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The chemical biology of RNA G-quadruplexes

Final Report Summary - RNAQUAD (The chemical biology of RNA G-quadruplexes)

We have introduced a reverse-transcriptase stalling (RTS), hybridisation ligation-mediated PCR (RTS-LMPCR) approach to probe G-quadruplex (G4) formation and location within full length low-abundance cellular RNAs. We have exemplified this approach through the detection and mapping of G4 structures in the biologically important cellular telomerase RNA component hTERC. Moreover, we have shown that the approach is applicable to detect and validate RNA targets for G4 ligands.
We have invented the first high-throughput experimental approach, rG4-seq, for sequence mapping of RNA G4 (rG4) formation transcriptome-wide at nucleotide resolution. The application of rG4-seq in vitro to probe rG4s in human HeLa RNA reveals that the rG4 is a pervasive RNA secondary structure in the human transcriptome with the presence of thousands of canonical and non-canonical rG4s. We find that rG4s are enriched in UTRs, associated with miRNA target sites and poly-adenylation signals. rG4s may therefore represent a general regulatory element for translation, miRNA-mediated gene regulation and alternative polyadenylation. The repertoire of rG4s identified provides a valuable resource for the RNA and gene regulation communities, while the significant influence of rG4s on predicted RNA secondary structures suggests that rG4s should be considered as important functional elements in biology.
We have developed SHALiPE to map rG4s at single nucleotide resolution. This approach exploits the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) with lithium ion-based primer extension (LiPE) with 2-methylnicotinic acid imidazolide.
The approach has numerous benefits over prior methods, and we have used it to discover a rG4 structure within the biologically important pre-miRNA 149. We also found that this rG4 structure inhibits DICER processing in vitro.
We have explored the proteins that regulate rG4 structures in cells using an unbiased affinity proteomic approach to identified cytoplasmic proteins interacting with a rG4 oligonucleotide derived from the NRAS oncogene 5’UTR sequence. We have identified several previously known rG4 interactors, such as DHX36 and hnRNPH1, and have discovered many new proteins including several helicases, such as DHX9, DDX17, DDX3X and GRSF. Two sets of rG4 interactors were apparent: one set enriched with arginine/glycine rich sequences (RGG/RG motifs) and the other devoid of these motifs. Overall, our approach expands our knowledge of rG4 interacting proteins and will suggest testable paths for their regulation in cells.
We have also developed polysome analysis and ribosome profiling techniques for investigation of the role of rG4 structures in mRNA translation. We have found that rG4-containing transcripts are translated less efficiently than unstructured mRNAs and helicases such as DHX36 contribute to the translation of rG4-containing mRNA. We have identified human mRNAs whose translation efficiency is modulated by the DHX36/DHX9-dependent folding/unfolding of rG4s within their 5'-UTRs. We uncovered a previously unknown mechanism for translation regulation in which unresolved rG4s within 5'-UTRs promote 80S ribosome formation on upstream start codons, causing inhibition of translation of the downstream main open reading frames. Our findings suggest that the interaction of helicases with rG4s could be targeted for future therapeutic intervention.

We have also identified the mechanism for the unwinding of rG4s by DHX36 and have provided a high-resolution X-ray structure of DHX36 bound to a G4.