Short non-coding RNA biology and neurodegenerative disorders: novel disease targets and therapeutics

Abstract

Genomic studies in model organisms and in humans have shown that complexity in biological systems arises not from the absolute number of genes, but from the differential use of combinations of genetic programmes and the myriad ways in which these are regulated spatially and temporally during development, senescence and in disease. Nowhere is this lesson in biological complexity likely to be more apparent than in the human nervous system. Increasingly, the role of genomic non-protein coding small regulatory RNAs, in particular the microRNAs (miRNAs), in regulating cellular pathways controlling fundamental functions in the nervous system and in neurodegenerative disease is being appreciated. Not only might dysregulated expression of miRNAs serve as potential disease biomarkers but increasingly such short regulatory RNAs are being implicated directly in the pathogenesis of complex, sporadic neurodegenerative disease. Moreover, the targeting and exploitation of short RNA silencing pathways, commonly known as RNA interference, and the development of related tools, offers novel therapeutic approaches to target upstream disease components with the promise of providing future disease modifying therapies for neurodegenerative disorders.

Figure 1.

Mammalian RNAi regulatory pathways. (A) miRNAs are encoded in pri-miRNAs (124,125). These ∼100 nt inverted repeat motifs are usually found embedded once or multiple times within coding or non-coding RNA Pol II-derived transcripts (126). Pri-miRNAs are first processed in the nucleus where their hairpin-like structures are recognized and cleaved by the RNase III enzyme Drosha together with DiGeorge critical region 8 protein (DGCR8), to produce shorter hairpin duplexes known as a 70–80 nt pre-miRNAs (127,128). For a small minority of miRNAs, short intronic sequences, referred to ‘mirtrons’, can be directly processed by the spliceosome into pre-miRNA-like hairpins without requiring Drosha cleavage (30,129). Spliced lariats are de-branched and likely produce functional pre-miRNAs for export. Pre-miRNAs are exported from the nucleus to the cytoplasm by the exportin-5 (130,131) followed by recognition and cleavage by a second RNase III enzyme, Dicer and its partner, TAR RNA-binding protein (TRBP), to produce a ∼22 bp, staggered miRNA/miRNA* duplex with 2 nt 3′ overhangs (–134). Dicer/TRBP, loads one of the strands, the ‘guide strand’, into a RISC consisting in its simplest form of Ago2 (135,136) directing cleavage of translational suppression of cognate RNA. (B) The mechanism of TGS is poorly understood in mammals but is thought to include a complex consisting (RITS) of Ago1, (and possibly Ago2) a polycomb group component, enhancer of zeste 2 (EZH2) and DNA methyltransferase 3a (Dnmt3a) (21,23,137). Moreover, TGS may require the presence of low-copy promoter-derived transcripts to direct silent heterochromatin marks (H3K9 and/or H3K27 methylation) and DNA methylation at the targeted locus (138). (C) Endogenous siRNAs are derived from long hairpin sequences and complementary transcripts which are processed by Dicer into siRNAs. piRNAs are 24–31 nt short RNAs processed from single-stranded precursors derived from transposons or genomic repeat elements in the germline (13). In the ‘ping-pong model’, primary piRNAs interact with the Piwi protein MILI to cleave a transcript that generates a piRNA for incorporation into MIWI2, which in turn cycles back to produce new MILI-interacting piRNAs (24,25).

Figure 2.

Exogenous RNAi-mediated gene silencing. RNA Pol II-derived transcripts introduce hairpin duplexes which structurally mimic mono or polycistronic pri-miRNAs which are recognized and processed by Drosha/DGCR8 into pre-miRNA-like hairpins. These hairpins are cleaved by Dicer/TRBP following export via exportin-5. The Pol II-generated U1 shRNA transcripts, which contain a 3′ terminal B-box, structurally mimic pre-miRNAs but are likely exported by the CRM1 pathway prior to Dicer/TRBP cleavage (139). RNA Pol III promoters express shRNAs and long hairpin RNAs (lhRNAs) with defined 5′ and 3′ termini. U6 or H1-derived shRNAs and lhRNAs, like pre-miRNAs, consist of 2 nt 3′ overhangs and exit the nucleus via exportin-5. lhRNAs, are processed sequentially by Dicer to produce up to three siRNAs (140,141). tRNA^Lys3 and tRNA^Val Pol III promoters can also be used to produced tRNA–shRNAs for processing in the nucleus by 5′ and 3′ tRNA processing enzymes prior to export (69,142). If unprocessed by RNase Z^L, tRNA–shRNAs may exit the nucleus via exportin-t (143). Synthetic siRNAs or pre-miRNA mimics can be introduced as Dicer substrates (typically >25 bp duplexes) or as 19 bp duplexes for direct loading into Ago2-RISC.

Figure 3.

Regulation and modulation of miRNA biogenesis and function. A number of endogenous and exogenous factors can control miRNA biogenesis, and these have the potential to be exploited as novel therapies. Transcription factors act on promoters to activate or suppress endogenous miRNA gene expression. Post-transcriptional control can occur at the level or pri-miRNA processing in the nucleus, or pre-miRNA cleavage in the cytoplasm. In the nucleus, Drosha/DGCR8 preferentially processes pri-miRNAs that are retained at the site of transcription (144), regulating pri-miRNAs co-transcriptionally (145). To stimulate the activity of a specific miRNA, exogenous pri-miRNAs can be introduced as gene-based cassettes or as synthetic pre-miRNAs. Factors which positively regulate specific pri-miRNAs include the SMAD proteins and hnRNAP A1 (112,113), with other positive or negative regulatory factors likely to be discovered in the future. Pri-miRNAs are additionally subject to RNA editing (adenosine to inosine RNA editing), resulting in an unprocessed pri-miRNA or in miRNAs with altered seed regions (146,147). miRNAs can be blocked by ‘looptomirs’ or short ASOs targeted to the loop of pri-miRNAs (112,113). In the cytoplasm, miRNAs can be blocked by ‘antagomirs’ or ASOs (120,121). Moreover, expressed sequences that contain multiple miRNA-targets are referred to as ‘sponges’, blocking translational suppression of endogenous mRNA targets (122). Small molecule inhibitors may be found to block miRNA: mRNA interactions, pre-miRNA maturation or earlier steps in the nucleus. miRNA dysregulation has the potential to affect multiple genes, whose products in turn may positively or negatively feedback to regulate specific miRNA gene transcription or biogenesis.