Metazoan MicroRNAs

Abstract

MicroRNAs (miRNAs) are ∼22 nt RNAs that direct posttranscriptional repression of mRNA targets in diverse eukaryotic lineages. In humans and other mammals, these small RNAs help sculpt the expression of most mRNAs. This article reviews advances in our understanding of the defining features of metazoan miRNAs and their biogenesis, genomics, and evolution. It then reviews how metazoan miRNAs are regulated, how they recognize and cause repression of their targets, and the biological functions of this repression, with a compilation of knockout phenotypes that shows that important biological functions have been identified for most of the broadly conserved miRNAs of mammals.

Figure 1. The Biogenesis and Function of Canonical miRNAs of Animals

(A) Biogenesis and function of a typical miRNA. Once transcribed by Pol II, the pri-miRNA folds back on itself to form at least one distinctive hairpin structure (further described in Figure 2), which is cleaved by Microprocessor (purple arrowheads, cleavage sites) to release a pre-miRNA (P, 5′ phosphate). The pre-miRNA hairpin is exported to the cytoplasm through the action of Exportin 5 and RAN–GTP. In the cytoplasm, the pre-miRNA is cleaved by Dicer (blue arrowheads, cleavage sites) to produce a ~20-bp miRNA duplex with a 5′ phosphate (P) and a 2-nt 3′ overhang on each end. One strand of the miRNA duplex, the mature miRNA (red), is loaded into the guide-strand channel of an Argonaute protein (Ago) to form a silencing complex, whereas the other strand, the miRNA* (blue), is degraded. Within the free silencing complex, miRNA nucleotides 2–5 (upward red vertical lines) are poised to initially interact with target RNAs (blue; filled circle, cap; AAAAA, poly(A) tail). This pairing usually extends to nucleotide 7 or 8 of the miRNA, and occasionally is more extensive (Figures 5 and 6). If pairing is very extensive, the target can be sliced (left; black arrowhead, cleavage site), whereas if it is not, the target can undergo other types of repression (right; further described in Figure 4). (B) Typical sources of canonical miRNAs. Most canonical miRNAs derive from the introns or exons of non-coding primary transcripts, some of which harbor hairpins for more than one miRNA. In addition, many canonical miRNAs derive from introns of pre-mRNAs.

Figure 2. The Menu of Structural and Primary-Sequence Features that Define Canonical miRNA Hairpins

Structural features include a 35-bp stem with an unstructured loop and unstructured flanking regions. At every position within the stem except for one, pairing is preferred; this is typically Watson–Crick pairing, although at many positions one or both of the G–U wobble pairs is as favorable as some of the Watson–Crick pairs. Natural miRNA hairpins typically also have a few mismatches or small bulges scattered at various locations within the stem, and the mismatches are each counted as base pairs when describing the optimal length of 35 ± 1 bp. Additional features can enhance processing and help specify the sites of cleavage (purple arrowheads); these include a basal UG motif, an apical UGU motif, a flanking CNNC motif (in which N is any of the four nucleotides), and a mismatched GHG motif (in which H is A, C, or U), each located at the indicated positions relative to the Drosha cleavage sites (Auyeung et al., 2013; Fang and Bartel, 2015). Drosha recognizes the base of the hairpin, including the basal UG and probably the mismatched GHG motif, whereas the DGCR8 dimer recognizes the apical region, including the apical UGU (Nguyen et al., 2015), and together Drosha and DGCR8 form a molecular caliper that measures the length of the stem. An auxiliary factor, such as SRp20 or p72, recognizes the flanking CNNC motif (Auyeung et al., 2013; Mori et al., 2014). The sites of Dicer cleavage are also shown (blue arrowheads).

Figure 3. Comparison of Canonical and Noncanonical Biogenesis Pathways

(A) The processing of canonical miRNA hairpins. (B–D) The processing of mirtrons (B), endogenous shRNAs (C), and chimeric hairpins (D), which each feed into the canonical pathway after bypassing Microprocessor. (E) The biogenesis of miR-451, which bypasses Dicer. All genes are transcribed by Pol II, except as noted. Drawing conventions are as in Figure 1.

Figure 4. The Dominant Mechanisms of miRNA-guided Repression in Bilaterian Animals

Guided by the miRNA, the silencing complex associates with the mRNA and recruits TNRC6, which interacts with PABPC and recruits either the PAN2–PAN3 deadenylase complex (not shown) or the CCR4–NOT deadenylase complex, either of which shortens the mRNA poly(A) tail. Alternative downstream consequences of poly(A)-tail shortening, which are not depicted in this figure, consummate this major mode of TNRC6-mediated repression; in early embryos tail shortening reduces translation initiation with little effect on mRNA stability, whereas in most other developmental contexts tail shortening hastens decapping and degradation of the mRNA with relatively little effect on translation initiation. Although not through tail shortening, recruitment of TNRC6 can nonetheless repress translation initiation in postembryonic cells through a parallel mechanism that involves CCR4–NOT-mediated recruitment of DDX6 and 4E-T. This translation initiation normally involves the recruitment of the 43S preinitiation complex (PIC) through the action of initiation factors (4A, 4B, 4E, 4G).

Figure 5. MicroRNA Target Sites

(A) Canonical sites of mammalian miRNAs. These canonical sites each have 6–7 contiguous Watson–Crick pairs (vertical lines) to the seed region of the miRNA (miRNA positions 2–8). Two of these sites also include an A at position 1. Relative site efficacy in mammalian cells is graphed to the right (log scale). The most effective canonical sites are 7–8 nt sites that include a perfect match to the miRNA seed (positions 2–7, red), whereas the 6 nt sites are the least effective. Not shown is the 6mer-A1 site (the same as the 7mer-A1 but lacking a pair at position 7), which is rarely conserved above background in mammalian mRNAs and has negligible efficacy in mammalian cells yet is sometimes included among the canonical sites because of its more robust conservation and efficacy in C. elegans (Jan et al., 2011). (B) The 3′-supplementary site, an atypical type of canonical site. A small fraction of the canonical sites (< 5%) benefit from pairing to the 3′ region of the miRNA. Productive 3′-supplementary pairing typically centers on nucleotides 13–16. (C) The 3′-compensatory site, a functional type of noncanonical site. The noncanonical sites do not have six contiguous Watson–Crick pairs to the seed region. Shown is a mismatch at position 6, but the imperfection (either a wobble, mismatch, or single-nucleotide bulge) can instead occur at another seed position. Compensating for the imperfect seed match is extensive pairing to the 3′ region of the miRNA, which typically centers on miRNA nucleotides 13–16.

Figure 6. A Unified Model for miRNA Target Recognition and Pairing Propagation

The miRNA is tightly bound within the silencing complex. A pocket within the Ago MID domain binds the miRNA 5′ terminus, and one within the PAZ domain binds its 3′ terminus. In addition, interactions with the seed region (red) pre-organize nucleotides 2–5 for initial pairing with the target (step 1). As pairing propagates through the seed region (step 2), α-helix 7 (α7), which initially imposes a kink in the seed and steric block to this propagation, shifts to a location at which it reinforces Watson–Crick paring at positions 6 and 7. Most canonical sites rely on this pairing to the seed region, often supplemented with an additional interaction between Ago and an A at target position 1, but with a steric and conformational block (B) preventing contiguous pairing from propagating beyond position 8. Some sites have a segment (green) that can pair to the miRNA 3′ region, optimally centering on miRNA nucleotides 13–16 (orange) to nucleate a second RNA helix (step 3). Because this second helix forms on the other side of the block, it can form without either steric hindrance or the conformational challenge of wrapping the miRNA around the mRNA. This second helix contributes the 3′-supplementary/compensatory pairing observed for some sites. For the very few sites that also have pairing through the center of the miRNA, pairing can then form between the second helix and the seed helix, propagating from either helix or from both simultaneously (step 4). To accommodate the torsional strain of forming this pairing between the helices, the second helix rotates around its helical axis, while the seed helix remains fixed. As the second helix begins to rotate and as pairing propagates from the second helix towards the miRNA 3′ terminus, the 3′ terminus is pulled from its binding pocket, relieving strain for further rotation of the second helix and full propagation of pairing (step 4). Propagation of pairing through the block and other RNA conformational changes that generate a long contiguous helix are coupled to protein conformational changes that help create the active site for target slicing (arrowhead). Each step prior to slicing is reversible, and thus this model suggests an analogous pathway for the stepwise release of the passenger strand during silencing-complex maturation, in which a miRNA duplex replaces the fully paired miRNA–target complex, and the pathway proceeds in reverse.