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MicroRNA are endogenous molecules between 16-24 nucleotides (nt) long that post- transcriptionally regulate messenger RNAs (mRNAs) by targeting them for cleavage or translational repression164. In the past decade or so, these molecules have gained significant

attention in the research community due to the important regulatory roles they have been found to play164. In fact, it is thought that approximately 1% of the human genome consists of miRNA genes165, though many of their functional roles still remain undiscovered. It is estimated that there are over 700 miRNAs encoded in the human genome, which are involved in regulating around one-third of all human genes166-168. Furthermore, their specific functions may be highly conserved across many species, with the majority of miRNA evolutionarily conserved in closely related species169. In this section, I will describe the discovery of miRNA in animal cells, the biogenesis of miRNA, and its mode of action.

1.4.1. Discovery of microRNA

MicroRNA were originally discovered in animals by Victor Ambros and colleagues118,170-172, when they found in the C. elegans species, that lin-4, a gene known to control the timing of larval development, did not code for a protein, rather it produced a pair of small RNAs, one 22 nt in length and the other 61 nt118. It was predicted that the longer of the two, folded into a stem loop and acted as the precursor to the smaller of the small RNAs. Furthermore, it was noticed that these two RNAs possessed antisense complementarity to sites in the 3’untranslated region (3’UTR) of the lin-14 gene. The importance of this regulation of lin-14 by the small lin-4 RNA was demonstrated by the significant reduction in the amount of LIN- 14 protein was seen without changes in the levels of lin-14 mRNA. The 22 nt length lin-14 small RNA is now recognised as the founding member of the regulatory miRNAs118,172.

1.4.2. Biogenesis of microRNA

The biogenesis and action of miRNA is illustrated in Figure 1.4. MiRNA are transcribed by RNA polymerase II (Pol II), which often binds to a promoter found near the DNA sequence and encoding the hairpin loop of the pre-miRNA. This transcript will then be capped with a

Figure 1.4. Simplified schematic of miRNA biogenesis and action. MiRNA biogenesis begins in the cell nucleus as a pri-miRNA with characteristic hairpin loops still present in the pre-miRNA form. The pre-miRNA is then transported into the cytoplasm before the hairpin loop is cleaved to form a duplex miRNA:miRNA* double stranded complex. Only one strand of the miRNA eventuates into the mature form where it is incorporated into RISC to target mRNA for translational repression of mRNA degradation.

specially modified nucleotide at the 5’ end and spliced. Mammalian miRNA are transcribed as part of one arm of an RNA stem-loop that is approximately 80 nt long, which in turn forms a part of a several hundred nucleotide-long hairpin loop, miRNA precursor structures called the primary miRNA (pri-miRNA)173. These pri-miRNA may contain as many as six miRNA precursors and the hairpins are flanked by sequences necessary for efficient nuclear processing. These double stranded hairpin pri-miRNA are first recognised by a nuclear protein, DiGeorge Syndrome Critical Region 8 (DGCR8). The enzyme Drosha, then associates with DGCR8 to form the RNase III endonuclease which mediates the cleavage of the pri-miRNA to form a miRNA precursor known as the pre-miRNA, approximately 60-70 nucleotides long174. The cleavage occurs at sites near the base of the primary stem loop and leaves the base of the pre-miRNA stem loop with a 5’ phosphate end and approximately a two nucleotide overhang at the 3’ end174,175. The pre-miRNA is then transported from the nucleus to the cytoplasm by nucleocystoplasmic shuttler Exportin-5 that recognises the two- nucleotide overhang at the 3’ end of the hairpin.

Once in the cytoplasm, another cleavage step occurs with RNase III enzyme, Dicer174,176-178. It recognises the double-stranded portion of the pre-miRNA and cuts both strands of the duplex at about two helical turns away from the base of the stem loop. This process results in the cleavage of the terminal base pairs and loop of the pre-miRNA, leaving the 5’ phosphate and two nucleotide overhang at the 3’ end. This results in an imperfect duplex made up of the mature miRNA and a similar-sized fragment known as the passenger strand, or miRNA*179. Technically, either strand of the duplex can act as a functional miRNA but only one strand is usually incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC), where the mature miRNA can then interact with its respective target mRNA.

1.4.3. Mode of miRNA action

Although either strand of the miRNA:miRNA* duplex can be loaded into the RISC180-185, normally the miRNA is incorporated, while the miRNA* was originally though to peel away and be degraded (Figure 1.4). However, it has been shown now that either strand can be functional186 with the nomenclature of miRNA:miRNA* changing to miRNA-5p:miRNA-3p. Which strand of the miRNA duplex is loaded into the RISC is determined by the relative stability of the two ends of the duplex. It has been found that the strand whose 5’ end is less tightly paired is nearly always the one loaded into the RISC187,188.

The RISC complex is made up of a number of proteins with members of the Argonaute (AGO) protein family being central to its function178,182,183. Every RISC contains at least one member of the AGO protein family. AGO proteins contain two RNA binding domains, in a bi-lobed structure that is largely conserved and vital for miRNA-induced silencing. Firstly, there is the N-terminal lobe containing the PIWI, AGO, and Zwille (PAZ) domain that can bind the single stranded 3’ end of mature miRNA. Secondly, there is the C-terminal lobe containing the middle domain, which binds to the 5’-phosphate end of the guide strand, and the PIWI domain which can bind the 5’-phosphate end of the guide strand via coordination of a divalent cation164. AGO proteins bind to the mature miRNA strand incorporated in the RISC and orients it in a conformation to facilitate mRNA target recognition164.

Once incorporated with a miRNA, the RISC has two possible gene repressing processes. The RISC can either work at the level of protein synthesis by repressing ribosomal translation, or at the transcript level and degrade the mRNA target, the latter of which is only known to occur in plants164. The miRNAs bind complementary Watson-Crick base pairs in primarily the 3’UTR region of the target mRNA. This base pairing between the miRNA and mRNA

chiefly occurs within a short region spanning nucleotides 2-8 known as the “seed region.” In fact, as little as 6 base pair (bp) of complementarity is sufficient for a mature miRNA to effectively silence its target mRNA. This is what allows a single miRNA sequence to have up to 200 different mRNA targets in its arsenal.