, providing the basis for molecular switching functions (3).…”

Section: Introductionmentioning

confidence: 99%

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

Mustoe¹,

Lama²,

Irving³

et al. 2019

Preprint

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2 ABSTRACT RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having notable deficiencies. Here we convert the common reagent dimethyl sulfate (DMS) into a useful probe of all four RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base pairing interactions in cells. PAIR-MaP has superior resolution and accuracy compared to alternative experiments, can resolve alternative pairing interactions of structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and two bacterial mRNA 5'-UTRs reveals new functionally important and complex structures undetectable by conventional analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells. !

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“…However, with the accumulation of knowledge about RNA structures and functions, especially those of different types of non-coding RNAs, modern views have expanded RNA function a lot to gene expression regulation and human diseases. RNA structure is a bridge from primary sequence to biological function and is crucial for correct functioning, such as RNA transcription [ 1 ], splicing [ 2 ], translation [ 3 ], localization [ 4 ], protein interaction, and gene expression regulation [ 5 ]. For example, riboswitches rely a lot on structural changes to respond to metabolites and regulate gene expression.…”

Section: Introductionmentioning

confidence: 99%

Targeting RNA structures in diseases with small molecules

Shao

Zhang

2020

Essays in Biochemistry

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RNA is crucial for gene expression and regulation. Recent advances in understanding of RNA biochemistry, structure and molecular biology have revealed the importance of RNA structure in cellular processes and diseases. Various approaches to discovering drug-like small molecules that target RNA structure have been developed. This review provides a brief introduction to RNA structural biology and how RNA structures function as disease regulators. We summarize approaches to targeting RNA with small molecules and highlight their advantages, shortcomings and therapeutic potential.

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Messenger RNA Structure Regulates Translation Initiation: A Mechanism Exploited from Bacteria to Humans

Cited by 56 publications

References 5 publications

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

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

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

Targeting RNA structures in diseases with small molecules

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