MicroRNA and microRNA Clusters in the Aging Heart Figure 16. Schematic representation of a model of miRNA expression, which is regulated by transcriptional regulation and other factors, such as Ago proteins. The major change in expression of the miRNA biogenesis components occurred during later adulthood. miRs: both miR and miR*. Pri-miRNAs: the primary miRNA transcripts. Agos: the Argonaute proteins. Month: the mouse age (from 1–24 months in the present study). Adult Aging is a process that spans many decades in human beings and, similarly, many months in rodents. To translate human aging with a much longer life span into rodent years may be fraught with complexities. However, in general, adult aging might be arbitrarily divided into an earlier phase (humans between approximately 20 and 39 years and in C57BL6 mouse strain between 4 and 10 mo), a middle phase (humans between approximately 40 and 59 years and in C57BL6 between approximately 11 and 22 mo), and a later phase (humans between 60 and 79 years and in C57BL6 between 23 and 35 mo). Senescence (the age at which 50% of the populations has died) occurs in humans at around 79–80 yrs, and in C57BL6 mice at around 35–36 months. Of course, with the progressively changing demographics and aging of the population, these arbitrary divisions of what might be considered ‘‘early’’ or ‘‘late’’ aging will likely undergo revision and change. doi:10.1371/journal.pone.0034688.g016 derived from pri-miRNAs expressed from intergenic regions where a stretch of DNA sequences located in the chromosomal region referred as junk DNA region that contain few or no protein-coding genes [59,60]. It is generally believed that intragenic miRNAs are co-transcribed with their host genes, while most intergenic miRNAs are transcribed from their own RNA polymerase II (Pol II) promoter. However, recent studies indicate that as much as 26% of the intragenic miRNAs may be transcribed from their own unique promoters even though they are physically located within protein coding genes. The miRNA gene promoters have similar features to those of protein coding genes, but miRNA transcript organization is more complex [60]. In the present study, we observed that the expression of pri- miRNA levels change during adult aging, which correlates with the level of miRNA, suggesting that transcription regulation likely impacts miRNA biogenesis. Argonaute proteins are key effectors of small RNA-mediated regulatory pathways that modulate gene expression and regulate various cellular functions, and are essential for development, differentiation and maturation [61,62]. The structure of Ago proteins is well conserved, consisting of an amino-terminal domain, the MID domain, and their signature PAZ and Piwi domains [61,62]. Argonaute proteins form the core of RNA- induced silencing complexes (RISCs). Mice and humans generate four Ago proteins, but only Ago2 appears to retain the ability to cleave RNA targets. Whether the functions of Ago1, Ago3, and Ago4 might differ is not clear [62,63]. Ago1 and Ago2 are the two proteins that have been more extensively studied. Ago1 selectively binds to microRNAs, while Ago2 binds to both microRNAs and siRNAs [15]. MicroRNA duplexes are intrinsically asymmetric, with the guide strand preferentially entering Ago1-RISC complex, while the miR* strand is usually loaded as functional species into Ago2-RISC complex [15]. It has been shown that the miR* strand can silence its messenger RNA targets both in vitro and in-vivo. Therefore, each microRNA precursor can potentially produce two mature strands (miR and miR*) that are differentially sorted into Ago1-mediated or Ago2-mediated RNA interference (RNAi) pathways, respectively. In addition to being key components in the RISC complex that are responsible for gene silencing, Ago proteins also play a role in maintaining a proper level of mature miRNA strands, which was initially revealed by gene targeting against Ago1 and Ago2 genes. When Ago1 was depleted, a significant reduction in miR strands and an unexpected concomitant increase in the levels of target messenger RNAs were observed. When Ago2 was depleted, a reduction of endogenous siRNA and miR* levels was observed, however, the miR strand levels were unaffected. Recent studies further indicate that Ago proteins stabilize miRNAs, either within PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e34688