Non-coding RNAs: Epigenetic regulators of bone development and homeostasis

Abstract

Non-coding RNAs (ncRNAs) have evolved in eukaryotes as epigenetic regulators of gene expression. The most abundant regulatory ncRNAs are the 20-24 nt small microRNAs (miRNAs) and long non-coding RNAs (lncRNAs, <200 nt). Each class of ncRNAs operates through distinct mechanisms, but their pathways to regulating gene expression are interrelated in ways that are just being recognized. While the importance of lncRNAs in epigenetic control of transcription, developmental processes and human traits is emerging, the identity of lncRNAs in skeletal biology is scarcely known. However, since the first profiling studies of miRNA at stages during osteoblast and osteoclast differentiation, over 1100 publications related to bone biology and pathologies can be found, as well as many recent comprehensive reviews summarizing miRNA in skeletal cells. Delineating the activities and targets of specific miRNAs regulating differentiation of osteogenic and resorptive bone cells, coupled with in vivo gain- and loss-of-function studies, discovered unique mechanisms that support bone development and bone homeostasis in adults. We present here "guiding principles" for addressing biological control of bone tissue formation by ncRNAs. This review emphasizes recent advances in understanding regulation of the process of miRNA biogenesis that impact on osteogenic lineage commitment, transcription factors and signaling pathways. Also discussed are the approaches to be pursued for an understanding of the role of lncRNAs in bone and the challenges in addressing their multiple and complex functions. Based on new knowledge of epigenetic control of gene expression to be gained for ncRNA regulation of the skeleton, new directions for translating the miRNAs and lncRNAs into therapeutic targets for skeletal disorders are possible. This article is part of a Special Issue entitled Epigenetics and Bone.

Keywords: LncRNAs; MicroRNA; Osteoblasts; miRNA biogenesis.

Fig. 1

MicroRNA biogenesis, maturation, function and decay. Nuclear events: (A) schematic model of microRNA (miRNA) transcription to synthesize primary miRNA (pri-miRNA) by RNA polymerase II (Pol II), and processing of pri-miRNA by the Drosha–DGCR8 microprocessor complex to generate precursor miRNA (pre-miRNA) in the nucleus. Exportin 5 (EXP5) RAN•GTP complex exports pre-miRNA from nucleus to cytoplasm; (B) examples of miRNA transcriptional control and processing: tumor suppressor p53, growth factor MYC and myoblast specific transcription factor MYOD1 transactivate miR-34, miR-17 and miR-1 clusters, respectively. Osteoblast specific factor Runx2, leukemic factor Runx1, MYC, and zinc finger transcription factors ZEB1 and ZEB2 transcriptionally suppress the miR-23a cluster, miR-15a cluster, and miR-200 cluster. DNA methyltransferases (DNMTs) and RE1-Silencing Transcription Factor (REST) epigenetically regulate miR-9 and miR-124, respectively, at the level of transcription. Numerous RNA-binding proteins, including p68, KH-type splicing regulatory protein (KSRP), heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) and LIN28, regulate the processing of primary miRNAs including miR-21, 199a, miR-21, let-7, miR-16, miR-18a, and let7. The phosphorylation and acetylation of Drosha and DGCR8 proteins control the processing activity of these proteins; (C) cytoplasmic events: illustration of pre-miRNA maturation and formation of pre-and mature RNA Induced Silencing Complex (RISC). Dicer associates with TRBP (TAR RNA-binding protein) and processes pre-miRNA to generate 22–24 nt mature duplex miRNA that subsequently loaded onto AGO2 to form pre-RISC complex. Heat shock protein 90 (HSP90) and heat shock cognate 70 (HSC70) form a complex that hydrolyses ATP to load the RNA duplex on to the RISC. The miRNA* (passenger strand) is further degraded and the mature miRNA ‘guide’ strand remains in the RISC complex. Post-translational modifications AGO proteins, including prolyl hydroxylation, poly-ADP ribosylation and phosphorylation influence its efficiency and ability to control the processing of Dicer, RISC formation and miRNA activity; (D) RNA helicases, including MOV10 [152], DDX6 [153], translational repressor FMR1 [154], GW182 and AGO2 [4] are present in the active RISC and mediate miRNA-dependent repression of translation of complementary mRNAs by Argonaute proteins. Succeeding translation repression cognate mRNA is degraded by CAF1–CCR4 deadenylase complex [155]; (E) target guided miRNA degradation by tailing and trimming mechanism [156].

Fig. 2

miRNA circuitry: pathways supporting bone formation. (A) Positive regulation of miR-218 for normal osteogenesis and in promoting metastasis. Wnt signaling is activated by miR-281 which increases Runx2 and multiple genes that promote matrix formation and mineralization during osteoblast differentiation. However high levels of miR-218 are associated with cancer and their osteomimetic properties promote metastasis to bone. (B) Negative regulation of the cluster MiR-23a by Runx2 transcriptional down-regulation of the cluster at a Runx site in its promoter. This action relieves the inhibition of both Runx2 and Satb2 which form a complex that drives differentiation. Restraints are placed on the feed forward path to bone formation by miR-23a targeting Runx2 and miR-27a by targeting Hoxa10 an activator of Runx2 in osteoprogenitors. (C) Positive regulation by Runx2 of two miRNAs, miR-3960 and miR-2861, targets an inhibitor of Runx2, HDAC5 and Hoxa2, respectively. The transcriptional activation of these miRNAs downregulates the inhibitors and generates a feed forward circuit for osteogenesis.