Natural Antisense Transcripts: Molecular Mechanisms and Implications in Breast Cancers

Abstract

Natural antisense transcripts are RNA sequences that can be transcribed from both DNA strands at the same locus but in the opposite direction from the gene transcript. Because strand-specific high-throughput sequencing of the antisense transcriptome has only been available for less than a decade, many natural antisense transcripts were first described as long non-coding RNAs. Although the precise biological roles of natural antisense transcripts are not known yet, an increasing number of studies report their implication in gene expression regulation. Their expression levels are altered in many physiological and pathological conditions, including breast cancers. Among the potential clinical utilities of the natural antisense transcripts, the non-coding|coding transcript pairs are of high interest for treatment. Indeed, these pairs can be targeted by antisense oligonucleotides to specifically tune the expression of the coding-gene. Here, we describe the current knowledge about natural antisense transcripts, their varying molecular mechanisms as gene expression regulators, and their potential as prognostic or predictive biomarkers in breast cancers.

Keywords: breast cancer; gene expression regulation; lncRNA; natural antisense transcript; natural antisense transcripts; next generation sequencing; non-coding RNA.

Figure 1

lncRNA classification according to their orientation and position in the genome. lincRNAs are located between two pcGenes, regardless of their orientation. Intronic lncRNAs are entirely encoded in pcGene introns, while sense lncRNAs overlap pcGene exons. Bidirectional lncRNA transcription starts less than 1 kb from a pcGene transcription start site and goes in its opposite direction. Cis-NATs (natural antisense transcript) are RNA sequences that are transcribed from the two strands of the same genomic locus, in the antisense direction. NAT pairs can be protein-coding sequences (pc, red colored) or non-coding sequences (nc, blue colored), forming nc|pc, nc|nc or pc|pc pairs. NAT pairs that are nc|pc or nc|nc sequences only belong to the lncRNA classification (purple colored sequences are pc or nc).

Figure 2

cis-NAT classification. cisNAT pairs can be protein coding sequences (pc) or non-coding sequences (nc), forming nc|pc, nc|nc or pc|pc pairs. In head-to-head orientation, sense and antisense transcripts overlap on their 5′ ends. Inversely, tail-to-tail describes an overlap of the 3′ ends. In a full overlap (or embedded overlap), one transcript is totally included in the other one.

Figure 3

Transcriptional Interference: (A) in the initiation phase, promoters of head-to-head NATs are competing for the use of RNA Pol II and common regulatory elements; (B) in the elongation phase, interference can occur after the following events: a collision between RNA Pol II complexes, leading to a machinery blockage; (C) a promoter occlusion by RNA Pol II during the antisense transcript; and (D) a RNA Pol II dislodgement by the RNA Pol II standing on the opposite strand, when the first one was too slow to start. Promoters of protein coding sequences are represented in red, and promoters of non-coding sequences in blue. RNA pol II enzyme is represented in dark grey when able to transcribe the sequence, and light grey when its binding and thus activity, is prevented.

Figure 4

ncNATs (non-coding natural antisense transcripts) may regulate the expression levels of the sense pcGenes (protein coding genes) by regulating chromatin modifications by the following. (A) A decoy mechanism: The NAT binds a protein complex that can trigger chromatin modifications and prevents, by competition, this complex from binding the sense transcript. This complex can also prevent the interaction of the sense gene with RNA Pol II (RNA polymerase II); (B) a tethering mechanism, such as ANRIL (antisense non-coding RNA in the INK4 locus): ANRIL recruits PRC2 (polycomb repressive complex) through interaction with SUZ12 (suppressor of zeste 12 homolog) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) components and PRC1 by binding CBX7 (chromobox homolog 7). Next, PRC2 silences the INK4 locus expression by inducing H3K27 tri-methylation, and PRC1 maintains a repressive chromatin structure by mono-ubiquitination of H2AK119. Protein coding sequences or promoters are represented in red, and non-coding in blue.