Identification and Targeting of RNA G-quadruplex Structures

Abstract

RNA G-quadruplexes (rG4s) are unique secondary structures formed by G-rich RNA sequences, playing crucial regulatory roles in several biological procedures, including replication, transcription, genomic instability, and epigenetic regulation. Although many rG4 structures have been identified in coding messenger RNAs (mRNA), little attention has been given to rG4s in non-coding RNAs (ncRNAs), especially in long non-coding RNAs (lncRNAs). Cumulative evidence indicates that lncRNAs play an essential role in cancer development. To understand the functions and regulations of lncRNAs in cancer cells, it is crucial to investigate the structural elements like rG4s and their interactions with proteins. Thus, the first objective of my Ph.D. research was to identify conserved rG4 motifs in lncRNAs and investigate the functional role of the rG4 structures and their interactions with proteins.

In Chapter 2, we identified hundreds of rG4-containing ncRNAs to address this issue and then focused on investigating one lncRNA of interest, MALAT1. Multiple conserved rG4 motifs were identified in MALAT1 lncRNA, which form thermostable rG4 structures with parallel topology. Then we showed that rG4s in MALAT1 lncRNA can interact with NONO protein with high specificity and affinity in vitro and in cell lysate. We also demonstrated that the NONO protein recognizes MALAT1 lncRNA via rG4 motifs in cells, and MALAT1 rG4-NONO protein interaction could be dissociated via targeting rG4 structures in MALAT1 lncRNA by the rG4-specific small molecule, peptide, and L-aptamer. Overall, this study uncovered new and vital rG4 motifs in MALAT1 lncRNAs, revealed their specific interactions with NONO protein, proposed different approaches for targeting MALAT1 and its RNA-protein complex through its rG4 motifs, and illustrated the prevalence and importance of rG4s in ncRNAs.

The second objective of my Ph.D. research was to investigate the factors involved in rG4 that drive the interaction between rG4s and their binding proteins/peptides. Furthermore, I aimed to develop a peptide selection and analysis platform to target rG4 structures and investigate the effect of novel short peptides in modulating G4-protein association and G4-linked gene function.

In Chapter 3, we studied and uncovered the impact of RNA sequence context and stereochemistry on G-quadruplex-peptide interaction. By studying the interaction between G4 and RHAU53, a well-known rG4 binding peptide, we found that the number of G-quartet, thermostability, flanking sequences, and base chirality affect rG4-RHAU53 binding. Moreover, our data indicated that RHAU53 binds to 5’ G-quartet over 3’ G-quartet preferentially and showcased that RHAU53 interacts strongly with unnatural L-rG4 for the first time. Altogether, our discoveries offered unique insights into the potential development of targeting tools for rG4 structure and rG4-binding peptide/protein recognization.

In Chapter 4, we first adapted and refined the mRNA display for the G4 target of interest (G4-mRNA display-Seq). Using an rG4 formed in the 5’ end of human telomerase RNA (hTERC) as the target, we selected a novel short peptide, pep11, which showed good binding to the hTERC rG4 target. Moreover, we rationally designed the tandem and cyclic version of pep11 for further characterization and found that both versions exhibited stronger binding affinity and great rG4 selectivity than the original pep11. In addition, we demonstrated that both the tandem and cyclic pep11 colocalized with hTERC rG4 but not hTERC rG4 mutant in living cells, supporting their interactions in cells. Notably, we carried out cellular experiments and found that both the tandem and cyclic pep11 can successfully negatively regulate the reporter gene expression of the hTERC G4 wildtype construct, but not the hTERC G4 mutant construct, supporting the G4-specific peptide can control gene activity. Overall, we presented an innovative platform to generate novel short peptides targeting different G4s with solid affinity and selectivity. We also showed innovative applications of the G4-targeting peptides in controlling G4-protein interaction, RNA metabolism, and gene expression.

In summary, our first work exemplified conserved and essential rG4 motifs in MALAT1 lncRNAs and uncovered their interactions and functional role with NONO protein. The methodology used in this work could apply to other lncRNA and inspire further exploration of lncRNA rG4 biology. And our second study revealed the critical factors of rG4-RHAU53 binding. It offered new insights into the rG4-peptide binding mode and specificity, which will facilitate the future development of tools that target G4 structure and G4-binding proteins. In the third work, we developed the G4-mRNA display-Seq platform, which provides a new and essential platform for evolving and selecting novel short peptides for targeting G4 structures. The peptides (Tandem pep11 and Cyclic pep11) we selected can be a valuable tool for determining G4 conformation, which will prompt further studies on the structure of G4 and its biological implication in vitro and in vivo. In addition, the platform will be applicable not only in G4 targeting studies but also potentially to biosensing, bioimaging, therapeutic and diagnostic applications. And we will further develop the system for other non-canonical structure targets.

Date of Award	8 May 2023
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Chun Kit KWOK (Supervisor)