CHARACTERIZING A NOVEL CLASS OF LONG NONCODING RNAS EXPRESSED IN CANCER

Long noncoding RNAs (lncRNAs) are an abundant class of molecules showing a wide range of biological functions. Many lncRNAs are known to be over-expressed in cancer but the significance of this finding remains to be elucidated. An interesting example of a cancer-linked lncRNA that has been recently identified in the Makeyev lab is referred to as pyrimidine-rich noncoding transcript (PNCTR). Unpublished work in the lab shows that PNCTR is localized to the nucleus where it interacts with an important RNA-binding protein called PTBP1. Importantly, PNCTR is predicted to contain a number of (CU)_n repeats believed to be involved in PTBP1 binding. However, because of the highly repetitious nature of this RNA molecule, it has been difficult to deduce its exact sequence from the reference genome information or next-generation RNA sequencing data. Therefore, the goal of this research project has been to obtain Sanger sequencing data for PNCTR, focusing especially on the (CU)_nrepeat regions. We began this work by designing reverse-transcription (RT)-PCR primers using primer3 software and hg38 human genome assembly. Hela cells were cultured in DMEM medium containing 10% FBS and antibiotics and harvested by trypsin digestion. Nucleo-cytoplasmic fractionation was then carried out and total RNA was extracted from the nuclear fraction. The RNA was subsequently reverse-transcribed into cDNA using a SuperScript enzyme (Life Technologies/Thermo Fisher Scientific) and random N10 oligonucleotides, and amplified using PCR with PNCTR-specific primers. Specific PCR products were finally ligated with a linearized pGEM-T Easy plasmid vector (Promega) and transformed into Sure2 E.coli using electroporation. Recombinant DNA was isolated using a Qiagen plasmid miniprep kit and sent for Sanger sequencing. Three PNCTR segments (each ~800 bp) comprising CU-rich sequences were sequenced, along with several smaller PNCTR segments devoid of the (CU)_n elements. Primer walking will be done using existing clones to resolve the rest of PNCTR sequence. Importantly, this project provides the groundwork to design antisense oligonucleotides in order to examine functional consequences of altered PNCTR expression on viability and proliferation of cancer cells.