microRNAs
Nucleic Acids Metabolism

What are MicroRNAs?

MicroRNAs are a class of post-transcriptional regulators. They are short ~22 nucleotide RNA sequences that bind to complementary sequences in the 3’ UTR of multiple target mRNAs, usually resulting in their silencing. MicroRNAs target ~60% of all genes, are abundantly present in all human cells and are able to repress hundreds of targets each. These features, coupled with their conservation in organisms ranging from the unicellular algae chlamydomonas reinhardtii to mitochondria, suggest they are a vital part of genetic regulation with ancient origins.
MicroRNAs were first discovered in 1993 by Victor Ambros, Rosalind Lee and Rhonda Feinbaum during a study into development in the nematode ''C. elegans'' regarding the gene lin-14. This screen led to the discovery that the lin-14 was able to be regulated by a short RNA product from lin-4, a gene that transcribed a 61 nucleotide precursor that matured to a 22 nucleotide mature RNA which contained 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. Retrospectively, this was the first microRNA to be identified, though at the time Ambros et al speculated it to be a nematode idiosyncrasy.
It was only in 2000 when let-7 was discovered to repress lin-41, lin-14, lin28, lin42 and daf12 mRNA during transition in developmental stages in c elegans and that this function was phylogenetically conserved in species beyond nematodes, that it became apparent the short non-coding RNA identified in 1993 was part of a wider phenomenon.
Since then over 4000 miRNAs have been discovered in all studied eukaryotes including mammals, fungi and plants. More than 700 miRNAs have so far been identified in humans and over 800 more are predicted to exist.
Comparing miRNAs between species can even be used to delineate molecular evolutionary history on the basis that the complexity of an organisms phenotype may reflect that of the microRNA found in the genotype.
When the human genome project mapped its first chromosome in 1999, it was predicted it would contain over 100,000 protein coding genes. However, only around 20,000 were eventually identified (International Human Genome Sequencing Consortium, 2004) and for a long time much of the non-protein-coding DNA was considered "junk", though conventional wisdom holds that much if not most of the genome is functional. Since then, the advent of sophisticated bioinformatics approaches combined with genome tiling studies examining the transcriptome, systematic sequencing of full length cDNA libraries and experimental validation (including the creation of miRNA derived antisense oligonucleotides called antagomirs) have revealed that many transcripts are for non protein coding RNA of which many new classes have been deducted such as snoRNA and miRNA. Unfortunately, the rate of validation of microRNA targets is substantially more time consuming than that of predicting sequences and targets.
Due to their abundant presence and far-reaching potential, miRNAs have all sorts of functions in physiology, from cell differentiation, proliferation, apoptosis to the endocrine system, haematopoiesis, fat metabolism, limb morphogenesis. They display different expression profiles from tissue to tissue, reflecting the diversity in cellular phenotypes and as such suggest a role in tissue differentiation and maintenance.

MicroRNA biogenesis

Most microRNA genes are found in intergenic regions or in anti-sense oritentation to certain genes and as such contain their own miRNA gene promoter and regulatory units. However, as much as 40% are said to lie in the introns of protein and non-protein coding genes or even rarely in exons. These are usually, though not exclusively, found in a sense orientation and thus usually show a concurrent transcription and regulation expression profile originating from a common promoter with their host genes .
Other microRNA genes showing a common promoter include the 42-48% of all miRNAs originating from polycistronic units contaning 2-7 discrete loops from which mature miRNAs are processed , though 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 class II (meaning transcribed by POL II) genes such as protein coding genes .
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 to those encoded by their DNA. This increases the diversity and scope of miRNA action beyond that implicated from the genome alone.
In the nucleus, polymerase II (POL II) is usually used to transcribe microRNA encoding parts of the genome often through binding to a promoter found near the sequence destined to be the hairpin loop of the pre-miRNA. This produces a transcript that is capped at the 5’ end, polyadenylated to give a (poly)A tail and spliced to form pri-miRNA several hundred to thousand bp in size . Curiously, some pri-miRNAs have been shown to be able to co-ordinately express both miRNAs and mRNAs, when the stem loop precursor is found in the 3’ UTR of an mRNA . Uncommonly, polymerase III (POL III) is speculated to be used instead of POL II when transcribing microRNA that have upstream -Alu, -tRNA, mammalian wide interspersed repeat (MWIR) promoter units .
Pri-miRNA are processed by the microprocessor complex consisting of drosha and its cofactor DGCR8 into pre-miRNAs.
Pri-miRNA contains at least 1 (up to 6 when transcribed from polycistronic units) ~70 nucleotide hairpin loop structures, there is a potential for a single pri-miRNA to house many miRNAs. The hairpin loops have >40 nucleotide flanking RNA sequences necessary for efficient processing. These are recognised by the Di George Syndrome Critical Region 8 (DGCR8), the cofactor to drosha. DGCR8 is a dsRNA binding nuclear protein that recognizes the hairpin loop of the pri-miRNA and orientates the catalytic RNAse III domain of drosha for cleavage. This cleavage occurs around 11 nucleotides from their base (2 helical RNA turns into the stem) by Drosha, a RNAse III type dsRNA specific endonuclease, to form pre-miRNA. Together, drosha and DGCR8 (the invertebrate equivalent is Pasha) form the microprocessor complex. The microprocessor complex introduces staggered cuts to the ends of the hairpin loop arms resulting in a 2 nucleotide overhand on the 3’ end and phosphate on the 5’ end to produce a pre-miRNA of ~ 70 nt in length. Mostly, one arm of the hairpin loop is destined to become the mature miRNA, though rarely a mature miRNA may be produced from either arm eg Mir-458-3p/mir-458-5p and mir-202/mir-202* with the asterisk applying to less predominantly expressed transcript.
There is evidence that pre-miRNAs can be produced without having to undergo the microprocessor machinery if they are directly spliced from the introns in which they reside. These miRNAs are called mirtrons and have traditionally been thought to only exist in drosphila and c elegans. Recently however, mammalian mirtons that even show conservation between species have recently been discovered.
Pri-miRNA can also be subject to RNA editing wherein the miRNA processing or specificity is altered through adenosine deaminase acting on RNA (ADAR) enzymes catalysing adenosine to inosine transitions, the most common form of RNA editing in metazoans. RNA editing has been shown to occur in 6% of miRNAs, even altering the specificity of miRNAs when it was observed in the seed region of miR-376, though this is only present in the CNS.
RNA editing of microRNA can also prevent their processing, as seen in the pri-miR-142 editing leading to degradation by the tudor SN protein (a RISC component) and thus avoiding of the drosha pathway. Overall, this offers many implications in expanding the already complicated role in genetic expression that are covered in more detail than this paper has to opportunity to do in an excellent review.
The nuclear membrane protein exportin 5 recognises the 2 nucleotide overhang on the 3 end of the pre-miRNA and then transports it into the cytoplasm using ran-guanine triphosphatase (Ran-GTP).
Dicer cleavage, cofactor binding and RISC formation
In the cytoplasm the pre-miRNA is cleaved by another RNAse III type double stranded endonuclease called Dicer. Dicer cleavage of pre-miRNA results in an imperfect miRNA:miRNA duplex around 20-25 nucleotides in size containing the mature miRNA strand and its opposite complementary miRNA strand. Dicer is associated with the cofactors immunodeficiency virus (HIV) transactivating response RNA binding protein (TRBP) and protein activator of the interferon induced protein kinase PACT that physically bring the TRBP-PACT-dicer complex into contact with Ago2 to form the RNA Induced Silencing (RISC) loading apparatus. Dicer processing of the pre-miRNA is thought to be coupled to the unwinding of the duplex to produce a mature miRNA which Ago2 binds to, forming the active miRISC complex. The mature miRNA then guides the RISC to target sites in order to induce silencing. The precise sequence of events is difficult to elucidate and still under debate.
Generally, only 1 strand of the miRNA duplex is incorporated into the miRISC and is selected on the basis of it being less stable thermodynamically and capable of weaker base-pairing than the other strand, though the position of the stem-loop within the pre-miRNA has been implicated. The other strand, called the passenger strand due to its lower levels in the steady state, is denoted by miRNA. In short interfering RNAs (siRNAs) it is often cleaved and degraded by the argonaute protein Ago2 in the RISC in order to integrate the guide strand into the RISC, but this is not necessary in miRNAs. The passenger strand is normally degraded and present in lower levels in cells in the steady state, though there have been instances where both strands of the duplex have been viable and become functional miRNA that target different mRNA populations. However, there is also evidence that the duplex as a whole is incorporated and operates in a Fragile X Mental Retardation Protein (FMRP) mediated strand-exchange system with the target mRNA during miRNA:mRNA assembly.
Homo sapiens has 8 argonaute proteins divided into 2 families based on sequence similarities: AGO (present in all mammalian cells and called E1F2C/hAgo in humans) and PIWI (conserved to the germ line and hematopoietic stem cells). In humans, RISC is composed of Ago family members 1-4 amongst other proteins, which seem to ultimately affect the outcome of miRISC: target binding. Ago proteins contain 2 conserved RNA binding domains: a PAZ domain that can bind the single stranded 3’ end of the microRNA and a PIWI domain that structurally resembles ribonuclease-H and functions to interact with the 5’ end of the guide strand. It is important to note that out of the 4 mammalian argonaute proteins, only Ago2 has endonucleolytic (also known as slicer) ability and is the only argonaute necessary for RNA silencing, though the others are involved in translational repression.
There are other proteins found in the miRISC, also referred to as the miRNP complex, that are associated with the AGOs but as yet not fully characterised and are thought to modulate the silencing effects of the miRISC; i.e. the SMN complex, fragile X mental retardation protein (FMRP), tudor staphylococcal nuclease-domain-containing protein (tudor-SN).

MicroRNA cellular functions

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. Perfect or near perfect base pairing with the target RNA promotes cleavage of the RNA. This is the primary mode of plant microRNAs.
In animals, microRNAs more often only partially base pair and inhibit protein translation of the target mRNA (this exists in plants as well but is less common). For partially complementary microRNA to recognise their targets, the nucleotides 2–7 of the miRNA ('seed region'), still have to be perfectly complementary.
miRNAs occasionally also causes DNA methylation of promoter sites and therefore affecting the expression of targeted genes. miRNAs function in association with a complement of proteins collectively termed the miRNP. Human miRNPs contain eIF2C2 (also known as Argonaute 2), DDX20, GEMIN4 and microRNA.
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.
This effect was first described for the worm ''C. elegans'' in 1993 by Victor Ambros and coworkers. As of 2002, miRNAs have been confirmed in various plants and animals, including ''C. elegans'', human and the plant ''Arabidopsis thaliana''. Work at the University of Louisville has resulted in the production of microarrays dubbed MMChips containing all then known miRNAs for human, mouse, rat, dog, ''C. elegans'' and ''Drosophila''.
Mirtrons are the type of microRNAs which are located in the introns of the mRNA encoding host genes. All the miRNAs in plants are derived from the sequential DCL1 cleavages from pri-miRNA to give pre-miRNA (or miRNA precursor). But the mirtrons bypass the DCL1 cleavage and enter as pre-miRNA in the miRNA maturation pathway.

MicroRNA and Disease

Just as miRNA is involved in the normal functioning of eukaryotic cells, so has dysregulation of miRNA been associated with disease. Disease association in turn has led to increased funding opportunities for academic research and financial incentives for development and commercialization of miRNA-based diagnostics and therapeutics. After early commercialization aimed at academic research support was established, the initial research focus based on products and services requested was on cancer and neuroscience research. During 2007, interests indicated by product and services requested broadened to include cardiac research, virology, cell biology in general and plant biology.
miRNA and cancer
Several miRNAs has been found to have links with some types of cancer.
A study of mice altered to produce excess c-myc — a protein 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.
Leukemia can be caused by the insertion of a virus next to the the 17-92 array of microRNAs leading to increased expression of this microRNA.
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.
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. In 2008, the companies Asuragen and Exiqon were working to commercialize this potential for miRNAs to act as cancer biomarkers.
miRNA and heart disease
The global role of miRNA function in the heart has been addressed by conditionally inhibiting miRNA maturation in the murine heart, and has revealed that miRNAs play an essential role during its development. miRNA expression profiling studies demonstrate that expression levels of specific miRNAs change in diseased human hearts, pointing to their involvement in cardiomyopathies. 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. In 2008, academic work on the relationship between miRNA and heart disease had advanced sufficiently to lead to the establishment of a company, miRagen Therapeutics, with a primary focus on "cardiovascular health and disease".

Databases

To learn more about miRNAs and to have more informations about the already known miRNAs, it is possible to consult the following databases:

mir2disease
mirbase
microrna
Ambion

Example for mir2disease database:
Users can search all entries in three ways: by miRNA ID, by disease name or experimentally verified target gene.
Detailed information shown in search results:
miRNA ID
Disease name
miRNA expression pattern in the disease state
Detection method for miRNA expression
A brief description of the miRNA-disease relationship
Title & publication year of the reference from which the entry was extracted
Experimentally validated target gene(s) extracted from the original reference and target gene(s) directly derived from Tarbase
If I'm searching for miRNAs and diseases related, for example, to BCL2, I've to put the term in the target gene section and this will be my result:
BCL2

Example for mirbase database:
miRBase provides the following services:
The miRBase database is a searchable database of published miRNA sequences and annotation. Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database), with information on the location and sequence of the mature miRNA sequence (termed miR). Both hairpin and mature sequences are available for searching and browsing, and entries can also be retrieved by name, keyword, references and annotation. All sequence and annotation data are also available for download.
The miRBase Registry provides miRNA gene hunters with unique names for novel miRNA genes prior to publication of results.
If I'm searching for the hsa-let-7a-3 miRNa, which is involved in BCL2 pathway, I just have to put that term in the search box, and I'll find the following informations:
hsa-let-7a-3

Example for microrna database:
The microRNA.org website is a comprehensive resource of microRNA target predictions and expression profiles. Target predictions are based on a development of the miRanda algorithm which incorporates current biological knowledge on target rules and on the use of an up-to-date compendium of mammalian microRNAs. The target sites predicted by miRanda are scored for likelihood of mRNA downregulation using mirSVR, a regression model that is trained on sequence and contextual features of the predicted miRNA::mRNA duplex. Expression profiles are derived from a comprehensive sequencing project of a large set of mammalian tissues and cell lines of normal and disease origin. This website enables users to explore:
The set of genes that are potentially regulated by a particular microRNA.
The co-occurrence of predicted target sites for multiple microRNAs in an mRNA.
MicroRNA expression profiles in various mammalian tissues.
If I'm searching for the hsa-let-7a-3 miRNa, which is involved in BCL2 pathway, I just have to put the term "let-7" in the search box, and I'll find the following informations:
hsa-let-7a-3

Example for Ambion Database:
Ambion database provides general informations and products related to miRNAs.

Something interesting to read...

Primary microRNA transcript retention at sites of transcription leads to enhanced microRNA production, 2008
MicroRNA biogenesis: isolation and characterization of the microprocessor complex, 2006
MicroRNAs: something new under the sun, 2002
MicroRNA maturation: stepwise processing and subcellular localization, 2002
MicroRNAs: hidden in the genome, 2002
MicroRNAs: tiny regulators with great potential, 2001
Introducing ... Mirna I. Rucci, 1997

Informations from: news-medical

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