Gene regulation by non-coding RNAs

Abstract

The past two decades have seen an explosion in research on non-coding RNAs and their physiological and pathological functions. Several classes of small (20-30 nucleotides) and long (>200 nucleotides) non-coding RNAs have been firmly established as key regulators of gene expression in myriad processes ranging from embryonic development to innate immunity. In this review, we focus on our current understanding of the molecular mechanisms underlying the biogenesis and function of small interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs). In addition, we briefly review the relevance of small and long non-coding RNAs to human physiology and pathology and their potential to be exploited as therapeutic agents.

Figure 1. siRNA-mediated gene regulation

siRNAs can be introduced experimentally into the cell or processed by Dicer and partners from long dsRNAs or shRNAs. siRNAs interact with Ago2 and other proteins to form the RISC complex. During RISC assembly the siRNA guide strand is loaded onto Ago2 while the passenger strand is degraded. Once the active RISC is formed, the siRNA guide strand pairs with, cleaves, and degrades the target mRNA, and. the RISC complex is recycled.

Figure 2. Biogenesis of miRNAs and their role in gene silencing

miRNAs are transcribed by RNA Pol II as long hairpin structures called primary miRNAs (pri-miRNAs), which are then processed into ~60–70-nt precursor miRNAs (pre-miRNAs) by the Drosha-DGCR8 complex. The pre-miRNAs are exported out of the nucleus by Exportin-5, and are processed into ~22-nt miRNA/miRNA* duplexes by Dicer and its partners. The 22-nt RNA duplexes associate with Ago and other proteins to form the miRISC complex. miRISCs bind at the 3′ UTR of the target mRNA, in which results translational repression of the target gene. The imperfect base pairing between the miRNA and the target mRNA leads to bulge structures that cannot be cleaved by Ago proteins.

Figure 3. piRNA biogenesis pathway

An unknown primary processing event generates antisense piRNAs from the piRNA loci and/or from active transposons. Antisense piRNAs associate with Aub or Piwi, which cleave and process the target mRNA to generate sense piRNAs. Sense piRNAs associate with Ago3, which then binds the antisense transcripts. This form of repetitive binding and cleavage creates an amplification loop that maintains a constant level of sense and antisense piRNAs. The flamenco-derived piRNAs, on the other hand, are generated by an unknown primary processing pathway that does not involve an amplification cycle. Flamenco piRNAs appear to be restricted to somatic follicle cells and are associated with Piwi.

Figure 4. Transcriptional control mediated by lncRNAs Gas-5 and lincRNA-p21.

The lncRNA Gas-5 functions as a molecular decoy to prevent binding of the nuclear transcription factor GR to response elements in the cIAP2 promoter region. LincRNA-p21 acts as a repressor at both the transcriptional and translational levels.

Figure 5. Post-transcriptional and translational regulation mediated by the lncRNAs MALAT-1 and BACE1-AS, respectively

MALAT-1 is retained in the nucleus and modulates alternative splicing by forming an RNP complex with serine/arginine (SR) splicing factors and their recruitment to the transcription site. BACE1 and BACE1-AS form RNA duplexes that stabilize the mRNA. Altering expression of BACE1-AS decreases or increases the stability of BACE1 mRNA.

Figure 6. Epigenetic regulation mediated by the lncRNAs HOTAIR and ANRIL.

HOTAIR, one of the best-characterized lncRNAs, is a 2.2 kb antisense transcript residing in the HOXC locus. HOTAIR mediates epigenetic silencing by interacting with the PRC2 and LSD1-CoREST complexes via its 5′ and 3′ domains, respectively. The EZH2 subunit of PRC2 has histone methyltransferase activity and trimethylates histone 3 at lysine 27, whereas LSD1 demethylates H3K4me2 and H3K4me1. The lncRNA ANRIL is located within the INK4b/ARF/INK4a locus that encodes three tumor suppressor genes: INK4b encodes p15/CDKN2B (cyclin-dependent kinase inhibitor 2B), ARF encodes p14/ARF (alternative reading frame), and INK4a encodes p16/CDKN2A (cyclin-dependent kinase 2A). ANRIL regulates transcriptional silencing of INK4a by recruiting the PcG protein Chromobox 7 (CBX7) to the INK4b/ARF/INK4a locus. CBX7 is a component of PRC1, which binds to the H3K27me3 repressive mark and is required for maintenance of epigenetic gene silencing.