1.1. What are micro RNAs?
Micro RNAs (miRNA) are small non-coding RNAs that are involved in regulating the translation of messenger RNA (mRNA). Over 1000 miRNAs have been identified which control approximately 60% of the protein coding genes1. The miRNAs are on average 23 nucleotides long, with nucleotides 2-7 acting as the seed region. The seed region is needed for specific mRNA interactions and mutations occur in this region it can disrupt the miRNA, mRNA Watson-Crick base pairing2.
mRNA is stabilized in the cytoplasm by adding a 5’ cap and a 3’ poly adenine tail, which prevents degradation by ribonucleases. The binding of miRNA can cause 3 events to occur; deadenylation, decapping, and 5’ to 3’ degradation1. Often in the 3’ untranslated region (UTR) there are AU rich elements (AREs) when the miRNA along with Argonaute 1, Argonaute 2 and Dicer1 is bound it allows rapid decay of the mRNA
1.2. miRNA biogenesis
miRNAs can be transcribed from their own genes; often there are clusters of these miRNA genes for example, C19MC, which is the largest human cluster3. Another place where miRNAs are coded for is within introns of other genes. The miRNAs are transcribed using either RNA polymerase II or III4,5, this is dependent on the specific promoter or terminator sequences for each gene. The new transcript is called the primary miRNA (pri-miRNA) and must undergo processing before it is able to regulate mRNA.
While in the nucleus the pri-miRNA undergoes the first cleavage, which is catalysed by Drosha, figure 1. Drosha is part of the RNase III family of enzymes and cleaves the transcript 11 base pairs from the double stranded/single stranded RNA junction3. For the cleavage to be accurate a molecular ruler in the form of Pasha (partner of Drosha) is used, this gives a pre-miRNA of approximately 70 nucleotides long6.
The exception to this rule is miRNA encoded in introns (mitrons), these are not cleaved but are instead processed using splicing from the immature mRNA transcript7.
After processing by Drosha, the pre-miRNA is then exported from the nucleus via Exportin5 (Exp5) which has Ran-GTP bound as co-factor. The binding of pre-miRNA is not a sequence specific event, the size of the double stranded stem and 3’ overhangs is the deciding factor to whether to transport the pre-miRNA into the cytoplasm3. This interaction with Exp5 protects the pre-miRNA from degradation, as when knocked out pre-miRNA levels do not accumulate in the nucleus3,8.
Once in the cytoplasm the pre-miRNA can then undergo the final cleavage step before becoming a mature miRNA. The RNase III Dicer cleaves the terminal loop of the pre-miRNA to leave a duplex, which is approximately 22 nucleotides in length3. Dicer is able to cleave without assistance, however the binding of TRBP (transactivating response RNA-binding protein) is thought to increase stability and improve the reactions efficiency9. In humans there is only one copy of a Dicer like protein,...