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MicroRNAs (miRNAs)are short ribonucleic acid (RNA) molecules, on average only 22 nucleotides long and are found in all eukaryotic cells. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing.[1][2] The human genome may encode over 1000 miRNAs,[3][4] which may target about 60% of mammalian genes[5] and are abundant in many human cell types.[6]
miRNAs show very different characteristics between plants and metazoans. In plants the miRNA complementarity to its mRNA target is nearly perfect, with no or few mismatched bases. In metazoans on the other hand miRNA complementarity is far from perfect and one miRNA can target many different sites on the same mRNA or on many different mRNAs. Another difference is the location of target sites on mRNAs. In metazoans the miRNA target sites are in the three prime untranslated regions (3'UTR) of the mRNA. In plants targets can be located in the 3' UTR but are more often in the coding region itself.[7] MiRNAs are well conserved in eukaryotic organism and are thought to be a vital and evolutionary ancient component of genetic regulation.[8][9][10][11]
The first miRNAs were characterized in the early 1990s, but miRNAs were not recognized as a distinct class of biologic regulators with conserved functions until the early 2000s. Since then, miRNA research has revealed multiple roles in negative regulation (transcript degradation and sequestering, translational suppression) and possible involvement in positive regulation (transcriptional and translational activation). By affecting gene regulation, miRNAs are likely to be involved in most biologic processes.[12][13][14][15][16][17][18]Different sets of expressed miRNAs are found in different cell types and tissues.[19]
Aberrant expression of miRNAs has been implicated in numerous disease states, and miRNA-based therapies are under investigation.[20][21][22]
MicroRNAs were discovered in 1993 by Victor Ambros, Rosalind Lee and Rhonda Feinbaum during a study of the genelin-14 in C. elegans development.[23] They found that LIN-14 protein abundance was regulated by a short RNA product encoded by the lin-4 gene. A 61 nucleotide precursor from lin-4 gene matured to a 22 nucleotide RNA containing sequences partially complementary to multiple sequences in the 3’ UTR of the lin-14 mRNA. This complementarity was sufficient and necessary to inhibit the translation of lin-14 mRNA into LIN-14 protein. Retrospectively, the lin-4 small RNA was the first microRNA to be identified, though at the time, it was thought to be a nematode idiosyncrasy. Only in 2000 was a second RNA characterized: let-7, which repressed lin-41,lin-14, lin-28, lin-42, and daf-12 expression during developmental stage transitions in C. elegans. let-7 was soon found to be conserved in many species,[24][25] indicating the existence of a wider phenomenon.
Under a standard nomenclature system, names are assigned to experimentally confirmed miRNAs before publication of their discovery.[26][27] The prefix "mir" is followed by a dash and a number, the latter often indicating order of naming. For example, mir-123 was named and likely discovered prior to mir-456. The uncapitalized "mir-" refers to the pre-miRNA, while a capitalized "miR-" refers to the mature form. miRNAs with nearly identical sequences bar one or two nucleotides are annotated with an additional lower case letter. For example, miR-123a would be closely related to miR-123b. Pre-miRNAs that lead to 100% identical mature miRNAs but that are located at different places in the genome are indicated with an additional dash-number suffix. For example, the pre-miRNAs hsa-mir-194-1 and hsa-mir-194-2 lead to an identical mature miRNA (hsa-miR-194) but are located in different regions of the genome. Species of origin is designated with a three-letter prefix, e.g., hsa-miR-123 would be from human (Homo sapiens) and oar-miR-123 would be a sheep (Ovis aries) miRNA. Other common prefixes include 'v' for viral (miRNA encoded by a viral genome) and 'd' for Drosophila miRNA (a fruit fly commonly studied in genetic research). When two mature microRNAs originate from opposite arms of the same pre-miRNA, they are denoted with a -3p or -5p suffix. (In the past, this distinction was also made with 's' (sense) and 'as' (antisense)). When relative expression levels are known, an asterisk following the name indicates an miRNA expressed at low levels relative to the miRNA in the opposite arm of a hairpin. For example, miR-123 and miR-123* would share a pre-miRNA hairpin, but more miR-123 would be found in the cell.
Most microRNA genes are found in intergenic regions or in anti-sense orientation to genes[28] and contain their own miRNA gene promoter and regulatory units.[28][29][30][31] As much as 40% of miRNA genes may lie in the introns of protein and non-protein coding genes or even in exons.[32] These are usually, though not exclusively, found in a sense orientation.[33][34] and thus usually are regulated together with their host genes.[32][35][36] Other miRNA genes showing a common promoter include the 42-48% of all miRNAs originating from polycistronic units containing 2-7 discrete loops from which mature miRNAs are processed,[29][37] although this does not necessarily mean the mature miRNAs of a family will be homologous in structure and function. The promoters mentioned have been shown to have some similarities in their motifs to promoters of other genes transcribed by RNA polymerase II such as protein coding genes.[29][38] The DNA template is not the final word on mature miRNA production: 6% of human miRNAs show RNA editing, the site-specific modification of RNA sequences to yield products different from those encoded by their DNA. This increases the diversity and scope of miRNA action beyond that implicated from the genome alone.
Transcription
miRNA genes are usually transcribed by RNA polymerase II (Pol II).[29][38] The polymerase often binds to a promoter found near the DNA sequence encoding what will become the hairpin loop of the pre-miRNA. The resulting transcript is capped with a specially-modified nucleotide at the 5’ end, polyadenylatedwith multiple adenosines (a poly(A) tail),[29][33] and spliced. The product, called a primary miRNA (pri-miRNA), may be hundreds or thousands of nucleotides in length and contain one or more miRNA stem loops.[29][33] When a stem loop precursor is found in the 3’ UTR, a transcript may serve as a pri-miRNA and a mRNA.[33] RNA polymerase III(Pol III) transcribes some miRNAs, especially those with upstream Alu sequences, transfer RNAs (tRNAs), and mammalian wide interspersed repeat (MWIR) promoter units.[39]
Nuclear processing
A single pri-miRNA may contain from one to six miRNA precursors. These hairpin loop structures are composed of about 70 nucleotides each. Each hairpin is flanked by sequences necessary for efficient processing. The double-stranded RNA structure of the hairpins in a pri-miRNA is recognized by a nuclear protein known as DiGeorge Syndrome Critical Region 8 (DGCR8 or "Pasha" in invertebrates), named for its association with DiGeorge Syndrome. DGCR8 associates with the enzyme Drosha, a protein that cuts RNA, to form the "Microprocessor" complex.[40] In this complex, DGCR8 orients the catalytic RNase III domain of Drosha to liberate hairpins from pri-miRNAs by cleaving RNA about eleven nucleotides from the hairpin base (two helical RNA turns into the stem). The resulting hairpin, known as a pre-miRNA, has a two-nucleotide overhang at its 3’ end; it has 3' hydroxyl and 5' phosphate groups.
pre-miRNAs that are spliced directly out of introns, bypassing the Microprocessor complex, are known as "mirtrons." Originally thought to exist only in Drosophila and C. elegans, mirtrons have now been found in mammals.[41]
Perhaps as many as 16% of pri-miRNAs may be altered through nuclear RNA editing.[42][43][44] Most commonly, enzymes known as adenosine deaminases acting on RNA (ADARs) catalyze adenosine to inosine (A to I) transitions. RNA editing can halt nuclear processing (for example, of pri-miR-142, leading to degradation by the ribonuclease Tudor-SN) and alter downstream processes including cytoplasmic miRNA processing and target specificity (e.g., by changing the seed region of miR-376 in the central nervous system).[42]
Nuclear export
pre-miRNA hairpins are exported from the nucleus in a process involving the nucleocytoplasmic shuttleExportin-5. This protein, a member of the karyopherin family, recognizes a two-nucleotide overhang left by the RNase III enzyme Drosha at the 3' end of the pre-miRNA hairpin. Exportin-5-mediated transport to the cytoplasm is energy-dependent, using GTP bound to the Ran protein.[45]
Cytoplasmic processing
In the cytoplasm, the pre-miRNA hairpin is cleaved by the RNase III enzyme Dicer.[46] This endoribonuclease interacts with the 3' end of the hairpin and cuts away the loop joining the 3' and 5' arms, yielding an imperfect miRNA:miRNA* duplex about 22 nucleotides in length.[46]Overall hairpin length and loop size influence the efficiency of Dicer processing, and the imperfect nature of the miRNA:miRNA* pairing also affects cleavage.[46][47] Although either strand of the duplex may potentially act as a functional miRNA, only one strand is usually incorporated into the RNA-induced silencing complex (RISC) where the miRNA and its mRNA target interact.
Biogenesis in plants
miRNA biogenesis in plants differs from metazoan biogenesis mainly in the steps of nuclear processing and export. Instead of being cleaved by two different enzymes, once inside and once outside the nucleus, both cleavages of the plant miRNA is performed by a Dicer homolog, called Dicer-like1 (DL1). DL1 is only expressed in the nucleus of plant cells, which indicates that both reactions take place inside the nucleus. Before plant miRNA:miRNA* duplexes are transported out of the nucleus its 3' overhangs are methylated by a RNA methyltransferase protein called Hua-Enhancer1 (HEN1). The duplex is then transported out of the nucleus to the cytoplasm by a protein called Hasty (HST), an Exportin 5 homolog, where they disassemble and the mature miRNA is incorporated into the RISC.[48]
The mature miRNA is part of an active RNA-induced silencing complex (RISC) containing Dicer and many associated proteins.[49] RISC is also known as a microRNA ribonucleoprotein complex (miRNP);[50] RISC with incorporated miRNA is sometimes referred to as "miRISC."
Dicer processing of the pre-miRNA is thought to be coupled with unwinding of the duplex. Generally, only one strand is incorporated into the miRISC, selected on the basis of its thermodynamic instability and weaker base-pairing relative to the other strand.(Krol et al., 2004)(Khvorova et al., 2003; Schwarz et al., 2003) The position of the stem-loop may also influence strand choice. (Lin et al., 2005) The other strand, called the passenger strand due to its lower levels in the steady state, is denoted with an asterisk (*) and is normally degraded. In some cases, both strands of the duplex are viable and become functional miRNA that target different mRNA populations.(Okamura et al., 2008)
Members of the argonaute (Ago) protein family are central to RISC function. Argonautes are needed for miRNA-induced silencing and contain two conserved RNA binding domains: a PAZ domain that can bind the single stranded 3’ end of the mature miRNA and a PIWI domain that structurally resembles ribonuclease-H and functions to interact with the 5’ end of the guide strand. They bind the mature miRNA and orient it for interaction with a target mRNA. Some argonautes, for example human Ago2, cleave target transcripts directly; argonautes may also recruit additional proteins to achieve translational repression.[51] The human genome encodes eight argonaute proteins divided by sequence similarities into two families: AGO (with four members present in all mammalian cells and called E1F2C/hAgo in humans), and PIWI (found in the germ line and hematopoietic stem cells).[51][52]
Additional RISC components include TRBP [human immunodeficiency virus (HIV) transactivating response RNA (TAR) binding protein],[53] PACT (protein activator of the interferon induced protein kinase (PACT), the SMN complex, fragile X mental retardation protein (FMRP), and Tudor staphylococcal nuclease-domain-containing protein (Tudor-SN).[54][55]
Mode of Silencing
Gene silencing may occur either via mRNA degradation or preventing mRNA from being translated. It has been demonstrated that if there is complete complementation between the miRNA and target mRNA sequence, Ago2 can cleave the mRNA and lead to direct mRNA degradation. Yet, if there isn't complete complementation the silencing is achieved by preventing translation.[citation needed]
Turnover of mature miRNA is needed for rapid changes in miRNA expression profiles. During miRNA maturation in the cytoplasm, uptake by the Argonaute protein is thought to stabilize the guide strand, while the opposite (* or "passenger") strand is preferentially destroyed. In what has been called a "Use it or lose it" strategy, Argonaute may preferentially retain miRNAs with many targets over miRNAs with few or no targets, leading to degradation of the non-targeting molecules.[56]
Decay of mature miRNAs in animals is mediated by the 5´-to-3´ exoribonuclease XRN2, also known as Rat1p.[57] In plants, SDN (small RNA degrading nuclease) family members degrade miRNAs in the opposite (3'-to-5') direction. Similar enzymes are encoded in animal genomes, but their roles have not yet been described.[56]
Several miRNA modifications affect miRNA stability. As indicated by work in the model organism Arabidopsis thaliana (thale cress), mature plant miRNAs appear to be stabilized by the addition of methyl moieties at the 3' end. The 2'-O-conjugated methyl groups block the addition of uracil (U) residues by uridyltransferase enzymes, a modification that may be associated with miRNA degradation. However, uridylation may also protect some miRNAs; the consequences of this modification are incompletely understood. Uridylation of some animal miRNAs has also been reported. Both plant and animal miRNAs may be altered by addition of adenine (A) residues to the 3' end of the miRNA. An extra A added to the end of mammalian miR-122, a liver-enriched miRNA important in Hepatitis C, stabilizes the molecule, and plant miRNAs ending with an adenine residue have slower decay rates.[56]
The function of miRNAs appears to be in gene regulation. For that purpose, a miRNA is complementary to a part of one or more messenger RNAs (mRNAs). Animal miRNAs are usually complementary to a site in the 3' UTR whereas plant miRNAs are usually complementary to coding regions of mRNAs.[58] Perfect or near perfect base pairing with the target RNA promotes cleavage of the RNA.[59] This is the primary mode of plant microRNAs.[60] In animals, microRNAs more often only partially base pair and inhibit protein translation of the target mRNA[61] (this exists in plants as well but is less common).[60]MicroRNAs that are partially complementary to the target can also speed up deadenylation, causing mRNAs to be degraded sooner.[62] For partially complementary microRNA to recognise their targets, the nucleotides 2–7 of the miRNA ('seed region') still have to be perfectly complementary.[63] miRNAs occasionally also causes histone modification and DNA methylation of promoter sites and therefore affecting the expression of targeted genes.[64][65]
Animal microRNAs target in particular developmental genes. In contrast, genes involved in functions common to all cells, such as gene expression, have very few microRNA target sites and seem to be under selection to avoid targeting by microRNAs.[66]
dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" orRNAa. dsRNAs targeting gene promoters can induce potent transcriptional activation of associated genes. This was demonstrated in human cells using synthetic dsRNAs termed small activating RNAs (saRNAs),[67] but has also been demonstrated for endogenous microRNA.[68]
MicroRNAs are significant phylogenetic markers because of their astonishingly low rate of evolution.[69] Their origin may have permitted the development of morphological innovation, and by making gene expression more specific and 'fine-tunable', permitted the genesis of complex organs[70] and perhaps, ultimately, complex life.[71] Indeed, rapid bursts of morphological innovation are generally associated with a high rate of microRNA accumulation.[69][70]
MicroRNAs originate predominantly by the random formation of hairpins in "non-coding" sections of DNA (i.e. introns or intergene regions), but also by the duplication and modification of existing microRNAs.[72] The rate of evolution (i.e. nucleotide substitution) in recently-originated microRNAs is comparable to that elsewhere in the non-coding DNA, implying evolution by neutral drift; however, older microRNAs have a much lower rate of change (often less than one substitution per hundred million years),[71] suggesting that once a microRNA gains a function it undergoes extreme purifying selection.[72] At this point, a microRNA is rarely lost from an animal's genome,[71] although microRNAs which are more recently derived (and thus presumably non-functional) are frequently lost.[72] This makes them a valuable phylogenetic marker, and they are being looked upon as a possible solution to such outstanding phylogenetic problems as the relationships of arthropods.[73]
MicroRNAs feature in the genomes of most eukaryotic organisms, from the brown algae[74] to the metazoa. Across all species, in excess of 5000 had been identified by March 2010.[75] Whilst short RNA sequences (50 – hundreds of base pairs) of a broadly comparable function occur in bacteria, bacteria lack true microRNAs.[76]
MicroRNA expression can be quantified in a two-step polymerase chain reaction process of modified RT-PCRfollowed by quantitative real-time PCR. Variations of this method achieve absolute or relative quantification.[77] miRNAs can also be hybridized to microarrays, slides or chips with probes to hundreds or thousands of miRNA targets, so that relative levels of miRNAs can be determined in different samples.[78] The activity of an miRNA can be experimentally inhibited using a locked nucleic acid oligo, a Morpholinooligo[79][80] or a 2'-O-methyl RNA oligo.[81] MicroRNA maturation can be inhibited at several points by steric-blocking oligos.[82] The miRNA target site of an mRNA transcript can also be blocked by a steric-blocking oligo.[83][84] Additionally, a specific miRNA can be silenced by a complementary antagomir.
Just as miRNA is involved in the normal functioning of eukaryotic cells, so has dysregulation of miRNA been associated with disease. A manually curated, publicly available database miR2Disease documents known relationships between miRNA dysregulation and human disease.[85]
miRNA and cancer
Several miRNAs have been found to have links with some types of cancer.[86][87]
A study of mice altered to produce excess c-Myc — a protein with mutated forms implicated in several cancers — shows that miRNA has an effect on the development of cancer. Mice that were engineered to produce a surplus of types of miRNA found in lymphoma cells developed the disease within 50 days and died two weeks later. In contrast, mice without the surplus miRNA lived over 100 days.[86] Leukemia can be caused by the insertion of a viral genome next to the 17-92 array of microRNAs leading to increased expression of this microRNA.[88]
Another study found that two types of miRNA inhibit the E2F1 protein, which regulates cell proliferation. miRNA appears to bind to messenger RNA before it can be translated to proteins that switch genes on and off.[89]
By measuring activity among 217 genes encoding miRNA, patterns of gene activity that can distinguish types of cancers can be discerned. miRNA signatures may enable classification of cancer. This will allow doctors to determine the original tissue type which spawned a cancer and to be able to target a treatment course based on the original tissue type. miRNA profiling has already been able to determine whether patients with chronic lymphocytic leukemia had slow growing or aggressive forms of the cancer.[90]
Transgenic mice that over-express or lack specific miRNAs have provided insight into the role of small RNAs in various malignancies.[91]
miRNA and heart disease
The global role of miRNA function in the heart has been addressed by conditionally inhibiting miRNA maturation in themurine heart, and has revealed that miRNAs play an essential role during its development.[92][93] miRNA expression profiling studies demonstrate that expression levels of specific miRNAs change in diseased human hearts, pointing to their involvement in cardiomyopathies.[94][95][96] Furthermore, studies on specific miRNAs in animal models have identified distinct roles for miRNAs both during heart development and under pathological conditions, including the regulation of key factors important for cardiogenesis, the hypertrophic growth response, and cardiac conductance.[93][97][98][99][100][101]
miRNA and the nervous system
miRNAs appear to regulate the nervous system.[102] Neural miRNAs are involved at various stages of synaptic development, including dendritogenesis (involving miR-132, miR-134 and miR-124), synapseformation and synapse maturation (where miR-134 and miR-138 are thought to be involved).[103] Some studies find altered miRNA expression in schizophrenia.
When the human genome project mapped its first chromosome in 1999, it was predicted the genome would contain over 100,000 protein coding genes. However, only around 20,000 were eventually identified (International Human Genome Sequencing Consortium, 2004).[106] Since then, the advent of bioinformatics approaches combined with genome tiling studies examining the transcriptome,[107] systematic sequencing of full length cDNA libraries,[108] and experimental validation[109] (including the creation of miRNA derived antisense oligonucleotides called antagomirs) have revealed that many transcripts are non protein-coding RNA, including several snoRNAs and miRNAs.[110]
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