Alternative RNA Conformations: Companion or Combatant

Abstract

RNA molecules, in one form or another, are involved in almost all aspects of cell physiology, as well as in disease development. The diversity of the functional roles of RNA comes from its intrinsic ability to adopt complex secondary and tertiary structures, rivaling the diversity of proteins. The RNA molecules form dynamic ensembles of many interconverting conformations at a timescale of seconds, which is a key for understanding how they execute their cellular functions. Given the crucial role of RNAs in various cellular processes, we need to understand the RNA molecules from a structural perspective. Central to this review are studies aimed at revealing the regulatory role of conformational equilibria in RNA in humans to understand genetic diseases such as cancer and neurodegenerative diseases, as well as in pathogens such as bacteria and viruses so as to understand the progression of infectious diseases. Furthermore, we also summarize the prior studies on the use of RNA structures as platforms for the rational design of small molecules for therapeutic applications.

Keywords: G-quadruplex; RNA conformational ensemble; gene regulation; pseudoknot; riboswitch.

Figure 1

(a) The hierarchical folding of aptamers in a flavin mononucleotide (FMN) riboswitch. The primary structure of the FMN aptamers involves a single-stranded nucleotide sequence that sequentially folds into the secondary structure consisting of 6 stems (P1–P6) and 5 loops (L2–L6). The FMN binding pocket is formed in a tertiary structure via loop–loop (L2–L6 and L3–L5) and loop–helix (L6-P2 and L3-P5) interactions (PDB ID: 3F2Q). (b) Mode of action of regulatory RNA via interaction with (i) DNA (e.g., R-loop formation), (ii) other RNAs (tRNA riboswitch), (iii) proteins (e.g., dicer) and (iv) metabolites (e.g., FMN riboswitch).

Figure 2

(a) HIV TAR RNA conformation ensemble consisting of diverse secondary structures with their respective populations. (b) Factors affecting the conformation ensemble: (i) molecular crowding in cells facilitates RNA folding in different conformations such as G-quadruplexes; (ii) metal ions like Mg²⁺ are required for ribozyme functioning to disrupt AU base pairing and the exposure of catalytic domains; (iii) post-transcriptional modifications such as N¹ methyl adenosine regulate the proper folding of tRNA; (iv) the single-nucleotide polymorphism in miRNA 1229 leads to a conformational shift towards the hairpin structure, leading to Alzheimer’s disease; (v) co-transcriptional folding of RNA to form secondary structures such as hairpins; (vi) the liquid–liquid phase separation in RNA granules maintains the compartmentalization responsible for gene regulation; (vii) conformational equilibria between hairpin and G-quadruplex structures due to flanking sequences in cancer cells.

Figure 3

RNA conformers involved in cancer: neurodegenerative, viral and bacterial diseases.

Figure 4

Targeting alternate RNA conformers using small molecules for therapeutics. (a) Small molecules shift the conformational equilibrium towards the G-quadruplex, causing suppression of the translation level in proto-oncogenes. (b) Intron 1 of the C9ORF72 gene contains a G-quadruplex-forming sequence, sequestering the hnRNPA1 protein and resulting in ALS/FTD. Small molecules that destabilize the G-quadruplex release the hnRNPA1 protein in its free form leading for normal development. (c) The pseudoknot structure formed in the ORF1 leads to −1 ribosomal frameshifting and the formation of a complete ORF1ab product that causes viral replication and infection. The addition of small molecules disrupts the pseudoknot structure and prevents −1 ribosomal frameshifting, inhibiting viral replication and infection. (d) Small molecules targeting the FMN riboswitch inhibit the riboflavin biosynthesis pathway and subsequently inhibit the growth of mycobacteria.