Understanding biological functions of repeat-containing noncoding RNAs

DNA has been traditionally thought to store information about development and function of our body by encoding numerous proteins with distinct enzymatic, regulatory and structural functions. Yet, only ~2% of the human genome is actually used for this purpose by producing RNA messengers subsequently translated into proteins. A large fraction of the remaining 98% - often referred to as genome's "dark matter" - has been recently shown to give rise to relatively long non-protein-coding RNAs (lncRNAs) with poorly understood functions.

Notably, this part of our genome is also enriched in repeated DNA sequences including so-called short tandem repeats (STRs). Expression of a few aberrantly long STR arrays expanded as a result of genetic mutations is known to lead to devastating degenerative diseases by recruiting multiple copies of important RNA-binding proteins (RBPs) to the repeated sequence units. On the other hand, the expression status and possible biological functions of most STRs encoded in healthy human genomes have not been investigated systematically.

We propose to test the hypothesis that STR-containing lncRNAs function as pervasive regulators of cellular RNA metabolism by forming multiple contacts with corresponding RBPs and controlling their activity and cellular localization. We will develop our program by pursuing two interrelated objectives.

In the first objective, we will estimate the number of STR-lncRNAs encoded in the genome and ask how these molecules can contribute to RBP regulation. For this purpose, we will identify STR-lncRNAs expressed in biomedically important samples - including cancer cells and developing neurons - by using an innovative combination of computational and experimental tools. The most interesting examples of the newly discovered STR-lncRNAs will be examined for their RBP interaction properties, localization, and possible role in shaping cellular gene expression.

In the second objective, we will focus on a specific STR-lncRNA called PNCTR that we uncovered in our preliminary work. We already know that PNCTR is over-produced in cancer cells where it focuses a critical RBP, polypyrimidine tract-binding protein (PTBP1), in a dot-like pattern characteristic of high-grade and metastatic tumors. Moreover, PNCTR might be required for cell survival and proliferation. We will continue this line of research by elucidating the mechanisms controlling PNCTR expression and underlying its functional contributions to cancer biology.

All in all, these studies should substantially improve our understanding of the "dark matter" of the human genome, provide fundamental insights into regulation of RNA metabolism, and ultimately open up new avenues in disease diagnosis and treatment.

Short tandem repeats (STRs) consist of 2-12 nt sequence units concatenated in a head-to-tail manner. Although STRs are widespread in a healthy human genome, their expression status and possible functions of the resultant RNAs have not been investigated systematically. Notably, transcription of aberrantly expanded STR arrays is known to participate in pathogenesis of several degenerative diseases by recruiting numerous copies of RNA-binding proteins (RBPs) with matching interaction site preferences. Here we propose to test the hypothesis that STR-containing long noncoding RNAs (STR-lncRNAs) play a substantially wider role in controlling RBP activity and cellular localization than currently thought. We will pursue two interrelated objectives. First, we will estimate the number of STR-lncRNAs encoded in mammalian genomes and ask how these transcripts contribute to regulation of cellular RNA metabolism. We will identify new STR-lncRNAs using an innovative bioinformatics approach followed by rigorous validation experiments. This will allow us to assemble an STR-lncRNA database and address molecular functions of a subset of these transcripts differentially expressed between normal and cancer cells or regulated in developing neurons. In the second objective, we will focus on the STR-lncRNA PNCTR uncovered in our preliminary work as a multivalent ligand of an important regulator of cellular RNA metabolism, polypyrimidine tract-binding protein (PTBP1). PNCTR is over-expressed in transformed cells where it recruits PTBP1 to the cancer-enriched perinucleolar compartment (PNC). We will continue this line of research by elucidating the mechanisms that control PNCTR expression and understanding functions of this STR-lncRNA in PTBP1 regulation, PNC assembly and cancer biology. Taken together, these studies will delineate biological functions of an emerging class of lncRNAs and provide new insights into regulation of RNA metabolism in biomedically important contexts.

Training workforce for the UK economy
The proposed research program will provide a framework for professional training of two postdoctoral researchers. They will master standard biology techniques as well as computational and high-throughput experimental approaches - a highly desirable set of skills for modern biomedical research. Both postdocs will additionally acquire advanced communication and managerial skills, as well as experience in supervising students. This comprehensive training will maximize their value as skilled employees capable of making important contributions to the UK academia and industry on the completion of their stints in the PI's lab. Of note, former members of our lab work in a range of sectors from academia and clinics to patent law and regulatory affairs.

Education and public engagement
The program will also allow us to make a lasting impact in the education sector. In addition to supervising KCL students, we will host summer projects of two students from the Judd School, Kent and a sixth-form student from a London state school. The first-hand experience in modern biomedical research will raise students' awareness of different science professions and enhance their educational opportunities. We will also collaborate with the Science Gallery London during their 2019 Dark Matter exhibition season. Together with an emerging artist and young people from the local community we will co-design and produce a small-scale installation exploring the "dark matter" of the human genome. The installation will raise people's awareness that a large part of our genome (e.g. the repeated elements that we will examine in our studies) is still underexplored, and that further research is needed to understand its functions. The Dark Matter season is expected to attract over 100,000 visitors - an excellent platform to enhance the reach and impact of our research. We will also come to the Gallery in our free time to help the visitors interact with the installation. This will provide opportunities for conversation, questions and deeper engagement with the artwork and the scientific problems it represents. All in all, these activities will contribute to scientific education in the UK and make functional genomics a part of a wide cultural landscape.

Medicine and forensics
Our research focus on short tandem repeats (STRs) should additionally deliver long-term impacts in medicine and forensics. Several degenerative diseases associated with considerable morbidity and mortality and a major economic burden (e.g., Amyotrophic Lateral Sclerosis, Myotonic Dystrophy and Fragile X-associated Tremor/Ataxia Syndrome) have been linked with transcription of expanded STRs. Interestingly, STRs also constitute a highly variable fraction of healthy human genomes that is extensively used in forensics for DNA-based testing. By shedding light on STR functions our work should generate valuable insights into molecular etiology of relevant disorders ultimately leading to improved therapies and diagnostic tools. Similarly, discovery of highly abundant STR-containing RNA in our studies may help forensic scientists increase sensitivity and accuracy of molecular identity tests. To maximize the impact of our work in these fields, we will initiate relevant collaborations with clinical and forensic scientists at the KCL and elsewhere in the UK and will publish our results as open-access papers in journals with widest readerships possible. The PI and the postdoctoral researchers involved in the program will also present their work in international meetings attended by both scientists and clinicians. An important deliverable of our program will be development and maintenance of a publicly available online database of experimentally validated STR-lncRNAs. We are convinced that, along with our papers and meeting presentations, this resource will facilitate future medical and forensic innovations.