Long noncoding RNA (lncRNA) are important regulatory RNA that are commonly misregulated in Disease. The lncRNA RPPH1 was previously associated with breast cancer metastasis (Zhang, Y., Tang, L., 2017). Surprisingly, for an RNA polymerase III transcription product, the RNAs present as polyadenylated isoforms. The ratio of two isoforms is cancer cell line specific, and the MCF10A progression series revealed that an increase in the long polyadenylated RPPH1 isoform correlated with increasing metastatic potential.
An investigation into mechanisms regulating RPPH1 polyadenylation revealed that the short isoform depended on the non-canonical poly(A) polymerase PAPD5 and thus represents a decay intermediate. Whereas, transcriptional inhibition with actinomycin D rapidly reduced the long RPPH1 isoform suggests its polyadenylation is linked to transcription. The 223bp RPPH1 promoter is shared with the RNA Polymerase II (RNAPII)-driven PARP2 gene (Ame, J.C., et. al. 2001). We hypothesised that a switch in RNAPII directionality generates the long polyadenylated RPPH1 and recruits the traditional 3’-end formation machinery to extend its poly(A)-tail. This idea was tested by use of a CDK9 inhibitor AZ5576 to prevent RNAPII promoter-proximal pause release. Application of this inhibitor resulted in increased long polyadenylated RPPH1 isoform and decreased PARP2 expression. We propose that this is due to inhibitor induced RNAPII stalling within PARP2 that results in increased bidirectional RNAPII activity and aberrant RNAPII-mediated RPPH1 transcription.
A shift toward RPPH1 long polyadenylated isoforms in cancer cells might reflect a change in RNA polymerase II activity that is particularly evident at this bidirectional promoter. Our research is still to uncover how this may be linked with the metastatic trajectory of tumours, but we are seeking to understand if the ratio of these adenylated RPPH1 isoforms might serve as functional cancer biomarkers.