**2. Biogenesis and function of miRNAs**

fications, making them important in the detection and treatment of multifactorial diseases

antagonist effects depending on whether or not they bind in the IRAP peptidase domain.

**Figure 1.** The renin-angiotensin system (RAS) and its components. This schematic depicts angiotensin ligands, receptors and the main enzymes involved; other peptidases and cathepsins also participate although to a lesser extent. All of the components of the RAS are present in the brain. RAS has two main axes: the pressor axis (tending towards an increase in blood pressure) comprising Ang II, ACE and AT1Rs and the counter-regulatory axis comprising Ang(1–7), ACE2 and MasR. Angiotensinogen is a substrate for renin to produce angiotensin I (Ang I), which is the inactive precursor of all angiotensin peptides. Conversion of Ang I to its most active ligand in the pressor axis, angiotensin II (Ang II), results from ACE-mediated hydrolysis [22]. Ang II is then sequentially converted to angiotensin III (Ang III) and angiotensin IV (Ang IV) by aminopeptidase A (APA) and aminopeptidase N (APN) respectively, which can be further cleaved by carboxypeptidase P (CP) and prolyl oligopeptidase (PO) to form angiotensin 3–7 (Ang3–7). Alternatively, Ang II can be converted, via the counter-regulatory axis to angiotensin 1–7 (Ang1–7) by carboxypeptidase P (CP) or ACE2, while both angiotensin A and Ang1–7 can be converted to alamandine by an ACE-mediated decarboxylation reaction [22–27]. Notably, angiotensin ligands acting on AT4R (also called insulin-regulated aminopeptidase (IRAP)) can have agonist or

Some of those epigenetic modifications are mediated by miRNAs, defined as single-stranded, non-coding RNA sequences approximately 21–23 nucleotides in length, expressed under physiological and pathological conditions [11, 12]. Deletion of complexes involved in miRNA biogenesis resulted in deleterious and non-viable phenotypes, highlighting their necessary involvement in the cellular development and differentiation [13, 14]. To date, 28,645 miRNAs have been reported in miRbase, a widely used resource for miRNA cataloging and nomenclature [15]. As epigenetic regulators of gene expression, functions of miRNAs include RNA degradation, inhibition of protein expression, regulation of methylation and histone modification on DNA [12, 14, 16]. miRNAs perform these functions by complementary base pairing

such as hypertension [7, 10].

174 Renin-Angiotensin System - Past, Present and Future

Canonical miRNA biogenesis starts with transcription of the primary miRNA sequence by RNA polymerase II and III [14]. Approximately half (52%) of human miRNAs are located in intergenic regions, 40% in intronic and 8% in exonic [28]. Intergenic miRNAs are independently expressed through promoter elements; yet related miRNAs that often have overlapping targets can be located on different chromosomes and expressed under different conditions. Intronic and exonic miRNAs that are clustered within 50 kilobases from each other show similar expression, while those spaced further apart tend not to [29]. However, there are some exceptions. Some miRNAs separated by more than 50 kilobases retain high correlation, likely as a result of co-expression [30]. The differential localization and expression of miRNAs suggest an evolutionary response to environmental insults and specific cell responses, a theory supported by observed higher numbers of miRNAs expressed in organisms of higher complexity [31–33].

**Figure 2A–F** summarizes the process of miRNA biogenesis. Primary miRNAs are cleaved in the nucleus by a nuclear microprocessor complex comprised of the RNase III endoribonuclease DROSHA and its double-stranded RNA-binding protein DGCR8—DiGeorge Critical

**Figure 2.** MicroRNA biogenesis and function. (A) Primary miRNAs are cleaved in the nucleus by the RNase III endoribonuclease DROSHA and DGCR8. (B) Once the primary miRNA is cleaved, the nuclear transport receptor exportin 5 binds the 3′ overhang structure of the pre-miRNA to export it to the cytoplasm. (C) The RNase III enzyme Dicer and TRBP and PACT target the pre-miRNA through the 3′ overhang, converting it into mature miRNA, liberating a duplex nucleotide structure with two nucleotides protruding at the 3′ end. (D) The guide strand is loaded into the RNAinduced silencing complex (RISC), and the passenger strand is degraded by RNases. (E) Complementary pairing with the seed region to mRNAs determines target binding and guides argonaute proteins to stop translation. Accumulation of untranslated mRNA in the cytoplasm allows recruitment of members of the GW182 protein family. (F) Deadenylase complexes cause destabilization of the transcript and further degradation by RNase activity.

Region 8, **Figure 2A** [34]. This cleavage by DROSHA/DGCR8 produces a 60 nucleotide stemloop structure with a 3′ overhang, the pre-miRNA [11, 34, 35]. The primary miRNA can also be further subjected to RNA editing by ADARs (adenosine deaminases acting on RNA) that modify adenosine to inosine producing miRNA isoforms called isomiRs [36].

Exportin 5 allows export of the pre-miRNA to the cytoplasm, **Figure 2B** [36], where Dicer and substrate stabilizing binding partners, TRBP (trans-activation response RNA-binding protein) and PACT (protein activator of RNA-activated protein kinase) facilitate conversion into mature miRNA, **Figure 2C** [12, 14]. Two strands result from the unwinding of the duplex, the guide (3p) and passenger (5p) strands. Most of miRNA effects are mediated by the 3′ form; the 5′ form comprises <10% of all miRNA reads in humans [36]. The guide strand is loaded into the RNA-induced silencing complex (RISC) and the passenger strand is degraded by RNases, **Figure 2D**. IsomiRs can also be produced at this step by trimming and capping of the mature miRNA.

Non-canonical miRNA biogenesis is independent of DROSHA/DGCR8 processing in the nucleus. Such biogenesis arises if an intron is spliced lacking the sequences ordinarily flanking the stem region of a primary miRNA and it is of sufficient size to generate a pre-miRNA and it can be exported to the cytoplasm and further processed as a pre-miRNA to form a mirtron. Alongside mirtrons, other RNA sequences derived from transfer RNA and small nucleolar RNA are loaded into an RISC complex and act as miRNAs [13, 14, 29].

The RISC is a ribonucleoprotein complex that mediates mRNA degradation, destabilization or translational inhibition, whatever the biogenesis mechanism and comprises the miRNA guide strand and argonaute proteins, **Figure 2E**. The complementary base pairing of the miRNA seed region (2nd to 8th position on the 5′ end) to mRNAs determines target binding and guides argonaute proteins [28, 37, 38]. miRNA levels are dependent on argonaute proteins [39, 40] that are also present in the nucleus and currently, only miRNA-29b has been shown to translocate and localize in the nucleus [14, 39, 41]. In humans, argonaute 2 (also called eukaryotic translation initiation factor 2C) cleaves target mRNAs [29] but can also block other translation initiator factors and ribosomal subunits [42].

After pairing of the miRNA seed region, protein translation can be inhibited, **Figure 2E**. Accumulation of untranslated mRNA in the cytoplasm allows argonaute 2 to recruit members of the GW182 protein family, which are enriched in cytoplasmic areas called processing bodies (p-bodies) [42]. Here, the mRNA is destabilized by deadenylase complexes and further degraded by RNases [43–45]. Finally, the effect miRNAs have on protein or mRNA levels depends on the position where the miRNA binds and five different classes of miRNA binding have been determined [12, 46, 47]. Most miRNA effects are mediated by binding at the 3′ UTR of mRNA and further processing as described previously, non-canonical binding sites represent <1% [48].
