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MicroRNA

Definition & Overview:

MicroRNAs (miRNAs) are small, RNA molecules encoded in the genomes of plants and animals (Figure 1). These highly conserved, ~21-mer RNAs regulate the expression of genes by binding to the 3'-untranslated regions (3'-UTR) of specific mRNAs.

Although the first published description of an miRNA appeared ten years ago (Lee 1993), only in the last two to three years has the breadth and diversity of this class of small, regulatory RNAs been appreciated. A great deal of effort has gone into understanding how, when, and where miRNAs are produced and function in cells, tissues, and organisms. Each miRNA is thought to regulate multiple genes, and since hundreds of miRNA genes are predicted to be present in higher eukaryotes (Lim 2003b) the potential regulatory circuitry afforded by miRNA is enormous. Several research groups have provided evidence that miRNAs may act as key regulators of processes as diverse as early development (Reinhart 2000), cell proliferation and cell death (Brennecke 2003), apoptosis and fat metabolism (Xu 2003), and cell differentiation (Dostie 2003, Chen 2003). Recent studies of miRNA expression implicate miRNAs in brain development (Krichevsky 2003), chronic lymphocytic leukemia (Calin 2004), colonic adenocarcinoma (Michael 2003), Burkitt’s Lymphoma (Metzler 2004), and viral infection (Pfeffer 2004) suggesting possible links between miRNAs and viral disease, neurodevelopment, and cancer. There is speculation that in higher eukaryotes, the role of miRNAs in regulating gene expression could be as important as that of transcription factors.

Figure 1. Transcription of miRNAs. Approximately 60% of miRNAs are expressed independently, 15% of miRNAs are expressed in clusters, and 25% are in introns.

Processing:
 
Several hundred miRNAs have been cloned and sequenced from mouse, human, Drosophila, C. elegans, and Arabidopsis (see www.sanger.ac.uk). Estimates suggest that 200–300 unique miRNA genes are present in the genomes of humans and mice (Lim 2003 b). The sequences of many of the miRNAs are homologous among organisms, suggesting that miRNAs represent a relatively old and important regulatory pathway (Grosshans 2002).

Most of the genome sequences encoding miRNAs occur in areas of the genome that are not associated with known genes; many are found in fragile sites in human chromosomes (Calin 2004) and appear to be independently transcribed (Lagos-Quintana 2001, Lau 2001, Lee 2001, Lim 2003a, 2003b). A number of miRNAs, are encoded in introns of primary mRNA transcripts. Typically they are encoded in the same orientation as the parent transcript, indicating that transcription of this class of miRNA gene is driven by an mRNA promoter (Aravin 2003, Lagos-Quintana 2003, Lai 2003, Lim 2003a). Unlike C. elegans and human, in Drosophila, most miRNA genes are found as clusters in the genome. There is good evidence that these clustered miRNA genes are expressed as multi-cistronic transcripts which are then processed to become mature miRNAs.

The excision and activation of active single-stranded miRNAs from precursor transcripts occurs through a multi-step process that is depicted in Figure 2, and is described below.

Figure 2 . miRNA Processing and Activity

1. Transcription
miRNAs are initially expressed as part of transcripts termed primary miRNAs (pri-miRNAs) (Lee 2002). They are apparently transcribed by RNA Polymerase II, and include 5' caps and 3' poly(A) tails (Smalheiser 2003, Cai 2004). The miRNA portion of the pri-miRNA transcript likely forms a hairpin with signals for dsRNA-specific nuclease cleavage.

2. Hairpin release in the nucleus
The dsRNA-specific ribonuclease Drosha digests the pri-miRNA in the nucleus to release hairpin, precursor miRNA (pre-miRNA) (Lee 2003). Pre-miRNAs appear to be approximately 70 nt RNAs with 1–4 nt 3' overhangs, 25–30 bp stems, and relatively small loops. Drosha also generates either the 5' or 3' end of the mature miRNA, depending on which strand of the pre-miRNA is selected by RISC (Lee 2003, Yi 2003).

3. Export to the cytoplasm
Exportin-5 (Exp5) seems to be responsible for export of pre-miRNAs from the nucleus to the cytoplasm. Exp5 has been shown to bind directly and specifically to correctly processed pre-miRNAs. It is required for miRNA biogenesis, with a probable role in coordination of nuclear and cytoplasmic processing steps. (Lund 2003, Yi 2003).

4. Dicer processing
Dicer is a member of the RNase III superfamily of bidentate nucleases that has been implicated in RNA interference in nematodes, insects, and plants. Once in the cytoplasm, Dicer cleaves the pre-miRNA approximately 19 bp from the Drosha cut site (Lee 2003, Yi 2003). The resulting double-stranded RNA has 1–4 nt 3' overhangs at either end (Lund 2003). Only one of the two strands is the mature miRNA; some mature miRNAs derive from the leading strand of the pri-miRNA transcript, and with other miRNAs the lagging strand is the mature miRNA.

5. Strand selection by RISC
To control the translation of target mRNAs, the double-stranded RNA produced by Dicer must strand separate, and the single-stranded mature miRNA must associate with the RISC (Hutvagner 2002). Selection of the active strand from the dsRNA appears to be based primarily on the stability of the termini of the two ends of the dsRNA (Schwarz 2003, Khvorova 2003). The strand with lower stability base pairing of the 2–4 nt at the 5' end of the duplex preferentially associates with RISC and thus becomes the active miRNA (Schwarz 2003).

Function:
 
Virtually all of the miRNAs that have been studied in animals reduce steady state protein levels for the targeted gene(s) without impacting the corresponding levels of mRNA (Olsen 1999). The mechanism by which miRNAs reduce protein levels is not fully understood, but one study involving the C. elegans lin-4 miRNA/lin-14 mRNA pair indicates that lin-4 miRNA does not affect the poly(A) tail length, transport to the cytoplasm, nor entry into polysomes of the lin-14 mRNA (Olsen, 1999). If this observation holds true for all animal miRNAs, then downstream steps such as translational elongation, translational termination, or protein stability are likely influenced by miRNAs. Mounting evidence suggests that miRNAs function via a similar enzyme complex as siRNAs. This evidence is summarized below:

The let-7 miRNA can associate with the RISC in vitro. This indicates that the structure of miRNA does not preclude it from entering the same complex as siRNAs (Hutvagner 2002).

Endogenous miRNAs can cause degradation of recombinant mRNAs with binding sites that are perfectly complementary to the expressed miRNA (Hutvagner 2002, Zeng 2003). This implies that natural miRNAs associate with RISC in cells. The key feature that distinguishes an miRNA from an siRNA is non-complementarity between the center of the miRNA and the targeted mRNA (Doench 2003, Zeng 2002).

Immunoprecipitation experiments with antibodies targeting known members of the RISC (eIF2C2, Gemin 3, and Gemin 4) recover endogenous let-7 (Hutvagner 2002), providing further evidence that miRNAs are indeed associated with RISC in cells.
Plant miRNAs differ from animal miRNAs in that many plant miRNAs have perfect homology to their target mRNAs, and they act through the RNAi pathway to cause mRNA degradation (Rhoades 2002). It is likely, however, that some plant miRNAs base-pair imperfectly with their miRNA target sites and act via a pathway similar to animal miRNAs (Figure 3). In plants and yeast there is also evidence that miRNAs are involved in repression of transcription by guiding chromatin methylation.

Figure 3 . Mode of Action of miRNAs in Plants and Animals

miRNAs Often Do Not Act Alone
A key observation made by two laboratories is that mRNAs containing multiple, non-overlapping miRNA binding sites are more responsive to miRNA-induced translational repression than those containing a single miRNA binding site (Doench 2003, Zeng 2003). Furthermore, comparisons of repression by miRNAs bound to 2, 4, and 6 binding sites on a reporter construct indicate that translation decreases with each additional site (Zeng 2003). This suggests that the expression of miRNA target genes can be fine-tuned in animals (and potentially plants) by altering the concentrations or identities of miRNAs within cells. This observation coupled with the predictions that many mRNAs have target sites for many different miRNAs suggests that gene expression in various tissues and cells can be greatly influenced by the miRNA populations in those cells. This could also explain why at least some miRNAs have such broad functionality, and conversely why translational control of some genes is so complex. If miRNAs indeed regulate the translation of, but not the stability of target mRNAs, this might at least partially explain why gene expression profiles based on mRNA analysis do not always correlate with protein expression data (Kern 2003).

Expression & Targets:
 
Like mRNAs, miRNA expression profiles appear to vary from tissue to tissue but are similar for identical tissues in different individuals (Lagos-Quintana 2002, Krichevsky 2003, Michael 2003). As would be expected, studies indicate that tissues in developing and mature organisms are characterized by unique profiles of miRNA expression. Comparisons of embryonic stem cells and differentiated cells revealed that miRNA expression profiles change during differentiation (Houbaviy 2003). Notably, at least four miRNAs are expressed at relatively high levels prior to differentiation but are not expressed at all in differentiated cells.

The mirVana™ miRNA Probe Set and mirVana™ miRNA Labeling Kit were used to compare the miRNA expression profiles of 25 human tissues (Figure 4). As expected, miRNA expression profiles vary from tissue to tissue (Lagos-Quintana 2002, Krichevsky 2003, Michael 2003). Interestingly, miRNA profiles are similar between related tissues and distinct between unrelated tissues. For instance, heart and skeletal muscle profiles are very similar, digestive tract tissues cluster, and reproductive organ tissues are similar (Figure 4). The brain miRNA profile, however, is clearly distinct from the other tissues that were analyzed. Follow-up experiments indicated that brain sub-regions exhibited unique miRNA profiles that were clustered according to the relatedness of the sub-regions. The expression data suggest that miRNAs are important factors in differentiating tissues in adult organisms.

Figure 4 . Data Obtained with the miRNA Expression System

The miRNA expression profiles (y axis) of 20-25 different human normal tissues (x axis) were compared to a pool of all samples in the experiment. Green in the heat map shows miRNAs that are down-regulated in the sample relative to the pool, and red shows miRNAs that are up-regulated in the sample relative to the pool.

The mirVana miRNA Probe Set and mirVana miRNA Labeling Kit were also used to compare the expression profiles of tumor and non-tumor samples from individual cancer patients. Interestingly, a number of miRNAs appear to be routinely under- or over-expressed in tumors. For instance miR-126, miR-143, and miR-145 were expressed at significantly lower levels in more than 80% of the tumor samples compared to their associated normal tissues. miR-21 was found to be over-expressed in 80% of the tumor samples. These miRNAs likely represent biomolecules that directly or indirectly influence oncogenesis. In addition, several miRNAs were found to be differentially expressed in specific types of cancers, suggesting that there are disease-specific miRNAs.

These studies are significant because they show that tissue and cell samples can be defined by their miRNA expression profiles. Categorizing miRNAs based on their co-regulation, or identifying miRNAs that are differentially expressed between different tissue or cell samples will enhance our understanding of miRNA and protein regulation and function. If miRNAs are involved in oncogenesis, inflammatory response, or other disease states, comparative miRNA expression studies might also reveal diagnostic markers or even therapeutic targets. Once the mRNA targets of miRNAs are identified, combining mRNA and miRNA expression profiles will provide a snapshot of genes being regulated at the transcriptional and translational levels and will suggest a more comprehensive list of genes that are important to the biological process being studied.

miRNA Target Sites
The expression of a large number of the predicted 200–300 human miRNA genes (this corresponds to 1% of the protein coding genes) has been confirmed, but the predicted miRNA targets remain to be identified and verified. Several groups have developed algorithms for identificaiton of mRNA sequences that could serve as target sites for known miRNAs. These algorithms take advantage of the observation that each of the known miRNA target sites in animals has perfect or near perfect homology with the first eight bases of the miRNA (Lai 2002). Since each of the known miRNA target sites are in the 3' UTR, algorithms also restrict sequence searches to the 3' UTRs of mRNAs. Another extremely important observation is that homology searches between two or more animal genomes with shared miRNAs confirms that miRNA target sites are conserved. This is a key way to narrow the number of putative target sites. Given that most of the miRNA sequences are conserved between organisms, one would expect that the miRNA target sites would likewise be maintained even though they lie in the typically poorly conserved 3' UTRs of genes. As more is learned about the mRNA targets of the different miRNAs, it will be possible to more accurately assess gene expression for a given sample by combining the profiles of mRNA and miRNA expression.

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