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RNAs can be broadly classified into two groups: coding RNAs, which code for proteins and are known as messenger RNAs (mRNAs); and non-coding RNAs (ncRNAs), which comprise the largest class of RNAs and include ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), long non-coding RNAs (lncRNAs, > 200 nucleotides) and small non-coding RNAs (< 200 nucleotides). miRNAs constitute the best studied class of small non-coding RNAs and act post-transcriptionally to regulate gene expression (Ebert and Sharp 2012). Mature miRNAs occur as single-stranded RNAs, are typically between 21 and 24 nucleotides in length, and are estimated to regulate about 50% of all mammalian protein-coding genes (Krol et al. 2010).

The biogenesis of miRNA has been comprehensively reviewed (Fabian and Sonenberg 2012; Ha and Kim 2014; Hammond 2015; Libri et al. 2013) and is shown diagrammatically in Figure. 4.2. The majority of miRNAs are encoded within introns or exons of non-coding RNAs or introns of pre-mRNA (Saliminejad et al. 2019). In the canonical pathway, miRNAs are transcribed by RNA polymerase II (pol II) into large primary miRNAs (pri-miRNAs); these contain embedded stem loop structures that are roughly 70 nucleotides long. Pri-miRNAs are cleaved within the nucleus by the microprocessor complex to form stem-loop structures called precursor miRNAs (pre-miRNAs). Pre-miRNAs are exported into the cytoplasm by Exportin 5 (XPO5) and are cleaved by Dicer to form a miRNA duplex (see Figure 4.2). miRNA duplexes are then loaded onto Argonaute protein (Ago), which stimulates the assembly of the RNA-induced silencing complex (RISC) (Ha and Kim 2014). The RISC facilitates the removal of the passenger strand RNA in order to generate mature miRNA (Michlewski and Caceres 2019).

Figure 4.2 Simplified overview of miRNA biogenesis and release of miRNA into the circulation. In the canonical pathway, miRNAs encoded within genes are transcribed by RNA Polymerase II (Pol II Transcription) into large primary miRNAs. Primary miRNAs are cleaved within the nucleus by the Drosha Microprocessing Complex (Drosha Complex Processing), which gives rise to precursor miRNAs. Precursor miRNAs are exported to the cytoplasm by Exportin 5. They are next cleaved by Dicer, followed by loading into RISC. The RISC facilitates removal of the passenger strand RNA to generate mature miRNA. Mature miRNAs can be actively secreted into circulation in association with RNA-binding carrier proteins (AGO2/HDL/LDL), or by packaging into microvesicles/exosomes (not shown). Alternatively, mature miRNA targets cognate mRNA in the cytoplasm through complimentary base pairing, which leads to regulation of gene expression.

Mature miRNAs regulate their target mRNAs by binding to 3ʹ untranslated regions (3ʹUTR) of mRNAs; however, miRNA binding sequences have also been identified within 5ʹ UTRs (Friedman et al. 2009). Mature miRNAs are guided to their cytosolic target mRNAs by complementary base pairing, and miRNA targeting is reliant on base pairing of the miRNA seed region, nucleotides 2-7, to mRNA targets. Perfect miRNA-mRNA base pairing leads to the degradation of the target mRNA, whereas imperfect base pairing leads to decreased mRNA translation, which can be restored once the repressing miRNA is degraded. Intriguingly, it has also been shown that miRNA-mRNA interaction with the 5ʹ UTR can increase the translation of the mRNA, and that some miRNAs can target promoters or enhancers (Fabbri 2018; Odame et al. 2021; Vasudevan et al. 2007). Biological functions of separate miRNAs have been extensively studied using a variety of miRNA-knockout/knockdown models and transgenic overexpression experiments (Hammond 2015; Saliminejad et al. 2019).