Natural antisense transcripts as drug targets

Abstract

The recent discovery of vast non-coding RNA-based regulatory networks that can be easily modulated by nucleic acid-based drugs has opened numerous new therapeutic possibilities. Long non-coding RNA, and natural antisense transcripts (NATs) in particular, play a significant role in networks that involve a wide variety of disease-relevant biological mechanisms such as transcription, splicing, translation, mRNA degradation and others. Currently, significant efforts are dedicated to harnessing these newly emerging NAT-mediated biological mechanisms for therapeutic purposes. This review will highlight the recent clinical and pre-clinical developments in this field and survey the advances in nucleic acid-based drug technologies that make these developments possible.

Keywords: anisense oligonucleotides; long nocoding RNA; natural antisense transcript (NAT); nucleic acid based therapeutics; posttranscriptional regulation.

FIGURE 1

Types of antisense transcripts. (A) Enhancer RNAs and PROMPTs are transcribed bidirectionally from enhancer and promoter regions respectively. Intronic and exonic NATs are transcribed from the DNA strand opposite to the protein-coding gene under the control of different promoters (bidirectional, independent, latent etc.). SINEUPS are encoded antisense to the 5′ end of the mRNA and have embedded transposable elements at their 3′ends. MIR-NATs are characterized by effector domains with retrotransposon-derived repeats, such as mammalian-wide interspersed repeats (MIR). (B) NATs are described as head-to-head, embedded and tail-to-tail depending on their position relative to the sense transcript. (C) Some NATs are modified with 5′-caps and polyA tails and undergo alternative splicing. NAT, natural antisense transcript; eRNA, enhancer RNA; PROMPT, promoter upstream transcript; UTR, untranslated region; SINEUP, inverted SINEB2 sequence-mediated upregulating molecule.

FIGURE 2

Transcriptional level biological processes regulated by NATs. (i) Histone and genomic DNA modification. NATs bound to specific loci in DNA scaffold epigenetic regulatory enzymes such as DNA methyltransferases (DNMT) and histone modifiers that mediate acetylation and methylation and initiate imprinting and changes in transcription rate. (ii) eRNA scaffolding 3D chromosomal interactions. eRNAs maintain the chromatin active state and mediate the enhancer-promoter looping by serving as decoys or otherwise modulating the functions of participating protein complexes such as BRD4, CBP, NELF, cohesin, Mediator complex and others leading to changes in transcription rate and mRNA abundance. (iii) PROMPT-mediated promoter-proximal pause release. PROMPTS initiate histone modifications by recruiting various epigenetic modifiers (KDM4B, KDM4C) triggering destabilization of protein complexes (HP1α, KAP1) involved in promoter-proximal Pol II pausing, resulting in release of Pol II and increased transcription. (iv) Imprinting. NATs scaffold/decoy/block different repressor and activator proteins catalyzing transcription repression of specific parental alleles. (v) DNA damage repair. NATs scaffold/decoy proteins involved in double-stranded DNA break repair. (vi) m6A RNA modification. NATs mediate m6A modification of specific RNA molecules by recruitment of regulators or enzymes associated with methyltransferase complexes (Mettl3/Mettl14) or m6A demethylases (ALKBH5). m6A modification alters availability and processing of RNAs. (vii) Alternative promoter activation. NATs alter the transcription rate of specific isoforms by antagonizing or activating alternative promoters. (viii) Alternative polyadenylation. NATs repress/activate polyA signals prompting transcription of alternative isoforms. ix) Modulation of splicing. NATs modulate the target binding and activity of splicing promoting/inhibiting factors (Sp) facilitating production of specific isoforms. DNMT, DNA methyl transferase; HMT, histone methyl transferase; eRNA, enhancer RNA; CBP/p300, CREB binding protein; PROMPT, promoter upstream transcripts; KDM4B/4C -lysine specific demethylase 4B/4C; UTR, untranslated region; m6A, N6-methyladenylation; TSS, transcription start site.

FIGURE 3

Post-transcriptional biological processes regulated by NATs. (i) miRNA sponging. NATs regulate mRNA degradation by sponging miRNA or blocking miRNA binding sites. (ii) Regulation of mRNA stability. NATs sponge proteins regulating mRNA degradation and stability. (iii) MIR-NAT-mediated translation repression. MIR-NATs overlapping 5′UTRs head-to-head compete with IRES for 40S ribosome subunit and repress translation. (iv) SINEUPs modulating translation initiation. The binding domain of SINEUP overlaps the translation initiation site and the effector domain recruits proteins (such as PTBP1) mediating assembly of translation initiation complex (TIC), resulting in translation upregulation. (v) Regulation by NAT-encoded mini-proteins. NATs can encode short peptides with downstream regulatory functions. (vi) Regulation of protein stability. NATs protect target proteins from degradation by sponging/inhibiting activity of proteins involved in ubiquitin-proteosome degradation pathway (such as SMURF1/2, MDM2). (vii) Protein translocation. NATs modulate subcellular distribution of proteins. UTR, untranslated region; CDS, coding sequence; MIR-NAT, mammalian-wide interspersed repeat natural antisense transcript; IRES, internal ribosome entry site; HuR, Hu-antigen R/ELAV-like RNA-binding protein 1; SINEUP, inverted SINEB2 sequence-mediated upregulating molecules; TIC, translation initiation complex; PTBP1, polypyrimidine tract binding protein-1; TIS, translation initiation site; USP14, ubiquitin-specific protease 14; SMURF1/2, SMAD-specific E3 ubiquitin protein ligase 1; MDM2, MDM2 proto-oncogene.