Regulation of microRNA function in animals

Abstract

Since their serendipitous discovery in nematodes, microRNAs (miRNAs) have emerged as key regulators of biological processes in animals. These small RNAs form complex networks that regulate cell differentiation, development and homeostasis. Deregulation of miRNA function is associated with an increasing number of human diseases, particularly cancer. Recent discoveries have expanded our understanding of the control of miRNA function. Here, we review the mechanisms that modulate miRNA activity, stability and cellular localization through alternative processing and maturation, sequence editing, post-translational modifications of Argonaute proteins, viral factors, transport from the cytoplasm and regulation of miRNA-target interactions. We conclude by discussing intriguing, unresolved research questions.

Figure 1:. Overview of miRNA function and its regulation.

(A) Mature miRNAs operate as functional units that include an Argonaute protein. Argonaute proteins have four domains, the amino-terminal domain (N), the Piwi-Argonaute-Zwille (PAZ) domain, the middle (MID) domain and the P-element induced wimpy testes (PIWI) domain, and two linker regions (L1 and L2). The MID and PIWI domain hold the 5’ end of the miRNA and arrange it in a helical conformation; the MID domain has a binding pocket for the 5’-terminal nucleotide. Another binding pocket, in the PAZ domain, holds the 3’-terminal nucleotide ^–. Nucleotides 2–8 from the 5’ end of the miRNA form the seed, which is crucial for target-mRNA recognition. Seed interactions involve nucleotides 2–8, 2–7 and 2–6 , and can be supplemented by the binding to the MID domain of an adenine in the target mRNA opposite miRNA nucleotide 1 (t1) ^,,, or through additional, base-pairing to nucleotides ~13–16 of the miRNA, which are termed ‘supplemental region’. (B) miRNAs silence gene expression by inhibiting translation at the initiation step, likely through release of eukaryotic initiation factor 4A-1 (eIF4A1) and eIF4A2^–, and by mediating mRNA decay through interactions with glycine-tryptophan protein of 182 kDa (GW182) proteins^–. GW182 binds polyadenylate-binding protein (PABPC) and the deadenylation complexes poly(A) nuclease 2 (PAN2)–PAN3 and carbon catabolite repressor protein 4 (CCR4)–NOT^–. Deadenylation is followed by decapping by the complex mRNA-decapping enzyme subunit 1 (DCP1)–DCP2 and 5’−3’ mRNA degradation (not shown) . (C) miRNAs form complex networks of interactions, as one miRNA can target many different mRNAs, and one mRNA can be regulated by many different miRNAs, with cooperative repression achieved by binding closely-spaced target sites^,,.

Figure 2:. Isomirs differ in length and sequence and expand the functional repertoire of miRNAs

Isomirs are classified as 5’, 3’ or polymorphic, with combinations possible. Depending on the arm of the miRNA precursors (5p or 3p; see inset) used to produce the mature miRNA, cleavage by either Drosha or Dicer can result in the formation of the isomir . 5’ isomirs have shifted seeds and can thereby target a different set of genes. The functions of 3’ isomirs are less clear, but there is increased evidence for their differential activity^,. Polymorphic isomirs are generated by RNA editing, mainly by ADAR. The editing can affect miRNA biogenesis, either by preventing it, or by leading to the formation of 5’ isomirs or 3’ isomirs; if editing alters the seed, it could retarget a miRNA to other mRNAs^,. nt, nucleotides.

Figure 3:. Non-templated nucleotide addition and miRNA turnover

(A) Non-templated nucleotide addition (NTA) by GLD2 can stabilize some miRNAs, but has no effect on others. Terminal 3’ adenylation of miR-21 by PAP-associated domain-containing protein 5 (PAPD5) was even found to promote exonucleolytic cleavage by poly(A)-specific ribonuclease (PARN) . Terminal 3’ uridylation by terminal uridylyltransferase 4 (TUT4) can reduce the activity of miR-26b, or prime miRNAs for degradation following T-cell activation. (B) Terminal 3’ adenylation stabilizes some miRNAs by counteracting 3’−5’ exonucleolytic activity by PARN. CUG triplet repeat RNA-binding protein 1 (CUGBP1) interacts with miR-122 and recruits PARN. (C) Interactions of an Argonaute (AGO)-bound miRNA with a target mRNA through the miRNA seed sequence result in translation repression and mRNA degradation. Targets with extensive pairing to the 3’ end promote tailing^, and trimming, and target-directed miRNA degradation^,,. A target mRNA that is fully complementary to a miRNA is cleaved when bound by a catalytic Argonaute, such as mammalian Ago2^,, but such pairing can also result in unloading of the miRNA from AGO. Cell-lines: A549 (adenocarcinomic human alveolar basal epithelial cell line), Huh-7 (human hepatocellular carcinoma cell line), human cellular carcinoma, THP1 (human leukemic monocyte cell line).

Figure 3:. Non-templated nucleotide addition and miRNA turnover

(A) Non-templated nucleotide addition (NTA) by GLD2 can stabilize some miRNAs, but has no effect on others. Terminal 3’ adenylation of miR-21 by PAP-associated domain-containing protein 5 (PAPD5) was even found to promote exonucleolytic cleavage by poly(A)-specific ribonuclease (PARN) . Terminal 3’ uridylation by terminal uridylyltransferase 4 (TUT4) can reduce the activity of miR-26b, or prime miRNAs for degradation following T-cell activation. (B) Terminal 3’ adenylation stabilizes some miRNAs by counteracting 3’−5’ exonucleolytic activity by PARN. CUG triplet repeat RNA-binding protein 1 (CUGBP1) interacts with miR-122 and recruits PARN. (C) Interactions of an Argonaute (AGO)-bound miRNA with a target mRNA through the miRNA seed sequence result in translation repression and mRNA degradation. Targets with extensive pairing to the 3’ end promote tailing^, and trimming, and target-directed miRNA degradation^,,. A target mRNA that is fully complementary to a miRNA is cleaved when bound by a catalytic Argonaute, such as mammalian Ago2^,, but such pairing can also result in unloading of the miRNA from AGO. Cell-lines: A549 (adenocarcinomic human alveolar basal epithelial cell line), Huh-7 (human hepatocellular carcinoma cell line), human cellular carcinoma, THP1 (human leukemic monocyte cell line).

Figure 4:. The activity and the stability of miRISC is modulated by post-translational modifications (PTMs) of Argonaute proteins.

(A) Phosphorylation of Ser387 (S387) in the L2 region of Argonaute (AGO) was found to be mediated by MAP kinase-activated protein kinase 2 (MAPKAPK2) and AKT3 in vitro. Ser387 phosphorylation increases miRNA activity by stimulating the assembly of miRNA-induced silencing complexes, and reduces translocation of Ago2 to multivesicular endosomes and secretion of exosomes. Phosphorylation of the nearby Tyr393 (Y393), also in the L2 region, decreases the miRNA–Ago2 association, thereby reducing miRNA activity^,. Tyr 529 (Y529) is located in the middle (MID) domain, near the miRNA 5’-nucleotide binding pocket, and its phosphorylation prevents miRNA loading. No function has yet been assigned to phosphorylation sites in the Piwi-Argonaute-Zwille (PAZ) domain (S253, T303, T307) and the P-element induced wimpy testes (PIWI) domain (S798) . Additional AGO PTMs include Pro700 (P700) 4-hydroxylation, which increases Ago2 stability. Lys402 (K402) SUMOylation, which was reported to either destabilize Ago2 or be required for full siRNA activity; and poly(ADP-ribosylation) (PARylation), which inhibits miRNA activity^,, presumably by decreasing target accessibility. (B) The S824–S834 cluster in the eukaryotic insertion region of human Ago2 undergoes a phosphorylation cycle, which regulates AGO–target interactions. Phosphorylation of the Ser residues in the cluster by casein kinase I isoform α (CSNK1A1) favors target release. Subsequent dephosphorylation by the serine/threonine-protein phosphatase 6 complex ANKRD52–PPP6C primes Ago2 for the next round of target binding^,.

Figure 5:. miRNA sequestration by endogenous and viral RNAs

(A) The competing endogenous RNA (ceRNA) hypothesis states that a newly expressed RNA can compete with the already present microRNA (miRNA) targets for cytoplasmic miRNA-induced silencing complexes (miRISCs), potentially leading to de-repression of certain genes. However, a ceRNA is unlikely to lead to gene de-repression when it is expressed at typical physiological levels^–. (B) Long non-coding RNAs (lncRNAs), pseudogenes and mRNAs can have ceRNA activity. In mice, long intergenic non-protein coding RNA of muscle differentiation 1 (Linc-md1) contains one binding site for miR-133 (blue) and two for miR-135 (green). By sequestering these miRNAs, the muscle-specific transcription factors mastermind-like protein 1(Maml1) and myocyte-specific enhancer factor 2C (Mef2c) are de-repressed, thereby promoting myoblast differentiation. The pseudogene phosphatase and tensin homolog pseudogene 1 (PTENP1) shares many miRNA target sites with the tumor suppressor PTEN, and can de-repress PTEN in human cells . Similarly, the mouse zinc finger E-box binding homeobox 2 (Zeb2) mRNA can de-repress PTEN. (C) Different viral mechanisms affect miRNA function. Hepatitis C virus (HCV) harbors two binding sites for miR-122 at the very end of the 5’ untranslated region (UTR) of its RNA genome. These recruit argonaute2 (AGO2)–miR-122 complexes to protect the viral RNA from the cellular antiviral response and the activity of exonucleases, and functionally sequester miR-122 and de-repress hepatic miR-122 target mRNAs. Bovine viral diarrhea virus (BVDV) is an RNA virus that contains a binding site for miR-17 in its 3’ UTR; miR-17 binding enhances the stability of the viral RNA. The site functionally sequesters the miRNA and de-represses its cellular targets. Herpesvirus saimiri (HVS) produces short non-coding RNAs termed herpesvirus saimiri uracyl-rich RNAs (HSURs), two of which are known to modulate miRNA function. HSUR1 binds miR-27a and promotes its TDMD, which leads to de-repression of miR-27a cellular targets and promotes T cell activation. HSUR2 binds miR-142–3p and miR-16 and tethers them to cellular target mRNAs, which prevents apoptosis.

Figure 5:. miRNA sequestration by endogenous and viral RNAs

(A) The competing endogenous RNA (ceRNA) hypothesis states that a newly expressed RNA can compete with the already present microRNA (miRNA) targets for cytoplasmic miRNA-induced silencing complexes (miRISCs), potentially leading to de-repression of certain genes. However, a ceRNA is unlikely to lead to gene de-repression when it is expressed at typical physiological levels^–. (B) Long non-coding RNAs (lncRNAs), pseudogenes and mRNAs can have ceRNA activity. In mice, long intergenic non-protein coding RNA of muscle differentiation 1 (Linc-md1) contains one binding site for miR-133 (blue) and two for miR-135 (green). By sequestering these miRNAs, the muscle-specific transcription factors mastermind-like protein 1(Maml1) and myocyte-specific enhancer factor 2C (Mef2c) are de-repressed, thereby promoting myoblast differentiation. The pseudogene phosphatase and tensin homolog pseudogene 1 (PTENP1) shares many miRNA target sites with the tumor suppressor PTEN, and can de-repress PTEN in human cells . Similarly, the mouse zinc finger E-box binding homeobox 2 (Zeb2) mRNA can de-repress PTEN. (C) Different viral mechanisms affect miRNA function. Hepatitis C virus (HCV) harbors two binding sites for miR-122 at the very end of the 5’ untranslated region (UTR) of its RNA genome. These recruit argonaute2 (AGO2)–miR-122 complexes to protect the viral RNA from the cellular antiviral response and the activity of exonucleases, and functionally sequester miR-122 and de-repress hepatic miR-122 target mRNAs. Bovine viral diarrhea virus (BVDV) is an RNA virus that contains a binding site for miR-17 in its 3’ UTR; miR-17 binding enhances the stability of the viral RNA. The site functionally sequesters the miRNA and de-represses its cellular targets. Herpesvirus saimiri (HVS) produces short non-coding RNAs termed herpesvirus saimiri uracyl-rich RNAs (HSURs), two of which are known to modulate miRNA function. HSUR1 binds miR-27a and promotes its TDMD, which leads to de-repression of miR-27a cellular targets and promotes T cell activation. HSUR2 binds miR-142–3p and miR-16 and tethers them to cellular target mRNAs, which prevents apoptosis.

Figure 6:. Mechanisms of sorting miRNAs into exosomes.

(A) miRNAs can be packaged into exosomes and thus may contribute to inter-cellular signaling. Uptake of exosomes can be receptor-mediated or receptor-indepedent. Upon entering a target cell, exosome-delivered miRNAs are speculated to regulate target mRNAs. (B) Although the biological function of exosomal miRNAs is still incompletely understood, multiple mechanisms direct miRNAs into exosomes. Argonaute 2 (Ago2)-dependent sorting of specific miRNAs has been reported in isogenic colon cancer cells, and phosphorylation of Ago2 Ser387 inhibited loading of some miRNAs into exosomes. Sorting based on miRNA-sequence complementary of exosomal long non-coding RNAs (lncRNA) was shown for miR-149–3p in prostate cancer cells. Exosomal RNA-binding proteins (RBP) can direct miRNAs into exosomes by binding ‘exomotifs’ at the miRNA 3’ ends. The exomotif GGAG promotes exosomal sorting of miR-198 by heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2B1) in human primary T cells, and the exomotif GGCU promotes hnRNPQ -mediated exosomal sorting of miRNAs in murine hepatocytes. No exomotifs are known for Y-box-binding protein 1 (YBX1)-mediated sorting of miR-223 in HEK293T cells and for major vault protein (MVP)-mediated sorting of miR-193a. Finally, in human B cells 3’-adenylated miRNAs are depleted in exosomes whereas 3’-uridylated miRNAs enriched in exosomes. Exosomes have been reported to carry proteins, different RNAs and miRNA biogenesis components , but also Ago2–miRNA complexes and AGO-free miRNAs.