MICRORNA INFORMATION CENTER

MicroRNAs (miRNAs) are short 20–22 nucleotide RNA molecules that function as negative regulators of gene expression in eukaryotic organisms. RNA mediated gene silencing pathways have essential roles in development, cell differentiation, cell proliferation, cell death, chromosome structure and virus resistance. Moreover, studies from the last three years have demonstrated that there is altered expression of miRNA genes in many human malignancies.

Recently, many small non-coding RNAs have been also identified in prokaryotic organisms and viruses.

These highly conserved RNAs regulating gene expression constitute about 1-5% of predicted genes in animals genomes, and more than 10% (up to 30%) protein-coding genes are probably regulated by miRNAs.

MicroRNA molecules are produced from larger transcripts that form hairpin precursors. Two RNase III endonucleases, Drosha and Dicer, cut both strands of the hairpin and generate ~22 nucleotide miRNA duplex. One arm of the duplex is chosen and mature miRNA is associated with RNA-induced silencing complex (RISC). RISC was identified before to repress the translation of mRNA in the presence of small interfering RNAs (siRNAs) by RNA interference (RNAi) mechanism. In animal cells, single-stranded miRNAs associated with RISC bind to target mRNAs through partly complementary sequences that are localized in their 3‘untranslated region (3‘UTR). The bound mRNA either remains untranslated, which is typical for miRNAs or can be degraded by a mechanism similar to siRNAs (see Fig. 2). In both cases it results in the decresed level of the protein encoded by the mRNA. A single miRNA can bind multiple genes and have a possible profound effect on cell physiology.

In this work I would like to review both the biogenesis and functions of microRNAs in animal cells and also focus on possible relations between microRNAs and human disease, mainly cancer. Additionally, a chapter about miRNAs and viruses shows miRNAs putative roles in host-virus interactions. The field of microRNAs is not fully understood and their research is continuously in a dynamic progress.

1. The Discovery of miRNAs

The first studied miRNA genes are lin-4 and let-7, which have been identified in C. elegans during genetic studies of defects in larval development. Worms with mutations in these genes failed to pass certain developmental switches resulting in the abnormal repetition of certain larval stages. The cloning of these genes showed that they are unusually small noncoding RNAs producing hairpin structures. The 22 nt lin-4 (discovered by Lee et al., 1993; see Fig. 1) and 21 nt let-7 (discovered by Reinhart et al., 2000) are both translational repressors of mRNAs. Products of these mRNAs have an important role in heterochronic developmental pathway in nematodes (Lee et al. 1993; Wightman et al., 1993; Moss et al., 1997; Slack et al., 2000).

lin-4 encodes a RNA that is partially complementary to sites located in the 3¢-untranslated region (UTR) of the lin-14 and lin-28 mRNAs (Lee et al., 1993; Wightman et al., 1991). The synthesis of LIN-14 and LIN-28 proteins is normally repressed by lin-4 during the early larval stages of C.elegans development (Bartel, 2004).

lin-14 encodes a nuclear protein, down-regulation of which at the end of the first larval stage initiates the developmental progression into the second larval stage (Lee et al., 1993; Ruvkun and Giusto, 1989). The base pairing between lin-4 and the lin-14 3¢UTR is essential for the ability of lin-4 to control LIN-14 expression through the regulation of protein synthesis (Olsen and Ambros, 1999; Ha et al., 1996 Wightman et al., 1993).

lin-28, another target of lin-4 RNA, is a cold-shock-domain protein that initiates the developmental transition between the L2 and L3 larval stages (Moss et al., 1997).

Fig. 1 The precursor structure and mature microRNA sequence of lin-4 (He and Hannon, 2004)

In the year 2000 the second miRNA, let-7, was discovered. let-7 encodes 21-nucleotide small RNA that controls the developmental transition from the L4 stage into the adult stage (Reinhart et al., 2000; Lin et al., 2003; Abrahante et al., 2003). let-7 binds to the 3¢UTR of lin-41 and hbl-1 (lin-57) and inhibits their translation (Reinhart et al., 2000; Lin et al., 2003 Abrahante et al., 2003; Bartel, 2004; Slack et al., 2000).

lin-4 and let-7 represented a new class of RNAs named small temporal RNAs (stRNAs) for their roles in developmental timing. Later all ~22nt small RNAs acting by translation repression where named microRNAs (miRNAs).

The first discovery providing evidence that gene regulation mediated by small RNAs of 22-nt may exist in species beyond worms came from Pasquinelli et al. (2000). They found that let-7 RNA expression can be detected in a wide range of animal species, including vertebrate, ascidians, hemichordate, mollusc, annelid and arthropod. Other 3 members of the let-7 family have been identified in C. elegans and at least 15 in human, but only one in Drosophila (Pasquinelli et al., 2000; Lai et al., 2003; Lim et al., 2003a). This extensive conservation strongly indicated a more general role of small RNAs in developmental regulation, as supported by the recent characterization of miRNA functions in metazoan organisms.

The second discovery, suggesting the widespread existence of miRNAs was the finding that small interfering RNAs of about 22nt lenght (siRNAs) are central to RNA interference (RNAi) (Sharp, 2001; Bartel, 2004). RNAi is an evolutionarily conserved mechanism that can degrade the mRNA in the presence of intracellular dsRNA corresponding to the targeted mRNA. lin-4 and lin-7 are not siRNAs (they do not trigger degradation of mRNA targets) but they are processed from their stem-loop precursors by the same enzyme, Dicer that generates siRNAs from dsRNAs (Hutvagner et al., 2001; Bernstein et al., 2001). Since Dicer and siRNAs are phylogenetically widespread, genes similar to lin-4 and let-7 are evolutionarily conserved.

Generally, each miRNA is thought to regulate multiple genes, hundreds of miRNA genes are predicted (Lim et al. 2003b) and several hundreds have been already cloned and sequenced from C.elegans, Drosophila, Arabidopsis, mice and human(see www.sanger.ac.uk). The large number of miRNAs and homologous sequences of many miRNAs among organisms suggest that these RNAs might constitute an abundant and conserved component of the gene regulatory machinery (Grosshans et al., 2002).

2. Biogenesis of miRNAs

After the transcription of a miRNA gene follows the nuclear cleavage of the pri-miRNA performed by the Drosha RNase III endonuclease (see Fig. 2 step No. 1 and 2). This enzyme cuts both strands of the pri-miRNA near the stem loop and generates ~60–70 nt stem loop miRNA precursor (pre-miRNA) (Lee et al., 2002). This pre-miRNA is transported to the cytoplasm by the export receptor Exportin-5 (Yi et al., 2003; Lund et al., 2004; see Fig. 2 step No. 3). The nuclear cut by Drosha defines one end of the mature miRNA and cytoplasmic cut by Dicer, also RNase III endonuclease, defines the opposite one (see Fig. 2 step No. 4). Dicer recognizes the pre-miRNA and cuts both of its strands at about two helical turns away from the base of the stem loop. Then one of this ~22 nucleotide miRNA duplex arms is chosen and mature miRNA is associated with RNA-induced silencing complex RISC (see Fig. 2 step No. 5). RISC acts to repress the translation of target mRNA by mechanisms of translational repression or mRNA cleavage (see Fig. 2 step No. 6).

Fig. 2 Processing events leading to miRNA functions in animal cells and similarities with biogenesis of small interfering RNAs (He and Hannon, 2004)

2.1. MicroRNA Genes and Their Transcription

2.1.1. MicroRNA Genes, Their Number and Discovery of New miRNAs

For seven years after the discovery of the lin-4 RNA there was no evidence for other lin-4-like RNAs. This changed with the discovery of let-7 in C. elegans (Reinhart et al., 2000; Slack et al., 2000).

Less than one year later, over one hundred additional genes for tiny noncoding RNAs were discovered (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001; Bartel, 2004). Unlike the first two discovered tiny noncoding RNAs, most of the new ones were expressed in particular cell types. The RNA products of all these genes are ~ 22 nt endogenously expressed RNAs, which are processed from one arm of a stem loop precursor. To refer to all the tiny RNAs with similar features, the term microRNA was used (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001).

The present number of discovered miRNAs in human genome is about 500 and the estimated number reaches 1000 miRNA genes. Nearly all of the cloned miRNAs are conserved in closely related animals, such as human and mouse, and many are also conserved more broadly among the animal lineages (Lagos-Quintana et al., 2003; Lim et al., 2003a; Lim et al., 2003b; Bartel, 2004). For example, about a third of the C. elegans miRNAs have homologs among the human miRNAs (Lim et al., 2003a).

A registry for catalogizing the miRNAs and labeling newly identified genes has been set up (see miR registry at www.sanger.ac.uk)

MicroRNAs are clearly not rare, even in contrast they appear to be one of the more abundant ribonucleoprotein complexes in the cell and could be identified by cloning approaches. Nonetheless, miRNAs, whose expression is restricted to less-abundant cell types or specific events in cell metabolism, could be missed in cloning efforts. Thus, computational approaches have been developed to identify new miRNAs.

One of them is searching for homologs of known miRNA genes (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). The disadvantage of this method is obvious because you find relatively few other miRNAs.

Scientists can also search the vicinity of known miRNA genes for other stem loops that might be miRNA genes of a genomic cluster (Lau et al., 2001; Aravin et al., 2003; Seitz et al., 2003; Ohler et al., 2004). This strategy is very useful because some of the miRNA genes are present within operon-like clusters.

There are also other approaches which are independent of homology or proximity to known miRNA genes (Ambros et al., 2003; Bartel, 2004; Lai et al., 2003; Lim et al., 2003a). Scientists start by identifying conserved genomic segments that are outside of predicted protein-coding regions and potentially could form stem loops. There are about 15,000 human genomic segments that fulfill this criteria and are at least loosely conserved among three vertebrate species (mouse, human and pufferfish Fugu rubripes) (Lim et al., 2003b). Then these putative miRNA stem loops are scored for the patterns of conservation and pairing that characterize known miRNAs genes. First of these methods, MiRscan, relies on the observation that the known miRNAs have phylogenetically conserved stem loop precursor RNAs with characteristic features (Bartel, 2004). MiRscan evaluates conserved stem loops as miRNA precursors by passing a 21-nt window along each stem loop, assigning a log-likelihood score to each window that measures how well its attributes resemble those of the experimentally verified C. elegans miRNAs with C. briggsae homologs (Lim et al., 2003b). Another one, miRseeker, has been applied systematically in a very similar way to insect candidates (Lai et al., 2003). For the list of other prediction algorithms see Tab. 1.

Both MiRscan and miRseeker have identified dozens of miRNAs and enabled to estimate the number of miRNA genes in the genomes of human (200–255 miRNA genes; Lim et al., 2003b), C. elegans (103–120 genes; Lim et al., 2003a; Ohler et al., 2004), and Drosophila (96–124 genes; Lai et al., 2003).

When estimating the miRNAs remaining to be identified, it is important to remember that gene number predicted by MiRscan and miRseeker rest on the presumption that the stem loops of the unknown miRNAs will have sequences similar to those of the easily cloned miRNAs (Bartel, 2004). Recent data suggest that more difficult-to-clone mammalian miRNAs are also less conserved in different species which makes their identification more difficult (Houbaviy et al., 2003; Seitz et al., 2003).

Nowadays, it is known that the real number of miRNAs is higher than predicted by Lim et al. (2003b). The public miRNA database contains about 320 human miRNA sequences and 235 of them have been experimentally verified. Cloning individual genes to determine whether they code for miRNAs is difficult and the bioinformatics approach requires experimental verification. Cummins et al. (2006) have developed an experimental approach called miRNA serial analysis of gene expression (miRAGE) to search for other miRNAs. Their study shows that previous predictions of the total number of human miRNAs were too low. Basically, this method involves isolating thousands of tiny RNA molecules from cells. These RNA molecules are reverse-transcribed into cDNA, then linked together into larger chain and sequenced. The researchers then used bioinformatics to analyze the DNA sequences (for similarity to miRNA genes in other species, ability to form a hairpin). The sequence analysis of nearly 274 000 small RNA tags allowed them to identify 200 already known miRNAs and 133 novel miRNA candidates (Cummins et al., 2006). With miRAGE method they increased the number of experimentally verified microRNAs by almost 50 percentages.

To summarize, in human there are about 500 discovered miRNAs and 1000 is the predicted number. MicroRNA genes represent probably 1-5% of the predicted genes in humans, a fraction similar to that seen for other very large gene families with regulatory roles, such as those encoding transcription-factor proteins. Because of their ability to target multiple mRNAs, about 10% - 30% protein-coding genes are predicted targets regulated by miRNAs.

Tab. 1 miRNA gene prediction algorithms (adapted from Kim and Nam, 2006)

2.1.2. Genomic Location of miRNA Genes

There are three groups of miRNA genes according to their genomic location (Fig. 3):

1. Intronic miRNA in protein coding transcription units

2. Intronic miRNA in noncoding transcription units

3. Exonic miRNA in noncoding transcription units

It was initially thought that most miRNA genes are located in intergenic regions (Lagos-Quintana et al., 2001). A recent analysis of miRNA gene locations and transcription units witch involved combining genome assemblies and expressed sequence tag databases demonstrated more than 70% of mammalian miRNA genes are located in defined transcription units (Rodriguez et al., 2004). Moreover, two thirds of miRNA genes are found in the introns in the sense orientation. About 80% of these 117 intronic miRNAs (out of 232 studied miRNAs) were in introns of protein-coding genes and just a small part of 117 miRNAs were in the introns of noncoding RNAs (ncRNAs). Interestingly miRNAs can be also present in either an exon or an intron depending on the alternative splicing pattern (14 miRNAs) (Rodriguez et al., 2004). This indicates that bioinformatic searches focused on intergenic regions might have missed some miRNA genes. Bioinformatic searches for miRNA-specific promoter elements upstream of miRNA sequences have not yet been successful.

Human miRNA genes are located in all chromosomes except Y chromosome and they are nonrandomly distributed in the human genome. Approximately 50% of known human miRNAs are found in clusters (Lagos-Quintana et al., 2001; Lau et al., 2001) and they are transcribed as polycistronic primary transcripts (Lee et al., 2002). There are usually two or three genes per cluster (Calin et al., 2004a) and the largest cluster at 13q31 is composed of seven genes (He et al., 2005). Also over half of the known Drosophila miRNAs are clustered (Aravin et al., 2003). A cluster can contain miRNAs related to each other, suggesting that it is a result of gene duplication, or contains unrelated miRNAs. Such clustered miRNAs can be functionally related by targeting the same gene or different genes in the same metabolic pathway. It is possible that even in cases where clustered genes have no sequence homology, they may share functional relationships.

Interestingly, some miRNA genes are located in the Hox clusters which play role in development. In the mammalian Hox clusters are the miR-10 and miR-196 families of miRNA genes (Lagos-Quintana et al., 2003; Lim et al., 2003b). miR-196a contains a nearly perfect complementary to HOXB8 3‘UTR and exhibits an expression pattern that is inverse to that of HOXB8 suggesting its possible role in developmental processes. Moreover, HOXB4, HOXC9, HOXC10, HOXD4, and HOXD8, all with miRNA neighbors, are deregulated in a variety of cancers (Cillo et al., 1999; Owens and Hawley, 2002)..

Overall, 98 of 186 (52.5%) of miR genes are in cancer-associated genomic regions or in fragile sites (FRA) (Calin et al., 2004a). A significant number of miRNAs are close to Human Papilloma Virus (HPV) integration sites (Calin et al., 2004a). FRAs are preferential sites of sister chromatid exchange, translocation, deletion, amplification or integration of plasmid DNA and tumor-associated viruses such as HPV. Infection with HPV16 or 18 is the major risk factor for developing cervical cancer (Thorland et al., 2003). Much more, looking at 113 isolated FRAs in human karyotype was found that 61 miRNAs are located in the same cytogenetic positions with FRAs (Calin et al., 2004a). Interestingly, chromosome region 13q14.3 which belongs to these regions is the site of the most common structural aberrations in both mantle cell lymphoma and B cell chronic lymphocytic leukemia. This site does not include any known or putative tumor suppressor and therefore the miR-15a-miR-16 cluster, which falls within this region, has candidate genes for this role (Calin et al., 2002).

The genomic location of miRNA genes as well as their putative targets and deregulation in cancer makes them potentially very important and conserved mechanism for gene expression regulation in eukaryotic cells.

Fig. 3 Genomic organization and structure of miRNA genes (Kim and Nam, 2006)

(a) Intronic miRNA in a protein-coding transcriptional unit (TU). As an example, miR-10 in HOX4B gene is shown. The green triangle indicates the location of a miRNA stem-loop and the exons are shown as rectangels .

(b) Intronic miRNAs in a noncoding transcript. The miR-15a-16-1 cluster is shown, which is found in the fourth intron of a previously defined noncoding RNA gene, DLEU2.

2.1.3. Transcription of miRNA Genes

Scientists are searching for pri-miRNAs transcripts and discussing their length. In case of lin-4 all the elements required for the regulation and initiation of transcription are located in a 693 bp genomic fragment (Lee et al., 1993). The pri-miRNA precursor for human miRNA cluster miR-23a-miR-27a-miR-24-2 and for isolated miR-21 are unspliced ~2,2 and ~3,4 kilobase long RNAs. Both are capped, polyadenylated non-coding RNAs (see Fig. 4). In contrast, human pri-miRNA for miR-155 contains two introns, two poly-A sites and can give two alternatively spliced pri-miRNA precursors of ~0,6 and ~1,4 kb (Cai et al., 2004; Lee et al., 2004; see Fig 4).

Fig. 4 Structure of pri-miRNAs (Cullen, 2004)

Little is known about transcriptional processes for this new class of small non-coding RNAs. MicroRNAs residing in introns share their regulatory elements and primary transcript with their pre-mRNA host genes. This gives a possible mechanism for coordinated miRNA and protein-coding gene expression. As expected for genes sharing the same promotors, both usually have similar expression profiles (Lagos-Quintana et al., 2001; Rodriguez et al., 2004; Lau et al., 2001). For the other miRNA genes, transcribed from their own promoters, few primary transcripts have been fully identified. Pri-miRNA transcripts are much longer than the conserved stem loops used to search and define miRNA genes (Lagos-Quintana et al., 2001; Lau et al., 2001).

There is a key question which RNA polymerase transcribes a miRNA gene. The two candidate RNA polymerases for pri-miRNA transcription are pol II and pol III. MicroRNAs processed from the introns of protein-coding host genes are undoubtedly transcribed by pol II. Pol III was initially believed to mediate the transcription of remaining miRNAs because it produces some of the shorter noncoding RNAs: tRNAs, 5S ribosomal RNA and U6 snRNA.

Recently, it has been suggested that pri-miRNAs with their own promotors are Pol II products, because:

1. Pri-miRNAs are sometimes several kilobases long - longer than typical pol III transcripts (Lee et al., 2002).

2. Many miRNAs are differentially expressed during development - typical for pol II but not pol III products (Lagos-Quintana et al., 2002; Krichevsky et al., 2003; Calin et al., 2004b; Miska et al., 2004).

3. Fully functional miRNA can be generated from a plasmid containing a pri-miRNA sequence under the control of pol II promotor (Cai et al., 2004).

4. Pri-miRNAs were shown to contain both CAP STRUCTURES (m7GpppN located at the 5‘end) and POLY(A) TAILS (25–200 adenine nucleotides at the 3‘end) (Cai et al., 2004; Lee et al., 2004).

5. MicroRNA transcription activity is sensitive to a-amanitin at a concentration that specifically inhibits pol II, but not pol III (Lee et al., 2004).

6. Association of pol II with the promoter of miR-23a~27a~24-2 and other miRNAs was demonstrated by chromatin immunoprecipitation analyses (Lee et al., 2004).

There are still many questions to answer about miRNAs transcription. For example, whether the intronic or exonic location affects miRNA biogenesis? Can a protein-coding pri-mRNA, containing both a protein-coding region and miRNA sequences, generate protein as well as miRNA?. May be only the miRNA pathway or the mRNA pathway has to be „chosen“. Currently, only a few miRNA promoters have been identified experimentally (Lee et al., 2004; Cai et al., 2004; Bracht et al., 2004). Those characterized promotors contain general RNA polymerase II transcriptional regulatory elements previously found in protein-coding genes.

2.2. MicroRNA Maturation

2.2.1. Nuclear Processing by Drosha

Droshas are 130–160 kDa proteins containing two RNase III catalytic domains and a dsRNA-binding domain (dsRBD) in the C-terminal half of the protein (Filipowicz et al., 2005; see Fig. 5). Some domains of unknown function are localized in the N-terminal half (Lee et al., 2003; Lamontagne et al., 2001). Interestingly plant genomes do not seem to encode Drosha homologs and in some plant species all miRNA biogenesis steps may be carried out by Dicer-like protein, DCL-1, localized in the nucleus (Kurihara and Watanabe, 2004). In animals only one Drosha homologue is found in all species.

Fig. 5 Domain organization of human Drosha (Kim, 2005)

The RIIID, RNase III domain

The dsRBD, double-stranded RNA binding domain

The “P-rich” indicates a proline-region, whose biochemical significance remains unknown.

The “RS-rich” is a region that is abundant in arginine and serine

Drosha is a RNase III enzyme that belongs to a family of double stranded RNA specific ribonucleases. Human cells express three members of this enzyme family. One of them functions in mitochondrial rRNA processing and other two (Drosha and Dicer) are necessary for miRNA maturation. Moreover, Dicer plays a role in rRNA processing (Wu et al., 2000) and Drosha was first recognized for its role in generating small interfering RNAs that mediate RNA interference (Bernstein et al., 2001). Drosha cleaves hundreds to thousands of nucleotides long pri-miRNAs to release ~70 nucleotide stem–loop pre-miRNAs. pri-miRNA duplex is cut in a way typical for RNase III endonucleases, and thus the base of the pre-miRNA stem loop has a 5¢ phosphate and 2nt 3¢ overhang (Basyuk et al., 2003; Lee et al., 2003). This overhang is probably necessary for transporting pre-miRNAs to cytoplasm by Exportin-5 (Yi et al., 2003; Lund et al., 2004). (see Fig. 6)

Fig. 6 The miRNA biogenesis pathway in animals (Tomari and Zamore, 2005)

Drosha acts together with dsRNA-binding protein known as Pasha (in flies) or its ortholog DGCR8 (in C. elegans and mammals) (Han et al., 2004; Denli et al., 2004). Pasha/DGCR8 binds to the central region and the RNase III domains (RIIIDs) of Drosha (Han et al., 2004; Filipowicz et al., 2005). This pri-miRNAs metabolizing complex of Drosha and Pasha/DGCR8 is a 500–650 kDa nuclear complex called microProcessor (Han et al., 2004; Denli et al., 2004; Gregory et al., 2004). Its large size may be due to dimerization of its components or the presence of additional proteins (Han et al., 2004). All mentioned proteins of microProcessor are required in vivo to convert pri-miRNA to pre-miRNA. Reducing the level of either Drosha or Pasha/DGCR8 by RNAi led to the reduction in both pre-miRNAs and mature miRNAs (Han et al., 2004; Denli et al., 2004; Gregory et al., 2004).

Drosha contains two catalytic sites in a single processing center, each of them breaks one phosphodiester bond (Lee et al., 2003). Recombinant human Drosha alone shows non-specific RNase activity, but the addition of DGCR8 affords its specifity for pri-miRNA processing (Gregory et al., 2004). Pasha/DGCR8 is a specific factor driving Drosha to bind to the pri-miRNAs. In a very similar way R2D2 organizes Dicer-2 binding to siRNA. The binding of Pasha/DGCR8 to pri-miRNA is believed to be essential for determinating the position od Drosha‘s catalytic center. Interestingly, the DGCR8 gene is one of the few genes located in a region (chrom. 22) deleted in a genetic disease termed DiGeorge syndrome (Gregory et al., 2004). Drosha cut both strands about 22 nucleotides (2 turns od dsRNA) from the loop that is required for recognition of pri-miRNA and must be greater than 10 nucleotides (Zeng et al., 2005).

2.2.2. Nuclear Export of pre-miRNAs

It is known that Exportin-5 (Exp5) mediates the nuclear export of adenovirus 160-nt noncoding RNA (Gwizdek et al. 2003; Brownawell and Macara 2002). Yi and colleagues (Yi et al., 2003) noticed that transport of this non-coding RNA by Exp5 requires a terminal dsRNA “mini-helix” bearing a base-paired 5‘ end and a 3‘ overhang of 3 nt (Gwizdek et al. 2003) which is a structure very similar to pre-miRNAs. Yi et al. (2003) asked whether Exp5 might also be required for nuclear export of small noncoding RNAs. They have demonstrated that the nuclear export is dependent on the Exportin 5 (Exp5) nuclear export factor which is a member of the karyopherin family of nucleocytoplasmic transport factors. They have also shown that Exp5 binds the pre-miRNA specifically, but only in the presence of Ran-GTP. Ran is a GTPase which binds in the GTP bound form to karyopherins (Exp5 in this case) and forms a nuclear heterotrimer with pre-miRNAs (Yi et al., 2003; Lund et al., 2004; see Fig. 7).

Experiments with knockdown of Exp5 showed little effect on the level of expression of the pri-miRNAs or of the pre-miRNA, but did significantly reduce the level of expression of the mature miRNA (Yi et al., 2003). It is interesting that no accumulation of the pre-miRNA intermediate was detected. Thus, Yi et al. (2003) proposed that Exp5 somehow also protects and stabilizes these short noncoding RNAs from exonucleolytic digestion. Loss of Exp5 expression leads not only to the nuclear retention of pre-miRNAs but also to their concurrent nuclear degradation (Yi et al., 2003). These results identify the major function of Exp5, which is pre-miRNA nuclear export and define a novel cofactor for miRNA biogenesis and function (Yi et al., 2003; Lund et al., 2004).

Fig. 7 Nuclear export of pre-miRNAs (adapted from Cullen, 2004)

2.2.3. Cytoplasmic Processing by Dicer

Pre-miRNAs transported to the cytoplasm are further cleaved by Dicer to yield 20-bp miRNA duplexes. Dicers are 200 kDa proteins containing generally an ATPase/RNA helicase, PAZ domains, DUF283, two catalytic RNase III domains (RIIIa and RIIIb) and a C-terminal dsRBD (Lamontagne et al., 2001; Filipowicz et al., 2005; see Fig. 8).

Fig. 8 Domain organization of human Dicer (Kim, 2005)

The RIIID, RNase III domain

The dsRBD, double-stranded RNA binding domain

Dicer enzymes process also dsRNA to siRNAs and thus have an important role in RNA interference in general. Dicer is a highly conserved protein and with one homologue in yeast (Dcr), one in human, one in nematode worm (DCR-1), two in Drosophila (DCR-1 and DCR-2), and four in Arabidopsis (DCL1, DCL2, DCL3, DCL4) (Lamontagne et al., 2001; Kim, 2005; Carmell and Hannon, 2004). Drosha, on the other hand, is conserved only among metazoans and only one homolog is found in each species (Lamontagne et al., 2001; Carmell and Hannon, 2004 see Tab. 2).

Dicer functions as a monomer and has a single processing center with intramolecular dimerization of the two RNase III domains. Each RNase domain cuts independently one RNA strand of the duplex and generates products with 2-nt 3‘ overhangs (Filipowicz et al., 2005). The structure of catalytic center and its function is very similar to that of Drosha. Dicer excises miRNAs from the end of pre-miRNA hairpins produced by Drosha (Lee et al., 2003; Zhang et al., 2002).

Dicer PAZ domain recognizes 3‘ overhangs of pre-miRNAs. Drosophila homolog of Dicer, Dcr-2, requires a functional helicase for dsRNA processing and ATP has a strong effect on the activity of both Dcr-2 and C. elegans Dicer in vitro (Liu et al., 2003). In contrast, ATP is not required for Dicer functions in human (Zhang et al., 2004). Drosophila expresses two Dicers, Dcr-1 and Dcr-2, with very distinct roles in cells. Dcr-1 functions in the miRNA machinery, whereas Dcr-2 in RNAi, both for the cleavage of dsRNA and for the assembly of the RISC (Lee et al., 2004; Filipowicz et al., 2005; Pham et al., 2004; see Tab. 2).

Similar to Drosha, Dicer associates with a partner containing dsRNA binding domain. Dcr-2 heterodimerizes with R2D2, a small protein containing two dsRBDs (Liu et al., 2003). Vertebrates and nematodes express only one Dicer protein that works in both miRNA and RNAi machinery. Thus, additional proteins must interact and modulate the specificity of these enzymes. In C. elegans, an R2D2-like protein, RDE-4, has been characterized. This protein forms a complex with Dicer, RDE-1 (the Argonaute protein) and others and is essential for RNAi but not miRNA function (Tabara et al., 2002). Dicer proteins also interact with Argonaute proteins (Carmell and Hannon, 2004).

It seems that Dicer cofactors are not strictly required for cleavage reactions because purified human Dicer can catalyze this reaction. Cofactors have probably various roles in miRNA stability and RISC assembly (Zhang et al., 2002; Zhang et al., 2004; Liu et al., 2003).

Tab. 2 Principal protein components of siRNA and miRNA pathway (adapted from Tang, 2005)

2.3. MicroRNA‘s Mechanism of Action

2.3.1. RNA Induced Silencing Complex (RISC) and Its Functions

After Dicer cleavage the miRNA pathway appears to be biochemically very much similar to the central steps of RNA silencing pathway known as RNA interference (RNAi) in animals. The RNAi pathway is briefly described here to show the similarity to miRNAs (see Fig 9).

RNAi pathway begins with long double-stranded RNA duplex or a hairpin that is introduced into a cell for gene knockdown experiment (Fire et al., 1998). Double-stranded RNA could be generated from sense and antisense genomic transcripts or as an intermediate of viral replication (Cogoni and Macino, 1999; Ketting et al., 1999; Aravin et al., 2001). The dsRNA is processed by Dicer into many ~22 nt siRNAs (Hamilton and Baulcombe, 1999; Hammond et al., 2000; Zamore et al., 2000). Some of them become incorporated as ssRNAs into a RNA-induced silencing complex (RISC) (Hammond et al., 2000; Elbashir et al., 2001; Martinez et al., 2002). The RISC identifies its targets by the perfect or nearly perfect complementarity between the siRNA and the mRNA. In the last step of the RNAi pathway, RISC cleaves the mRNA with its endonuclease activity (Bartel, 2004).

In contrast to siRNAs, microRNAs can direct the RISC to down-regulate gene expression by translational repression (based on lower complementarity between miRNA and mRNA). Interestingly, miRNAs can also function as siRNAs and can guide a typical siRNA mRNA cleavage. According to the current knowledge, the choice of post-transcriptional mechanisms is not determined by the origin of silencing RNA - siRNA or a miRNA. If the miRNA is perfectly or nearly complementary to its target, it can specifically cleave the mRNA (Hutvagner and Zamore, 2002; Zeng et al., 2002; Doench et al., 2003). When a miRNA guides cleavage, the cut is at the same site as in the case of siRNA-guided cleavage, i.e. between the nucleotides pairing to residues 10 and 11 of the miRNA (Elbashir et al., 2001; Hutvagner and Zamore, 2002; Llave et al., 2002). One example of miRNA directed cleavage is miR-196, which directs the cleavage of the HOXB8 transcript (Mansfield et al., 2004; Cuellar and McManus, 2005; Yekta et al., 2004). Nonetheless, it is believed that miRNAs typically mediate translational repression rather than mRNA cleavage. This is supported by a finding that most miRNAs repress the expression of proteins without decreasing mRNA levels (Brennecke et al., 2003)

There arises a question to answer, which strand of miRNA–siRNA duplexes resulting from Dicer cleavage will be incorporated into RISC. During this process, the duplex is unwound by an unknown helicase-like enzyme and one strand, known as miRNA*, is degraded, whereas the other strand is incorporated in the RISC. The strand which enters the RISC is nearly always the one whose 5‘ end is less tightly paired (Khvorova et al., 2003; Schwarz et al., 2003; Bartel, 2004). The complementary sites for miRNAs (associated with RISC) reside in the 3‘untranslated regions (UTRs) of target mRNAs (He and Hannon, 2004). It has been shown the presence of multiple miRNA complementary sites in 3‘ UTRs (Lee et al., 1993; Wightman et al., 1993; Reinhart et al., 2000; Abrahante et al., 2003; He and Hannon, 2004; Lin et al., 2003). These multiple binding sites provide an efficient translational inhibition mechanism of RISC action (Doench et al., 2003 see Fig. 10). MicroRNAs can also potentially bind to complementary sequences in the open reading frame (ORF) or 5‘ UTR (Doench et al., 2003; Cuellar and McManus, 2005).

Fig. 10 Se quence complementarity between lin-4 and the 3‘ UTR of lin-14 mRNA. lin-4 is partially complementary to 7 sites in the lin-14 3‘ UTR; its binding to these sites of complementarity repress LIN-14 protein synthesis (He and Hannon, 2004).

Interestingly, based on computional predictions, different miRNAs are believed to regulate the same targets (Reinhart et al., 2000; Abrahante et al., 2003; Lin et al., 2003). Some miRNAs might not only post-transcriptionaly repress translation, but also target DNA for transcriptional silencing, DNA methylation and heterochromatin formation in plants and fungi (Mette et al., 2000; Hamilton et al., 2002; Hall et al., 2002; Volpe et al., 2002; Grewal and Rice, 2004; see Fig. 11). In these cases miRNAs work probably in a kind of unknown nuclear RISC-like complex.

To summarize, in animal cells it is believed that the main mechanism of miRNA function is post-transcriptional repression of mRNA translation. Pillai et al. (2005) demonstrated that miRNAs in human cells act by inhibiting protein synthesis at the early step of initiation and that repressed mRNAs are relocated into specific intracellular organelles known as processing bodies (P-bodies) for storage. miRNAs and proteins associated with them also localize to P-bodies.

Fig. 11 A simplified model for the processing and functions of microRNAs (miRNAs) and some other small RNAs (siRNAs). miRNA biogenesis is shown on the left and siRNA biogenesis is shown on the right (Xu et al., 2004).

2.3.2. Components of RNA-Induced Silencing Complex (RISC)

RISCs (also referred to as miRNPs) are ribonucleoprotein complexes that contain members of the Argonaute (Ago) family of proteins (with PAZ–Piwi-domain; see Fig. 12), siRNAs/miRNAs, miRNA/siRNA-complementary mRNAs and a number of little known accessory factors (Kim, 2005; Sontheimer, 2005; Filipowicz et al., 2005).

Fig. 12 Simplified structure of Argonaute (Ago) family proteins containing PAZ domain and PIWI domain (Kim, 2005)

The number of Argonaute paralogs varies in different organisms from 1 in S. pombe to 27 in C. elegans (Carmell et al., 2002; see Tab. 2). Other proteins often found in RISC and miRNP include the Vasa intronic gene (VIG) protein, possible endonuclease Tudor-SN1 and RNA-binding protein dFXR, the Drosophila ortholog of human fragile X mental retardation protein (FMRP) (Sontheimer, 2005; Caudy et al., 2002; Filipowicz et al., 2005; Caudy et al., 2003). These proteins or their orthologs seem to form also a part of miRNPs of other organisms. In human cells, miRNAs were found in an 15S complex containing additional helicase proteins Gemin 3 and Gemin 4 (Mourelatos et al., 2002). Some dsRNA-binding proteins facilitate the transfer of miRNAs to RISC. For example RDE-4 in C.elegans and its Drosophila homolog R2D2 probably have such function (Yan et al., 2003; Tabara et al., 2002; Filipowicz et al., 2005; see Tab. 2).

The precise biochemical mechanisms of RISCs functions are unknown. In animal cells, two miRNA guided RISC activities are mRNA cleavage and translational suppression. In mammals, mRNA cleavage is thought to occur by perfect or nearly perfect interactions of miRNAs with their target mRNAs. This triggers the action of „Slicer“, Argonaute 2 (Ago-2) endonuclease (Liu et al. 2004; Meister et al. 2004; Okamura et al. 2004; Cuellar and McManus, 2005; see Fig. 13). In contrast, translational suppression is mediated by imperfect complementarity to mRNA and by Agronaute 1 (Ago-1) protein (Zeng et al., 2002; Okamura et al. 2004; see Fig. 13). The complex structure and functions of RISC are largely unknown and intensively studied.

Fig. 13 MicroRNA biogenesis pathway (Cuellar and McManus, 2005)

Two major processing events lead to the production of mature microRNAs (for simplicity, only catalytic components of the pathway are depicted in this diagram). They are first processed by the RNAse III enzyme, Drosha, and are then exported out of the nucleus. Once in the cytoplasm, they are subjected to another processing step by another RNAse III enzyme, Dicer. These mature microRNAs can then undergo unwinding and a single strand can enter the RISC complex, where they can act by repressing the translation of their mRNA targets or by inducing their degradation, mediating RNA interference.

3.MicroRNA Functions in Animal Cells

3.1. MicroRNAs in Developmental Timing

Research of genes controlling developmental timing in C. elegans led to the identification of the first microRNA, lin-4 (Lee et al., 1993) and its target, lin-14 mRNA (Wightman et al., 1993). Introduction to this theme was given in the chapter „The Discovery of miRNAs“. Here I would like to describe some recent and more detailed information about pathway regulating stage-specific processes during C. elegans larval development.

There are some new information about heterochronic gene activity in the worm epidermis in specialized ‘seam’ cells. These cells are situated on the lateral midlines of the worm. They terminally differentiate during the transition to the adult stage of the worm and help with the synthesis of cuticle. The seam cells divide in a typical way during the four larval molts of the nematode (Ambros and Horvitz, 1984)

C. elegans cells with mutated lin-4 repeat the cell division pattern that characterises the first larval stage (L1) of epidermis and fail to differentiate (Lee et al., 1993; see chapter 1). This results in the developmental ‘retardation’.

LIN-14 and LIN-28, both targets of lin-4, are core components of the mechanism that programs epidermal cell fate transitions. Their levels decrease during typical development events; LIN-14 disappears from the epidermis by the end of the L1 stage and LIN-28 by the end of the L2 stage (Lee et al., 1993). The temporal fall of both these protein levels is a key factor for seam cell to pass through the early larval fates.

The regulation of the early larval stage development is more complex and additional levels of control are present. It is not just a simple down-regulation of lin-14 and lin-28 by the lin-4 miRNA. Various experiments indicate that hbl-1 and daf-12, which are discussed in more details later, act in this early time development in the epidermis (Abrahante et al., 2003; Alvarez-Garcia and Miska, 2005). Moreover, other gene (lin-42) has unknown multiple or repeated roles during postembryonic development (Pepper et al., 2004) (see Fig. 14).

The second identified microRNA, let-7 (Reinhart et al., 2000), controls the transition from the fourth larval stage to the adult stage (see chapter 1) and targets lin-41, hbl-1 and transcription factor gene daf-12 (Abrahante et al., 2003; Lin et al., 2003; Slack et al., 2000; Grosshans et al., 2005; Alvarez-Garcia and Miska, 2005). DAF-12 binds hormones and could provide possible integration of nutritional signals and coordinate the progression of temporal cell fates throughout the animal (Gerisch and Antebi, 2004; Mak and Ruvkun, 2004). MicroRNAs of the let-7 family, miR-48, miR-84 and miR-241 act to control the developmental transition from the L2 to the L3 stage (Abbott et al., 2005; Lau et al., 2001; Lin et al., 2005; Reinhart et al., 2000; Ruvkun and Mello, 2001). Hbl-1 in C. elegans is a putative target of miR-48, miR-84 and miR-241. The presence of multiple let-7 family members in worms that share perfect identity in the 5‘ seed implicates their redundancy and similar functions (Alvarez-Garcia and Miska, 2005). Like let-7 all the new let-7 family members show also temporally expression patterns (Lau et al., 2001; Lim et al., 2003a). Victor Ambros and colleges are now generating C. elegans strains that are null for all these miRNA genes. Their further work will probably explain the role of let-7 family members.

To conclude, at least two microRNA families control developmental timing in C. elegans (see Fig. 14). Both of them are conserved and might play similar roles in other organisms. Moreover, let-7 is aberrantly regulated in human cancer and could regulate RAS oncogene (Johnson et al., 2005; Takamizawa et al., 2004; see chapter 4.1)

For the list of other proposed in vivo roles for microRNAs in animals see Tab. 3.

Fig. 14 The expression patterns of selected heterochronic genes. Arrows indicate positive regulatory interactions and bars indicate negative regulatory interactions (Rougvie, 2005)

3.2. MicroRNAs in Embryogenesis

Organisms with defective microRNA biogenesis are the first tool for investigating the biological roles of microRNAs. They allow us to study the first set of microRNAs transcribed during embryogenesis and development and RNAi related proteins in general.

Dicer knockout was first analyzed in C. elegans where authors described a null mutation in Dicer-1 (Knight and Bass, 2001). They found that dcr-1(-/-) animals have defects in RNAi and germ line defects that lead to sterility. Dcr-1 mutants are sterile and homozygous animals had to be derived from heterozygous mothers. It is possible that maternal cytoplasmatic transfer of DCR-1 masks the earliest abnormal phenotypes. This is supported by experimental RNA inactivation of homolog of Drosophila Dicer that caused more complex abnormal phenotypes and heterochronic phenotypes similar to lin-4 and let-7 mutations (Alvarez-Garcia and Miska, 2005). Grishok and his colleagues used RNAi to knockdown transcripts for the two C. elegans argonaute proteins required for miRNA biogenesis. RNAi-treated worms showed a mixed phenotype with severe developmental defects (Grishok et al., 2001).

Dicer-1 in D. melanogaster, protein required for microRNA biogenesis, is also required for wild-type development of both somatic tissues and the germline (Hatfield et al., 2005; Lee et al., 2004). Moreover, Hatfield et al. (2005) showed on the basis of cell cycle markers that dcr-1 mutant germ stem cells are delayed in the G1 to S transition.

In zebrafish (Danio rerio) homozygous Dicer mutants showed an initial build-up of miRNA, produced by maternal Dicer1, but miRNA accumulation stopped after a few days. This resulted in developmental arrest around day 10 post-fertilization (Wienholds et al., 2003). Indeed, complete removal of the maternal Dicer by generating maternal-zygotic dicer mutants for Dicer leads to zero processing of precursor miRNAs into mature miRNAs and mutants display abnormal morphogenesis during gastrulation, brain formation, somatogenesis and heart development (Giraldez et al., 2005).

Interestingly, Bernstein et al. (2003) were unable to generate viable Dicer1-null M. musculus embryonic stem cell (ES). Loss of Dicer1 lead to lethality early in development and mutants die around 7.5 days of gestation (Alvarez-Garcia and Miska, 2005). This observation could be explained by smaller size of mouse eggs and thus smaller maternal contribution of Dicer (Bernstein et al., 2003). In mouse, removal of Dicer leads to enhanced programmed cell death in the developing limb mesoderm (Harfe et al., 2005). Moreover, in mouse, Dicer is required for embryonic stem cell differentiation in vitro (Kanellopoulou et al., 2005) and in mutant mouse embryos the pool of pluripotent stem cells in blastocysts is diminished (Bernstein et al., 2001; Alvarez-Garcia and Miska, 2005). Studies in ES cell lines and mouse embryoid bodies have identified miR-302 family as specifically expressed in ES cells but not in adult mouse tissues (Houbaviy et al., 2003; Suh et al., 2004).

Unfortunately, when only Dicer mutants are analysed, one cannot easily distinguish between observed defects that are due to a loss of microRNA processing, loss of endogenous RNAi or other pathways mediated by Dicer.

Denli et al. (2004) observed mutants of other enzyme required for microRNA biogenesis, Drosha. They also suppressed Pasha protein, a partner of Drosha and dsRNA binding protein. Suppression of Pasha and Drosha expression in C. elegans interferes with pri-miRNA processing, leading to an accumulation of pri-miRNAs and a reduction in mature miRNAs (Denli et al., 2004).

For investigating the role of individual miRNAs during embryogenesis in D. melanogaster, Leaman et al. (2005) used 2‘O-Methyl antisense oligoribonucleotides (2‘OM-ORNs). In this study, embryos injected with miR-9 antisense 2‘OM-ORNs rarely form any cuticle and show virtually no internal differentiation. In contrast to miR-9, miR-31 depleted embryos development but showed severe segmentation defects. Embryos with eliminated miR-310/311/312/313/92 family show morphogenetic defects in later development. Another large microRNA family in Drosophila (miR-2/6/11/13/308) is required for suppressing embryonic apoptosis and miR-2 family regulates cell survival by translational repression of proapoptotic factors.

It would be interesting to explain the importance of microRNAs in human development. Mammalian miRNA, miR-196, that is located in a HOX cluster and can cleave HOXB8 mRNA, could be a candidate gene for such function (Mansfield et al., 2004; Yekta et al., 2004).

For the list of other proposed in vivo roles for microRNAs in animals see Tab. 3.

3.3. MicroRNAs in Organogenesis and Differentiation

The phenotypes of the lin-4 and the let-7 mutants of miRNAs in C. elegans are examples of cell differentiation defects. Another evidence for let-7 miRNA function has come from studies of vulva organogenesis, a reproductive organ of C. elegans. Vulval induction requires RAS/LET-60 signaling (Beitel et al., 1990) and this may be directly regulated by the let-7 family of microRNAs (Johnson et al., 2005). In worm, Johnston and Hobert (2003) showed that a previously undefined microRNA termed lsy-6 controls neuronal left/right asymmetry of chemosensory receptor expression. Genetic interaction and GFP reporter studies showed that lsy-6 is a negative regulator of the homeobox gene cog-1 and is the first worm microRNA known to play a role in neuronal patterning (Johnston and Hobert, 2003; Chang et al., 2003; Alvarez-Garcia and Miska, 2005). Interestingly, a second microRNA, miR-273, might act upstream of lsy-6 in the same pathway as a regulator of die-1, which encodes transcription factor (Chang et al., 2004).

As described in a previous chapter, the analysis of Dicer mutant animals suggests that microRNAs have important roles in differentiation and organogenesis. It is unclear whether all defects are due to the lack of microRNAs or other Dicer-involving processes (Alvarez-Garcia and Miska, 2005). Surprisingly, the injection of a single miRNA, miR-430, in maternal-zygotic Dicer mutant embryos of zebrafish rescued some aspects of observed mutant phenotype. Injection of miR-430 resulted in normal brain ventricles a rescue of gastrulation, retinal development, but not of heart or ear development. This demonstrated that some abnormal phenotypes of these mutant animals are due to the loss of microRNAs (Giraldez et al., 2005).

In D. melanogaster, Notch signalling pathway is an evolutionary conserved cascade required for patterning and normal development (Lai, 2004). Recently, several miRNAs were predicted to regulate some important factors in this cascade (Brennecke et al., 2005; Lai et al., 2005; Alvarez-Garcia and Miska, 2005).

In mice, Dicer1 inactivation during T-cell development blocked peripheral CD8+ T-cell development and CD4+ T cells proliferated poorly after stimulation (Muljo et al., 2005).

The microRNA miR-1 is specifically expressed in mouse cardiac and skeletal muscle precursor cells (Lagos-Quintana et al., 2001; Zhao et al., 2005). Zhao et al (2005) showed that miR-1 targets transcription factor Hand2, which promotes ventricular cardiomyocyte expansion.

One microRNA, miR-181, has been directly implicated in B-cell development (Chen et al., 2004). This microRNA is preferentially expressed in the B-lymphoid cells of mouse bone marrow and promotes B-cell differentiation when expressed in hematopoietic stem/progenitor cells (Chen et al., 2004). For the list of other proposed in vivo roles for microRNAs in animals see Tab. 3.

Tab. 3 Proposed in vivo roles for some microRNAs in animals (adapted from Miska, 2005).

miR-430 function was inferred from rescue experiments in a Dicer-mutant background.

2Family of Notch target transcription factors with GY-, Brd- and K-box motifs. Abbreviations: bHLH, basis helix–loop–helix; PFV-1, primate foamy virus type 1.

3.4. MicroRNAs in Programmed Cell Death and Growth Control

The first evidence for growth controlled by miRNAs came from the studies of the D. melanogaster bantam gene that was identified as a gene affecting tissue growth (Hipfner et al., 2002) Over-expression of the bantam locus causes an increase in cell number and tissue overgrowth. Conversely, flies homozygous for bantam deletion grow poorly in comparison to wild-type animals and have reduced cell numbers (Brennecke et al., 2003). The bantam region does not have the capacity to encode a protein and subsequent cloning of bantam locus identified it as a microRNA-coding gene (Brennecke et al., 2003). It was proved that the bantam gene promotes tissue growth. Moreover, bantam microRNA simultaneously stimulates cell proliferation and prevents apoptosis. Its over-expression interferes with apoptosis induced by over-expression of the transcription factor E2A element-binding factor (E2F) and its dimerisation partner (DP). Furthermore, apoptosis-inducing gene hid was shown to be the target of bantam mRNA (Brennecke et al., 2003; see Fig. 15).

A second studied D. melanogaster microRNA, miR-14, was showed to suppress death induced by expression of Rpr, Hid, Grim or the apical caspase Dronc (Xu et al., 2003; see Fig. 15). MiR-14 also regulates fat metabolism and deletion of miR-14 results in animals with increased levels of triacylglycerol and diacylglycerol. Targets for miR-14 as a regulator of fat metabolism may be distinct from those that mediate its role as a cell death inhibitor and it could be an example of fat metabolism and apoptotic related signaling (Xu et al., 2003).

Abnormal levels of programmed cell death in D. melanogaster were also found in depletion experiments using 2´O-Methyl antisense oligoribonucleotides (Leaman et al., 2005; Alvarez-Garcia and Miska, 2005; see chapter 3.2). All the deletion studies of Dicer in Drosophila, zebrafish and mouse suggest an important role for microRNAs in growth control and cell death (Hatfield et al., 2005; Wienholds et al., 2003; Alvarez-Garcia and Miska, 2005; Harfe et al., 2005; see chapter 3.2)

Fig. 15 MicroRNAs control programmed cell death. The D. melanogaster microRNA bantam and miR-14 inhibit programmed cell death (Mraz, 2006)

4.MicroRNAs and Human Disease

4.1. MicroRNAs and Cancer

Many studies showed that miRNAs are aberrantly expressed in cancer, suggesting their role as a novel class of oncogenes or tumor suppressor genes (see Fig. 16). The findings that miRNAs have a role in cancer are supported by the fact that about 50% of miRNA genes are localised in cancer-associated genomic regions or in fragile sites (Calin et al., 2004a). Regulation mediated by these genes has possibly a large impact on gene expression because, according to computional predictions, a single miRNA can target dozens of genes. Many authors have reported that each cancer tissue has a specific microRNA signature and microRNA based cancer classification is a very effective and potential tool (Lu et al., 2005).

4.1.1. Hematological Malignancies

First evidence of involvement of miRNAs in cancer came from molecular studies characterizing the 13q14 deletion in human chronic lymphocytic leukemia (CLL). Deletion of the 13q14 region occurs in more than half cases of B cell chronic lymphocytic leukemias and also in 50% of mantle cell lymphomas, in 16–40% of multiple myelomas and in 60% of prostate cancers, suggesting that tumor suppressor(s) gene(s) at 13q14 are involved in the pathogenesis of these tumors (Dohner et al., 1999, Bigoni et al., 1997, Stilgenbauer et al., 1998, Desikan et al., 2000). Calin et al. (2002) have shown that two miRNA genes are located at 13q14.3 within a 30-kb region of minimal loss in CLL between two exons of the LEU2 gene. Both of these genes, miR-15a and miR-16-1, are down-regulated in more than 60% of CLL cases (detected using Northern blot analyses) (Calin et al., 2002). A very similar cluster (miR-15b, miR-16-2), but with a different promoter, was found on chromosome 3q25–26.1 (Lagos-Quintana et al., 2002). It seems that these miRNAs are less intensivelly expressed in normal cells (Calin et al., 2002), but may play a role in the cases of 13q14 deletions. Putative target of miR-16 is the arginyl-tRNA synthetase gene (RARS) because it has a homology of 85% on the 20-nt overlap and the levels of expression of the RARS gene correlate with the levels of expression of miR-16 (Calin et al., 2002). Another evidence about the target gene for miR-16 came during the studies of pituitary adenomas, where miR-15a and miR-16-1 are expressed at lower levels as compared to normal pituitary tissue and their expression inversely correlates with RARS expression (Bottoni et al., 2005). Cimmino et al. (2005) have demonstrated a different possible target for miR-15a and miR-16-1, whose expression is inversely correlated to BCL2 expression in CLL and they have uncovered that both miRNAs negatively regulate BCL2 at a post-transcriptional level. Moreover, in a leukemic cell line model BCL2 repression by these microRNAs induces apoptosis (Cimmino et al., 2005). Deregulation of antiapoptotic BCL2 in CLL cells seems to be a key event in cancerogenesis.

Recently, it became possible to analyze the entire miRNome by microarrays containing all known human miRNAs. The use of miRNA microarrays made possible to confirm miR-16 deregulation in human CLL and also recognize miRNA expression signatures associated with defined clinicopathologic features. miR-16-1 and miR-15a , which were previously reported to be down-regulated in the majority (68%) of CLL cases by Northern analysis, were found to be expressed at low levels in 45% (miR-16-1) and in 25% (miR-15a) of CLL samples (Calin et al., 2004b). These findings, that down-regulation of miR-16-1 and miR-15a expression correlates with allelic loss at 13q14, can be important for clinical classification of CLL. Patients with a normal karyotype or deletion of 13q14 as the sole genetic abnormality have a better prognosis than those with a complex karyotype or frequent deletion of 11q23 or 17p13 (Dohner et al., 2000; Oscier et al., 2002; Juliusson et al., 1990). Expression profiling of miRNAs in human B-CLL identified significant differences in miRNome expression between CLL samples and normal CD5+ B lymphocytes. At the top of list of differently expressed miRNAs are several miRNAs located exactly inside fragile sites. In some miRNA genomic clusters all members are aberrantly regulated. In others only some members were abnormally expressed such as the largest known miRNA cluster - miR-17-92 (Calin et al., 2004b) (see Tab. 4). Two miRNA expression clusters in CLL samples that associate with the presence (20% as a cutoff) or absence of Zap-70 expression (Calin et al., 2004b) could be identified. ZAP-70 is a tyrosine kinase and low level of its expression is a predictor associated with good prognosis (Wiesner et al., 2003; Orchard et al., 2004). Moreover, five differentially expressed miRNAs distinguish CLL samples that express unmutated IgVh locus from those that express mutated IgVh locus – a favorable prognostic factor (Calin et al., 2004b) (see Tab. 4). A signature composed of 13 microRNAs could well discriminate between a group of CLL samples that expresses ZAP-70 and unmutated IgVh (patients with worse prognosis) and the group that has no expression of ZAP and mutated IgVh (patients with better prognosis) (see Tab. 4). Furthermore, members of the 13-member prognostic signature can well differentiate patients with a short interval from diagnosis to initial treatment (treatment begins with the development of the symptomatic or progressive disease) from patients with a longer interval (Calin et al., 2005) (see Tab. 4). To summarize, the miRNA expression profile is associated with progression in CLL and can serve as a possible prognostic marker.

Following the initial finding about miR-15 and miR-16 in CLL, miRNA expression deregulation has been proven in other tumors. The analyses of lymphoma samples and cell lines showed that an elevation in the amount of miR-155/ BIC RNA occurs in a wide range of lymphomas derived from B cells. Increased miR-155 levels (2,000–10,000 copies per cell vs. 150 in normal circulating B cells) were observed in diffuse large B cell lymphoma (DLBCL), CLL, marginal zone lymphomas and in other non-Hodgkin and Hodgkin lymphomas (Eis et al., 2005). Thus, miR-155 may play a role in the pathogenesis of B cell lymphomas in general. In clinical isolates of DLBCL, higher levels of miR-155 were present in cells with the activated B cell phenotype (patients with worse prognosis) than in cells with the germinal center phenotype (patients with better prognosis). The levels of miR-155 (and BIC RNA) appear to correspond with clinically significant subtypes of DLBCLs and quantification of miR-155 levels may be a useful prognostic marker (Eis et al., 2005). Interestingly, Metzler et al. (2004) have demonstrated over-expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma, but Kluiver et al. (2005) have found a lack of BIC and miR-155 expression in this type of lymphoma.

Microarray-based expression studies have indicated another specific alterations in human miRNA expression profiles that correlate with B cell lymphomas. It was found that levels of the primary or mature microRNAs derived from the miR-17–92 locus are often substantially increased in these cancers (He et al., 2005) (see Tab. 4). The miR-17–92 cluster is located at 13q31.3, a genomic locus that is amplified in cases of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, primary cutaneous B-cell lymphoma and other tumour types (Ota et al., 2004; Knuutila et al., 1998). The mRr-17–92 cluster named „OncomiR-1“ is located at 13q31.3, a genomic locus that is amplified in cases of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, primary cutaneous B-cell lymphoma and several other tumour types (Ota et al., 2004; Knuutila et al., 1998). The transcript of this cluster appears to be the functional precursor of seven microRNAs: miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b-1 and miR-92-1 . Additionally, this cluster is related to the homologous miR-106a–92 cluster on chromosome X and the miR-106b–25 cluster on chromosome 7 (Tanzer and Stadler, 2004). Co-expression of the miR-17–92 cluster acted with c-myc expression to accelerate tumour development in a mouse B-cell lymphoma model (He et al., 2005). O‘Donnell et al. (2005) discovered that c-Myc negatively regulates the transcription of miR-17-92 cluster and binds directly the genomic locus encoding these miRNAs. Dysregulated expression or function of c-Myc is one of the most common abnormalities in human malignancy. They also showed that two miRNAs of miR-17-92 cluster (miR-17-5p and miR-20a) target an important proproliferative /proapoptotic transcription factor E2F1 (O‘Donnell et al., 2005). This cluster is also overexpressed in lung cancers, especially in the most agressive small-cell lung cancer and has an increased gene copy numbers in a fraction of lung cancer cell lines with his over-expression (Hayashita et al., 2005).

Another miRNA located at a site of rearrangement linked to human leukemia is miR-142, whose gene is at the breakpoint junction of a t(8;17) translocation, which causes an aggressive B cell leukemia due to up-regulation of a translocated c-MYC gene (Gauwerky et al., 1989; Lagos-Quintana et al., 2002). Chen et al. (2004) have studied the role of miRNAs in hematopoietic lineage differentiation (mouse model) and have found that miR-142 expression is higher in B-lymphoid and myeloid lineages compared to other hematopoetic tissues. They were also able to identify several miRNAs that are specifically expressed and dynamically regulated during early hematopoiesis (miR-181 and miR-223) (Chen et al., 2004).

Fig. 16 Possible “tumor suppressing” and “oncogenic miRNAs” (Mraz, 2006)

4.1.2. Colorectal Neoplasia

At the adenomatous and cancer stages of colorectal neoplasia mature miRNAs, miR-143 and miR-145, consistently display reduced levels in cells. Northern blot analyses have shown that these miRNAs are down-regulated also in cell lines derived from breast, prostate, cervical, and lymphoid cancers as well as colorectal tumors. Altered transcription occurs despite the maintenance of constant levels of unprocessed hairpin precursors in both normal and tumor tissues, suggesting that this reduction is due to posttranscriptional processes (Michael et al., 2003).

4.1.3. Breast Cancer

MicroRNAs are also aberrantly expressed in human breast cancer. Among the differentially expressed miRNAs, miR-10b, miR-125b, miR-145, miR-21and miR-155 emerged as the most consistently deregulated in breast cancer. Two of them, miR-21 and miR-155, were up-regulated (Iorio et al., 2005) and the remaining three were down-regulated. miR-125b gene is located at chromosome 11q23-24, one of the regions most frequently deleted in breast, ovarian, and lung tumors (Negrini et al., 1995, Rasio et al., 1995) and it appears to be putative homologue of lin-4 in C. elegans (Lee et al., 2005). It was possible to identify miRNAs whose expression was correlated with specific breast cancer biological features, such as tumor stage, vascular invasion or lymph node metastasis. miR-145 was progressively down-regulated from normal breast tissue to cancer with high proliferation index. Similarly, but in opposite direction, miR-21 was progressively up-regulated from normal breast tissue to cancers with high tumor stage. miR-9-3 was down-regulated in breast cancers with either high vascular invasion or presence of lymph node metastasis. The expression of various let-7 miRNAs was down-regulated in breast cancer samples with either lymph node metastasis or higher proliferation index (Iorio et al., 2005).

4.1.4. Lung Cancer

In lung cancer has been shown for the first time that alterations in the miRNA expression may have a direct prognostic impact. let-7 expression is frequently reduced in lung cancers and this is associated with decreased postoperative survival (Takamizawa et al. 2004). In another study, high miR-155 and low let-7a-2 expression correlated with poor survival of lung adenocarcinomas (Yanaihara et al., 2006). An in vitro experiment has demonstrated that over-expression of let-7 results in the inhibition of lung cancer cell growth (Takamizawa et al. 2004). Expression of this miRNA is lower in lung tumors than in normal lung tissue, while RAS protein is significantly higher in lung tumors, providing a possible mechanism for let-7 as a negative regulator of the RAS oncogene family (Johnson et al., 2005). Moreover, Dicer protein expression is reduced in a fraction of lung cancers with a prognostic impact on the survival of surgically treated patients (Karube et al., 2005).

4.1.5. Glioblastoma

The analysis of both glioblastoma tissues and glioblastoma cell lines enabled the identification a group of microRNAs whose expression is altered in this frequent malignant primary brain tumor. Two miRNAs, miR-221 (Ciafré et al., 2005) and miR-21 are strongly over-expressed in glioblastoma. miR-21, knockdown in cultured glioblastoma cells, triggers activation of caspases and leads to increased apoptotic cell death (Chan et al., 2005). This miRNA is also up-regulated in breast cancer (Iorio et al., 2005), suggesting that its gene target(s) belong(s) to the class of tumor suppressors. Additionaly, a group of brain-enriched miRNAs, miR-128, miR-181a, miR-181b, and miR-181c, were down-regulated in glioblastoma (Ciafré et al., 2005). Genes encoding microRNAs that were found to be modulated in glioblastoma, do not reside in chromosomal locations commonly deleted, amplified or rearranged in this type of brain tumor (Holland, 2001; Zhu and Parada, 2002). This is in contrast with other miRNAs whose possition is very often located in common regions of deletion or chromosomal rearrangements. The concurrent down-regulation of miR-181a, b and c occurs probably due to modulation of their expression rather than to rearrangements or deletions because they are located on three distinct chromosomes (Lim et al., 2003b; Weber 2005).

Tab. 4 MicroRNAs associated with cancer disease (Mraz, 2006)

4.2 MicroRNAs and Other Human Disease

Several human diseases have been pinpointed in which miRNAs or their processing might be implicated. Deletion or loss-of-function mutations of survival of motor neuron gene (SMN) cause spinal muscular atrophy, a paediatric neurodegenerative disease. Two proteins, Gemin 3 and Gemin 4 that are part of miRNPs are also a part of the SMN complex. It remains to be discovered whether miRNA biogenesis is deregulated in this disease (Dostie et al., 2003).

Ishizuka et al.(2002) showed the possibility that defects in an RNAi-related machinery may cause mental retardation. They showed that Drosophila homolog of human Fragile Mental Retardation protein, dFMR1, is part of the RNAi-related apparatus. Fragile X mental retardation (FXMR) is caused by the absence or mutation of Fragile Mental Retardation protein 1 (Siomi et al. 1993; Verheij et al. 1993).

. MicroRNA processing might be also involved in DiGeorge syndrome, the most common human genetic deletion syndrome. Deletion region at 22q11.2 include about 30 genes, one of them is DGCR8 component of microProcessor complex (Shiohama et al., 2003). Haploinsufficiency of this region accounts for over 90% of individuals with DiGeorge syndrome, a disorder that affects 1 in 3,000 live births and results in heterogeneous defects including heart defects, immunodeficiency, schizophrenia and many others (Antshel et al., 2005; Sullivan, 2004).

Interestingly, the gene locus of miR-224 is a candidate region for two neurologic diseases: early-onset parkinsonism (Waisman syndrome; Gregg et al. 1991) and X-linked mental retardation (MRX3; Gedeon et al. 1991).

Recently, it was shown that over-expression of miR-375 suppressed glucose-induced insulin secretion. miR-375 has a direct effect on insulin exocytosis through targeting myotrophin. Thus, miR-375 as a regulator of insulin secretion could be a novel pharmacological target (Poy et al., 2004).

MicroRNAs might also be involved in immune defence against viruses. A cellular microRNA, miR-32, can regulate proliferation of primate foamy virus infected cell culture (Lecellier et al., 2005). In addition, large DNA viruses of the herpesvirus family encode viral microRNA genes (Pfeffer et al., 2004; Sullivan et al., 2005a) (see chapter 4.3). The function of these viral microRNAs is currently not understood, but the small size of miRNA precursors makes them potentially ideal for use by viruses as inhibitors of host cell defense pathways.

4.3. MicroRNAs and Viruses

Recently, many viral-encoded miRNAs have been discovered and their functions have been documented or proposed for viral miRNAs from three different viral families - herpesviruses, polyomaviruses and retrovirus (Sullivan et al., 2005a). Approximately 40 miRNAs and 10 RNAi suppressors encoded by diverse mammalian viruses have been already identified (Li and Ding, 2005).

Human cytomegalovirus (HCMV) expresses miRNAs during its productive lytic infection of human cells (Dunn et al., 2005). Interestingly, sequences of the miRNAs expressed from cytomegalovirus genome were conserved among all HCMV strains examined and also in chimpanzee cytomegalovirus (Dunn et al., 2005). HCMV encodes multiple conserved miRNAs and suggests that human cytomegalovirus may utilize a miRNA strategy to regulate cellular and viral gene function (Grey et al., 2005).

Epstein-Barr virus (EBV), large DNA virus of the Herpesviridae family, preferentially infecting human B cells, expresses several miRNA genes. These miRNAs originated from five different double-stranded RNA precursors clustered in two regions of the EBV genome. Epstein-Barr virus uses RNA silencing as a method for gene regulation of host and viral genes in a non-immunogenic manner (Pfeffer et al., 2004).

The simian virus 40 (SV40) encodes miRNAs significant for viral infection. These miRNAs accumulate in late stages of the infection, are perfectly complementary to early viral mRNAs and target those mRNAs for cleavage. This reduces the expression of viral T antigens (Sullivan et al., 2005b).

Bennasser et al. (2002) found, using computer-directed analyses, that HIV-1 putatively encodes five candidate pre-miRNAs and suggested that a large number of cellular transcripts could potentially be targeted if these 5 pre-miRNAs were processed into 10 predicted mature miRNAs.

Kaposi's sarcoma-associated herpesvirus encodes 11 distinct miRNAs, all of which are expressed in infected cells (Cai et al., 2005).

Moreover, some oncogenic viruses use miRNA genes as a preferred integration site. For example, Human Papilloma Virus 16 (HPV16), associated with cervical cancer, integrates into miRNA genes at a rate 3 times higher than to the rest of the genome (Calin et al., 2004a).

These observations show some connections between miRNAs encoded by possibly oncogenic viruses (EBV, HPV16, SV40, HCMV) and cancer.

Conclusion

It is becoming clear that microRNAs can play a very important role in regulation of gene expression. They probably constitute as many as 1000 miRNA genes in human genome and have a specific microRNA signature in each normal or cancer cell type. MicroRNAs are expressed at high levels in animal cells and are dynamically regulated during cell differentiation, apoptosis, proliferation, development and metabolism. Surprisingly, recent studies have led to the identification of numerous small regulatory RNAs also in bacteria and viruses. Scientists begin to unterstand the importance of the gene regulatory networks operated by miRNAs. Understanding the basic mechanism of miRNA biogenesis is one of the central aims of molecular biologists for the future. Many additional factors involved in miRNA biogenesis remain to be identified. It is necesary to understand how miRNA biogenesis interfaces with other aspects of RNA metabolism and how the miRNA pathway is related to the other small-RNA pathways. Only for really few individual miRNAs the exact target ist known. Understanting of these topics is necesary also for optimizing the shRNA design in RNAi experiment. The biological role of miRNAs in animals, plants, viruses and bacteria remains a big guestion.

There has been demonstrated a possibility to use these microRNA signatures for a specific cancer classification with potential predictive and therapeutic value. MicroRNAs are aberrantly expressed in all studied cancer tissues, are located in cancer-associated genomic regions and their putative targets are very often tumor suppressors or oncogenes. For instance, miRNAs miR-17–92, miR-155, miR-21, whose expression is enhanced in tumors, might be considered as oncogenes and their targets as tumor suppressors. Under-expressed miRNAs, such as let-7, probably act as tumor-suppressor genes and their modulation more likely reflects the loss of differentiation typical for tumor cells (see Fig. 16). Substantial number of eukaryotic microRNAs has to be discovered and identification of their target genes is a big challenge for bioinformatics and molecular biologists because of their imperfect base-pairing with the target mRNA. The known data provide an evidence that microRNAs could disclose new ways for cancer diagnosis, prognosis estimation, and therapy.

References

other information about micrornas

Basic PowerPoint presentation about microRNAs

full text review about microRNAs: MicroRNA BIOGENESIS, FUNCTIONALITY AND CANCER RELEVANCE