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 (Altuvia et al., 2005). The hairpin loops have >40 nucleotide flanking RNA sequences necessary for efficient processing (Zeng and Cullen, 2005). These are recognised by the DiGeorge Syndrome Critical Region 8 (DGCR8), the cofactor to drosha, (Han et al., 2006). 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 (Han et al., 2004). This cleavage occurs around 11 nucleotides from their base (2 helical RNA turns into the stem) (Zeng et al., 2005) by Drosha, a RNAse III type dsRNA specific endonuclease, to form pre-miRNA (Lee et al., 2003)(47,48,49,50). Together, drosha and DGCR8 (the invertebrate equivalent is Pasha (Landthaler et al., 2004)) form the microprocessor complex (Denli et al., 2004; Gregory et al., 2004). 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 (Denli et al., 2004) to produce a pre-miRNA of ~ 70 nt in length (Denli et al., 2004; Gregory et al., 2004; Han et al., 2004; Han et al., 2006; Landthaler et al., 2004; Lee et al., 2003; Zeng and Cullen, 2005; Zeng et al., 2005). 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 (Okamura et al., 2007; Ruby et al., 2007). 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 (Berezikov et al., 2007).
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 (Valente and Nishikura, 2005). RNA editing has been shown to occur in 6% of miRNAs (Blow et al., 2006), 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 (Kawahara et al., 2007).
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 (Yang et al., 2006). 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 (Ohman, 2007).
The nuclear membrane protein exportin 5 recognises the 2 nucleotide overhang on the 3 end of the pre-miRNA (Zeng and Cullen, 2004) and then transports it into the cytoplasm using ran-guanine triphosphatase (Ran-GTP) (Bohnsack et al., 2004) (Yi et al., 2003) (Yi et al., 2003).
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 (Bernstein et al., 2001; Hutvagner et al., 2001; Ketting et al., 2001). Dicer cleavage of pre-miRNA results in an imperfect miRNA:miRNA duplex around 20-25 nucleotides in size (Hutvagner et al., 2001; Ketting et al., 2001) containing the mature miRNA strand and its opposite complementary miRNA strand (Lim et al., 2003)(Schwarz et al., 2003). Dicer is associated with the cofactors immunodeficiency virus (HIV) transactivating response RNA binding protein (TRBP)(Chendrimada et al., 2005; Haase et al., 2005) and protein activator of the interferon induced protein kinase PACT (Lee et al., 2006) that physically bring the TRBP-PACT-dicer complex into contact with Ago2 (Kok et al., 2007) 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 (Maniataki and Mourelatos, 2005). The mature miRNA then guides the RISC to target sites in order to induce silencing (Gregory et al., 2005; Martinez et al., 2002). The precise sequence of events is difficult to elucidate and still under debate (MacRae et al., 2008).
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 (Krol et al., 2004)(Khvorova et al., 2003; Schwarz et al., 2003), though the position of the stem-loop within the pre-miRNA has been implicated (Lin et al., 2005). The other strand, called the passenger strand due to its lower levels in the steady state, is denoted by miRNA (Lau et al., 2001). 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 (Matranga et al., 2005) (Rand et al., 2005). 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 (Okamura et al., 2008). 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 (Plante and Provost, 2006).
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) (Sasaki et al., 2003; Sharma et al., 2001). 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 (Hammond et al., 2001) (Meister et al., 2004). 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 (Lingel and Sattler, 2005). 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 (Meister et al., 2004).
There are other proteins found in the miRISC, also referred to as the miRNP complex (Mourelatos et al., 2002), that are associated with the AGOs but as yet not fully characterised (94) and are thought to modulate the silencing effects of the miRISC (MacRae et al., 2008). They are covered in detail in other excellent review papers (MacRae et al., 2008; Mourelatos et al., 2002), though I include here a few that have been mentioned and will be discussed later on in more detail: the SMN complex (Mourelatos et al., 2002), fragile X mental retardation protein (FMRP) (Caudy et al., 2002; O'Donnell and Warren, 2002), tudor staphylococcal nuclease-domain-containing protein (tudor-SN) (Yang et al., 2006).
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