Poster abstracts
Poster number 140 submitted by Nikhil Vijai
Elucidating the structure of the 3′ end of premature MALAT1 using SHAPE
Nikhil Vijai (Department of Chemistry and Biochemistry, University of Notre Dame), Mika J. Schievelbein (Department of Chemistry and Biochemistry, University of Notre Dame), Jessica A. Brown (Department of Chemistry and Biochemistry, University of Notre Dame)
Abstract:
Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is a long non-coding RNA that contains an RNA triple helix at its 3′ end. Additionally, MALAT1 has been associated with cancer progression and is upregulated in many forms of cancer. MALAT1 undergoes a unique 3′ end processing mechanism, whereby it is cleaved by ribonucleases P (RNase P) and Z to form mature MALAT1 and a tRNA-like structure known as MALAT1-associated cytoplasmic RNA (mascRNA). The RNase P cleavage event enables the formation of the triple helix. However, the structure of MALAT1 before it is cleaved by RNase P is unknown. UV thermal denaturation and METTL16-binding assays indicate that a partial triple helix forms at the 3′ end of premature MALAT1, but the extent to which one forms is unclear. To provide insight into the putative triple helix structure of premature MALAT1, we employed selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). First, we generated an RNA in which the 3′ end of the premature MALAT1 sequence is inserted in a SHAPE cassette with a 3′ reverse transcriptase primer-binding site. Next, we optimized concentrations of RNA and primer for primer binding, reaction conditions for reverse transcription, ddNTP concentrations for sequencing, and NMIA concentrations for acylation. We then submitted some initial reactions for Sanger sequencing and analyzed the results using the QuSHAPE software. By elucidating the structure of premature MALAT1, we hope to better understand the life of the MALAT1 triple helix. Additionally, the structure of premature MALAT1 could represent a novel anti-cancer therapeutic target.
Keywords: MALAT1, SHAPE, Triple Helix