Analysis of Long‐noncoding RNA Structure‐Function Relationship in Prostate Cancer

Abstract

The noncoding RNA (ncRNA) revolution has revealed myriad RNA species that play critical roles at all stages of life, including developmental biology and disease progression. For example, two long ncRNAs (lncRNA), Metastasis Associated Lung Adenocarcinoma Transcript‐1 (MALAT1; ~6.7 kb) and Second Chromosome Locus Associated with Prostate‐1 (SChLAP1; ~1.5 kb), are basally expressed in normal prostate tissue but are dysregulated in prostate cancer. MALAT1 acts in trans at nuclear speckles during mRNA post‐transcriptional processing while SChLAP1 acts in cis to influence oncogenic gene expression. There is compelling evidence that the 3′‐end of MALAT1 is a triple helix structure that acts as a molecular knot, driving transcript accumulation in cancer cells and furthering its metastatic potential, but we currently lack any biophysical data detailing the relationship between SChLAP1 structure and function. In general, the relationship among lncRNA structure, dynamics, and function is not well understood; for example, even with high‐resolution structures of the MALAT1 triple helix, questions remain regarding the role of intrinsic dynamics in transcript stability or protein binding. As lncRNA represent an underexplored therapeutic avenue, this work aims to investigate the role of lncRNA structure and dynamics in driving prostate cancer metastasis.Current work includes: 1) Understanding the role of the MALAT1 3′‐end triple helix structure and dynamics in global transcript stability and 2) Conducting the first biophysical analyses of SChLAP1 and identifying structural elements that are critical to metastasis. For the MALAT1 project, we have performed chemical probing experiments using both SHAPE‐ and DMS‐MaP chemical probing on the MALAT1 triple helix and 5′‐end extensions, revealing different conformations of the triple helix region. Additionally, we have performed exonuclease degradation assays on the triple helix and 5′‐end extensions in triplex de/stabilizing buffer conditions, showing that transcript stability is indeed tied to triple helix structural dynamic. For the SChLAP1 project, we have determined the first secondary structure model of full‐length SChLAP1 and have data supporting the formation of independent folding domains that when deleted from the full‐length transcript lower the invasive phenotype of prostate cells. We are currently working to investigate the tertiary structure of SChLAP1 and identify structure ensembles. Future work aims to design novel in‐cell mutational studies to identify regions of SChLAP1 that are critical for driving cell migration and invasion, two hallmarks of aggressive prostate cancer. This work will deepen our current understanding of the lncRNA MALAT1 triple helix dynamics in driving prostate cancer metastasis and provide the first biophysical and biochemical assessment of the lncRNA SChLAP1. Through this pursuit, we will provide the basis for assessing novel lncRNA structure‐function relationships and therapeutically targeting lncRNA in cancer.Support or Funding InformationEJM funded through grants award to AEH: Prostate Cancer Foundation YIA and NIH MIRA