Investigating the post-transcriptional splicing of a long non-coding RNA in cancer cells.

Long non-coding RNAs play important roles in a variety of biological process including cancer. Studies have shown that the downregulation of lncRNAs is widely observed in human tumors. Removal of introns from messenger RNA precursors (pre-mRNA) by splicing is an essential step for the expression of most eukaryotic genes. Much of the splicing reactions occur in parallel to transcription, termed "co-transcriptional splicing", and only after the transcripts are spliced, are they released from the gene. Some introns in the pre-mRNA can also undergo "post-transcriptional splicing" occurring after the RNA is fully transcribed and released from the gene. In our study we are focusing on a specific lncRNA that is involved and overexpressed in several types of tumors. Using RNA FISH we detect the lncRNAs and quantify the number of single transcripts produced from the gene. By performing RNA FISH with specific probes directed to the lncRNA exon we find an unusual significant accumulation of the lncRNA transcripts at the site of transcription, which persists even after transcription inhibition. RNA FISH with specific probes to the lncRNA intron reveals distribution of unspliced lncRNA transcripts in the nucleoplasm of cancer cells and not only at the active gene, while spliced transcripts are detected in the cytoplasm as well. Since the unspliced transcripts are localized in close proximity to the site of transcription and are not found in the cytoplasm, we assume that the transcripts undergo post-transcriptional splicing and test this using a variety of treatments.