miRNA

2.1. Biogenesis of miRNA

The biogenesis of miRNA begins with the transcription of miRNA in the nucleus. The pri-miRNA contains a 60- to 80-nucleotide hairpin stem-loop structure. As shown in Figure 1, its biogenesis involves the cleavage of this hairpin structure by a protein complex consisting of Drosha and DGCR8 (DiGeorge critical region 8, also known as Pasha). This results in the pre-miRNA, which includes a 22-bp stem, a loop, and a 2-nucleotide 3^-overhang (11–13). Drosha is an RNase III enzyme, and DGCR8 is its binding partner. The pre-miRNA is exported from the nucleus to the cytoplasm by Exportin-5 (XPO5) (14). In the cytoplasm, the pre-miRNA is further cleaved by another RNase III enzyme, Dicer, which removes the loop to yield the∼22-nucleotide miRNA duplex (15, 16). After being unwound by an unidentified helicase, one strand of miRNA (whose 5^end binds more weakly to the complementary strand) is destined to be the mature miRNA and is termed the guide strand. The complementary strand, termed the passenger strand or miRNA^∗, is rapidly degraded (17, 18). Together with the Argonaute (Ago) family of proteins, the mature miRNA is then packed into a ribonucleoprotein complex known as miRISC (miRNA-induced silencing complex), which mediates gene silencing. The endonuclease activity of Ago cleaves the double-strand miRNA-mRNA complex but not the single-strand mRNA. Ago may also mediate the repression of protein synthesis through mechanisms that are unclear. The above provides a general scheme of miRNA biogenesis, but some miRNAs may be processed differently. For example, whereas most miRNAs mature in the cytoplasm, the biogenesis of human miR-29b occurs only in the nucleus (19).

Annu. Rev. Biomed. Eng. 2010.12:1-27. Downloaded from www.annualreviews.org by National Chiao Tung University on 04/24/14. For personal use only.

miRNA genes

RNA pol II or III

pre-miRNA

Dicer

miRNA duplex

Exportin-5

pre-miRNA

miRNA* miRNA Unwind

mRNA Targeting Degradation

pri-miRNA Nucleus

Cytoplasm

3' overhang Stem

Loop DGCR8

Drosha

Figure 1

Biogenesis and functional targeting of miRNA. pri-miRNA (a 60–80-nucleotide hairpin stem-loop) is transcribed from the miRNA gene by RNA pol II or III and then cleaved by the Drosha/DGCR8 complex to result in the pre-miRNA (with a 22-bp stem and a 2-nucleotide 3^overhang) in the nucleus. After being exported from the nucleus to the cytoplasm by Exportin-5, pre-miRNA is further cleaved by Dicer to remove the terminal loop. After unwinding, one strand of miRNA acts as the functional guide strand and binds to the target mRNA. The complementary strand (i.e., miRNA^∗) is rapidly degraded.

There are active investigations regarding the regulatory mechanisms in each of the steps in-volved in miRNA biogenesis. The important issues that remain to be elucidated are (a) the molec-ular mechanisms by which Drosha, Dicer, DGCR8, and Ago are regulated; (b) the mechanisms of distinctive cellular localization of miRNAs; and (c) the molecular bases of miRNA transcrip-tion, degradatranscrip-tion, and turnover. Conceptually, the homeostasis of miRNA, as that of all elements in the living system, is highly regulated by a set of intricate mechanisms. Understanding the structure-function basis of each component involved in miRNA biogenesis would help design the systems-biology approach to elucidate miRNA regulation and functions.

2.2. Mechanisms of miRNA Targeting to Cognate mRNA

The primary action of miRNA is to target the cognate mRNA, a process governed by base pairing.

Depending on the extent of complementarity, miRNA may exert one of two effects on the tar-geted mRNA: (a) mRNA cleavage and degradation, or (b) translation repression. In plants, most miRNAs pair to the target mRNAs in a nearly perfect match, leading to mRNA cleavage and sub-sequent degradation (Figure 2) (20). However, such a high degree of miRNA-mRNA matching is rare in animal cells, where miRNAs typically make imperfect pairings with their mRNA targets.

Investigators have extensively studied the mechanisms of animal miRNA targeting by using bioin-formatics approaches in conjunction with experimental validation (21–25). The most important Annu. Rev. Biomed. Eng. 2010.12:1-27. Downloaded from www.annualreviews.org by National Chiao Tung University on 04/24/14. For personal use only.

AGO

Mechanism of miRNA targeting. The 7-nucleotide seed region, which starts at the second nucleotide from the 5^end of the miRNA, is required for miRNA-mRNA interaction. The miRNA-mRNA targeting occurs predominantly at the 3^UTR of the target mRNA, located at the 3^downstream of the open reading frame (ORF). Bulges or mismatches may be present in the middle part of the miRNA-mRNA duplex. As shown in the oval on the lower left, perfect base pairing between miRNA and mRNA (mostly in plant cells) results in miRISC-mediated endonucleolytic cleavage and hence mRNA degradation. Despite the lack of complete complementarity in animal cells shown on the lower right, base pairing (particularly the 13–16 nucleotides of miRNA) is still important in stabilizing the miRNA-mRNA interaction, which leads to translational repression. Frequently, multiple miRNAs (red and blue) can target the same 3^UTR. Conversely, a unique miRNA may target multiple binding sites on the same 3^UTR in animal cells.

Seed region: a feature of the mechanism is the presence of a seed region comprising a segment of miRNA in

which nucleotides 2–8 precisely match their complementary sequences within the 3^untranslated region (3^UTR) of the targeted mRNA. Another feature is that mismatches and bulges may be present in the miRNA-mRNA duplex but not in the region where miRNA associates with Ago.

The third feature is the universal existence of complementarity between the 3^half of the miRNA (typically at nucleotides 13–16) and the 3^UTR of the mRNA to stabilize the duplex. This is nec-essary because the base pairing between the seed sequence and the target sites on the mRNA is usually not sufficient for repression (22, 24) (Figure 2).

Animal and plant miRNAs differ not only in their miRNA-mRNA base pairing but also in their modes of action. In plant cells, the high miRNA-mRNA complementarity recruits miRISC, leading to the cleavage of mRNA transcript by RNase activity associated with Ago. In animal cells, miRNAs fine-tune protein translation rather than degrade their mRNA targets as seen in plant cells (26). The 3^UTRs of animal mRNAs often contain more than one site targeted by one or more miRNAs, suggesting the possibility of cooperative repression imposed by multiple miRNAs (22–25). Several databases and Web resources provide information on miRNA-target interactions;

miRNA databases, algorithms, and tools for target prediction are reviewed in Section 5.

In animal cells, miRNA can suppress protein translation by both direct and indirect effects (Figure 3). The direct effects are exerted at various phases of protein translation, including the repression at the initiation (27–30), prevention of ribosome assembly for the initiation of trans-lation (31), repression at postinitiation steps (32), and inhibition of elongation or termination of translation process (33, 34). (For basic knowledge of protein translation, see Reference 2, pp. 132–39.) The indirect translational repression by miRNA occurs at the mRNA level and is Annu. Rev. Biomed. Eng. 2010.12:1-27. Downloaded from www.annualreviews.org by National Chiao Tung University on 04/24/14. For personal use only.

AGO AGO 60S

a

b

c

Deadenylation

mRNA degradation mRNA sequestration

P body

Direct translational repression Indirect translational repression

40S

miRNA-mediated translational repression in animal cells. The miRISC-mRNA interaction can lead to several modes of direct translational repression (a–c). (a) Initiation block: The recruitment of 40S and/or 60S ribosomes near the 5^cap of mRNA is inhibited.

(b) Ribosomal drop-off: The 40S/60S ribosomes are dissociated from mRNA. (c) Stalled elongation: The 40S/60S ribosomes are prohibited from joining during the elongation process. (d ) The indirect translational repression by miRISC occurs via deadenylation, by which the 3^poly-A tail of the mRNA is removed, leading to increased mRNA degradation. Alternatively, the destabilized mRNA resulting from deadenylation is localized in P bodies and hence sequestered from translational machinery.

Processing bodies

caused by the destabilization and subsequent degradation—as well as the compartmentalization and sequestration—of the target mRNAs. In this mode of action, miRNAs destabilize their target mRNAs mainly through deadenylation (35–37). The compartmentalization and sequestration rely on the cytoplasmic foci known as processing bodies (P bodies), where the repressed mRNAs are sequestered with enriched translational repressors (38, 39).

2.3. Transcriptional Regulation of miRNA

Like all classes of RNA, miRNAs are transcribed from DNA. However, the mechanism underlying transcriptional regulation of miRNA had not been understood until recently. Key transcription factors (TFs), including hypoxia-induced factor, the oncogene c-myc, the tumor suppressor p53, and NF-κB, have been shown to up- or downregulate clusters of miRNAs (40–43). The major challenge for the analyses of TFs is to delineate their exact binding sites in the miRNA promoter regions, which may range from a few kb to more than 50 kb upstream of the miRNA genes.

As a result, only a few of the TF binding sites have been identified experimentally. In silico Annu. Rev. Biomed. Eng. 2010.12:1-27. Downloaded from www.annualreviews.org by National Chiao Tung University on 04/24/14. For personal use only.

TRANSFAC:

analysis of the miRNA promoter regions will be able to facilitate this identification (44). Because of the highly conserved nature of miRNAs across species, the binding of TFs to promoter regions of conserved candidate miRNAs can be assessed by TRANSFAC and Match. TRANSFAC is a database containing information on TFs, their experimentally proven binding sites, and their target genes (45). Match is a software program specifically designed for identifying TF binding sites in the promoter sequences of the miRNA gene (46).

To date, TF regulation of miRNAs has been studied mainly in cancer cells. How various miRNAs are transcriptionally controlled under physiological and pathophysiological conditions and how other transcription regulators such as coactivators and corepressors may affect miRNA expression remain to be investigated. A given TF may regulate a cluster of miRNAs, which may in turn modulate other TFs as their target genes, thus forming genetic circuits. The hierarchical relationships of the TF regulation of miRNA and the miRNA regulation of TF are largely un-known. For bioengineers with expertise in computer-assisted design and analysis, elucidation of transcriptional regulation of miRNA can be a fruitful area of research.