P2-175: Tau gene-specific natural antisense transcript expression and splicing in sporadic tauopathies

Abstract

Natural antisense transcripts (NATs) are endogenous RNAs that are complementary in sequence to other transcripts and potentially interfere with their expression. Common genetic variation in MAPT, a gene which encodes for the tau protein, is a strong modulator of risk of sporadic tauopathies. In progressive supranuclear palsy (PSP), increased expression of total tau as well as of tau isoforms containing four microtubule binding repeat domains are strongly associated with the disease development. Recently, NATs with direct head-to-head overlap with the tau gene promoter has been identified. In this study, we investigated the complex pattern of splicing of these NATs and their possible role in MAPT regulation. Total RNA was extracted from SH-SY5Y neuroblastoma cells and several regions from flash frozen PSP and control brains. NAT transcripts were amplified with PCR primers designed for NAT expressed sequences obtained from public nucleotide and EST databases. MAPT and NAT expression levels were quantified by real-time PCR. To investigate the effect of NAT on MAPT expression, NAT variants were cloned into mammalian expression vector and cotransfected into neuroblastoma cells with pGL4-luciferase vectors containing MAPT core promoter sequences. The NAT is transcribed both in human brain and neuroblastoma cells with a highly complex pattern of alternative splicing. We identified several novel exons and found that the majority of transcripts overlap with conserved regulatory regions of MAPT. Both MAPT and NAT were expressed in all brain regions investigated and variable levels of transcripts were detected. Based on the transcripts characterised and those published in the EST and transcript databases, the transcripts lack any open reading frames. This supports the notion that the transcript is non-coding and functions by way of its RNA transcripts. Data from cell cultures showed that NATs reduced the transcriptional activity of the MAPT promoter. In this study, we identified several NATs against the MAPT gene. Sequencing of the transcripts showed extensive alternative splicing and real time quantitative PCR demonstrated their widespread expression. Preliminary luciferase reporter assays suggest that NAT transcripts modulate MAPT promoter transcriptional activity. This could be an important mechanism in the regulation of MAPT gene expression.