G-quadruplex (G4) DNA and paraspeckles have recently emerged as important regulatory features within the mammalian cell nucleus, with strong correlations to breast cancer and linked to poor clinical prognoses.
When guanine-rich DNA becomes single-stranded these sequences can fold into alternative structures known as G4s. G4s differ from canonical B-DNA, and form a four-stranded structure held by Hoogsteen hydrogen bonds between guanines of a G-tetrad (1). The formation of G4s within the genome has major impacts on replication and transcription, affecting how proteins bind and recognise DNA, and stalling molecular machinery (2). Moreover, G4s are overrepresented in regulatory regions with many G4s forming within the promoters of oncogenes, such as c-MYC and hTERT (3,4,5).
Paraspeckles are RNA-protein subnuclear bodies (6). Whilst little is known about the biological function of the paraspeckle, they have been demonstrated to have major roles in various biochemical processes, including mammary development. These paraspeckles have been shown to form under cellular stress conditions and act by sequestering regulatory proteins and nascent mRNA species, preventing their action/transportation (7,8). The major structural determinant of paraspeckles, a lncRNA NEAT1, has been shown to be overexpressed in many cancers and leads to poor clinical prognosis (9).
Thus, this project sought to evaluate the formation of both G4s and paraspeckles within healthy breast and breast cancer cells. Using various imaging techniques and qPCR, we demonstrated a marked increase in both G4 and paraspeckle formation within breast cancer cells. Furthermore, stabilising G4s within the promoters of oncogenes via small molecules significantly decreased gene expression of previously described “undruggable targets,” highlighting their therapeutic potential. Additionally, this project aimed to examine whether G4-stabilisation by small molecules could influence paraspeckle formation, which saw a significant increase in paraspeckle formation. Furthermore, a potential G4 was discovered within the NEAT1 promoter, able to form within biologically relevant conditions. This is the first study to examine the relationship between G4s and paraspeckles, and the first to identify and characterise a G4 within the NEAT1 promoter.