Breast cancer cells build a tiny, droplet-like structure called the PNC around a specific piece of RNA, and this project will work out exactly how and why they do it. The problem is that cancer cells often accumulate scrambled DNA, particularly in the regions that produce ribosomal RNA. This genetic instability makes cancers harder to treat. The researchers have found that a noncoding RNA called PNCTR, which is made from these unstable DNA regions, helps cancer cells survive by gathering up a key protein. But no one knows how the cancer cells manage to produce PNCTR from damaged DNA, or how the PNC structure actually assembles. If this research succeeds, it will reveal a fundamental mechanism linking DNA damage to the production of a cancer-promoting RNA. The team will sequence PNCTR from patient samples to see if it always comes from rearranged DNA, and they will use live imaging to watch the PNC assemble in real time. This is fundamental science—there is no immediate diagnostic or treatment here. But understanding how genome instability drives noncoding RNA production could eventually point toward new ways to disrupt cancer cell survival, much as earlier work on RNA biology unexpectedly enabled mRNA vaccines.
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Genetic information is encoded in DNA as an ordered sequence of nucleotide units. Cells can retrieve this information by transcribing DNA into protein-coding messenger and noncoding RNAs. We have previously identified the pyrimidine-rich noncoding transcript PNCTR and showed that it is widely expressed in cancer cells. This RNA originates from an intergenic spacer between repeated genes encoding 47S precursors of ribosomal RNAs in genomic regions called ribosomal DNA (rDNA) arrays. PNCTR can promote cancer cell survival, at least in part, by sequestering multiple copies of the RNA-binding protein PTBP1 in a membraneless perinucleolar compartment (PNC). Cancers cells tend to accumulate mutations, which often make cancers more challenging to treat. Such genetic instability is commonly observed, for example, in breast cancers, the most common cancer type in the UK. Notably, many breast cancers produce the PNC through yet-to-be-understood mechanisms. Our new data suggest that cancer cells may transcribe PNCTR from genetically rearranged rDNA sequences, emerging as a result of error-prone repair of DNA double-strand breaks (DSBs). We also hypothesize that the assembly of the PNC around nascent PNCTR molecules segregates genetically compromised rDNA loci away from the nucleolus. Finally, we propose that PNCTR plays a key part in the breast cancer biology, and that its knockdown may reduce the ability of cancer cells to thrive and metastasize. We will explore these intriguing possibilities by pursuing three distinct but interrelated objectives. 1. Elucidating the role of rDNA rearrangements in PNCTR expression: We will test if PNCTR is commonly produced from genetically rearranged rDNA by sequencing PNCTR-enriched RNA fractions from breast cancer cell lines and patient samples and mining publicly available sequencing datasets. The proposed analyses will also illuminate the role of recurrent rDNA DSBs and different DSB repair pathways in the emergence of PNCTR-encoding loci. 2. Dissecting the mechanisms of PNC assembly: Our new data suggest that the PNC assembles near the PNCTR transcription site. We will test this prediction using proximity DNA labeling and chromatin immunoprecipitation with PTBP1-specific and control antibodies. To examine the PNC assembly dynamics, we will perform live imaging of cancer cells retrofitted with fluorescent markers. By combining these experiments with appropriate knockdown and knockout approaches, we will be able to distinguish between co- and post-transcriptional mechanisms of PNC assembly and find out if this process facilitates the segregation of rearranged rDNA loci to the nucleolar periphery. 3. Understanding PNCTR functions: We will employ appropriate knockdown or/and knockout approaches in both 2D and organoid cultures to investigate the role of PNCTR in sustaining the viability and metastatic properties of breast cancer cells. To understand the underlying mechanisms, we will evaluate the impact of PNCTR/PNC on PTBP1 activity and the integrity of the nucleolus. These studies will yield fundamental insights into the role of genome instability in noncoding RNA production and subnuclear compartmentalization. Furthermore, they are expected to contribute to the development of innovative research tools for the broader biomedical community. We anticipate that this research trajectory will ultimately pave the way for transformative diagnostic approaches and precision therapies for cancer patients.
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