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Regulatory network of G-quadruplex dependent Post-Transcriptional mRNA Operons (PTROs)

Periodic Reporting for period 1 - G4-PTROs (Regulatory network of G-quadruplex dependent Post-Transcriptional mRNA Operons (PTROs))

Reporting period: 2016-05-01 to 2018-04-30

Understanding post-transcriptional control is crucial to gain detailed knowledge of gene expression. G-quadruplexes (G4s) are important secondary structures in RNAs and represent integral signaling components that modulate post-transcriptional activity of rG4 containing mRNAs. Especially the finding that rG4s are located in mRNAs of several cancer and neurologically relevant genes points towards largely unexplored opportunities to identify new therapeutically avenues.
In essence, G4-specific RNA binding proteins are recruited to rG4s and determine the ultimate fate of G4-containing mRNAs. These interactions deserve to be further studied. Therefore, the overall objective of my project was to further shed light on rG4 guided mechanisms and how the functional consequences of protein-rG4 interactions impact mRNA fate.
Throughout the two-year duration of this fellowship, I have investigated and utilized different techniques to understand the function of rG4s. I have confirmed that presence of rG4s in 5’UTR restricts translation in cells. In a collaborative effort, we have identified several novel rG4 interacting proteins that represent potential therapeutic targets modulating a defined set of mRNAs in a rG4 dependent manner. Furthermore, I managed to establish a robust reporter system to study rG4 function in 5’UTRs. This reporter system will allow me to identify transient factors that impact the translation of rG4 containing mRNAs. All aspects of this work are currently still ongoing and I am looking forward to finish planned experiments as it is an exciting time to explore rG4 driven mechanisms.
The first objective of my proposal was to characterize rG4 function in translation in a genome wide manner. Overall I could confirm previous in vitro data that the presence of rG4s hamper translation when located in 5’UTRs in cells, in a transcriptome wide manner. Then I moved on and in a collaborative project we characterized novel cytoplasmic interactors of the 5’UTR NRAS rG4 and identified several interesting interactors. One of these new interactors was DDX3X which binds mRNAs encoding key mitochondrial proteins in an rG4 dependent manner. This result is especially exciting as DDX3X is known to regulate mRNA translation and stability. Importantly, mutated DDX3X has been detected in medulloblastoma, an aggressive childhood cancer. This disease connection could declare DDX3X dependent mechanisms suitable therapeutic invention.
The results of this work were already disseminated at the “Trinity Science Society” and the “Trinity Forum” at Trinity College Cambridge. I spoke at the internal Cancer Research UK Cambridge Institute weekly seminar series, which resulted in a collaboration with an in house group already working on DDX3X and medulloblastoma. Furthermore, I gave a talk at the famous “RNA club” at the Gordon Institute in Cambridge and presented a poster at the international meeting “7th Cambridge Symposium – Nucleic Acids Chemistry and Biology” in Cambridge. Lastly, this work will be presented at the upcoming EMBO Workshop “RNA: Structure meets function” in Stockholm.
Prediction and in vitro mapping of rG4s in transcripts has suggested presence of rG4s in 5’UTRs and 3’UTRs of several cancer related transcripts. This important disease relationship needs to be further explored and rG4 dependent mechanisms regulating gene expression should be identified. In the field, only few reports of rG4s affecting translation, stability or transport of mRNAs are available and most work on translation are based on in vitro results. Only few rG4 interacting proteins have been identified and hardly anything is known about the consequences of these interactions on the mRNA life cycle. Hence, unbiased, genome wide approaches are needed to develop a deeper understanding of rG4 function. Exploring some of these avenues was the main goal of this project. Here I have shown, in a unbiased manner, that a single rG4 in the 5’UTR hampers translation of mRNAs while single rG4s in other positions in mRNAs did not have this effect. Next, in a collaborative effect I started to catalogue the protein interaction partners of the NRAS rG4. This lead to the identification of multiple interactors and further study of one of these novel rG4 interactors revealed a strong disease relationship with a possible mechanistic link to disease development. This work can be considered as strong basis for future projects that will result in more detailed description of rG4 function. Lastly, development of a reporter system that is able to identify new upstream regulators of rG4 function has great potential to determine whole pathways regulating rG4 dependent mRNA fate. This reporter system can also easily be used to screen for small molecules that can specifically target rG4 dependent gene expression.
Overall my work has opened up new perspectives on rG4 mediated function and highlighted new disease relationships.