Impact of age-associated increase in 2′-O-methylation of miRNAs on aging and neurodegeneration in Drosophila

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) small RNAs that regulate gene expression post-transcriptionally through translational repression and/or mRNA cleavage. Starting from the discovery of the first miRNA lin-4 in Caenorhabditis elegans, hundreds of miRNAs have now been identified in various species. miRNAs were classically studied for their role in developmental timing (Lee et al. 1993; Reinhart et al. 2000; Bartel 2004). By now, functional analyses have identified some miRNAs, such as lin-4 in C. elegans (Boehm and Slack 2005; Kenyon 2010; Smith-Vikos and Slack 2012) and miR-34 in Drosophila (Liu et al. 2012), as miRNAs with critical roles in organismal and brain aging, respectively. Although most studies have focused on changes in the abundance of miRNAs with age (Ibanez-Ventoso et al. 2006; de Lencastre et al. 2010; Kato et al. 2011), evidence suggests that miRNAs also show heterogeneity in length and sequence in different cell types and biological contexts (Burroughs et al. 2010; Marti et al. 2010; Li et al. 2012; Westholm et al. 2012; Llorens et al. 2013). Such heterogeneity is mediated by imprecise Drosha and Dicer cleavages (Calabrese et al. 2007; Azuma-Mukai et al. 2008; Wyman et al. 2011), 3′ end adenylation/uridylation (Ruby et al. 2006; Landgraf et al. 2007; Burroughs et al. 2010; Wyman et al. 2011), and RNA-editing events (Kawahara et al. 2007; Nishikura 2010; Alon et al. 2012; Ekdahl et al. 2012). Importantly, effects of this heterogeneity on miRNA target silencing are also reported (Azuma-Mukai et al. 2008; Seitz et al. 2008; Fukunaga et al. 2012; Lee and Doudna 2012), suggesting a functional impact of regulating miRNA heterogeneity in animals. Our study on the role of miR-34 in aging and age-associated neurodegeneration in Drosophila revealed an intriguing pattern of miR-34 isoforms: Although multiple-length isoforms of miR-34 are generated, only the short isoform accumulates with age (Liu et al. 2012). Furthermore, the generation of isoforms of miR-34 requires 3′ end trimming by a novel 3′-to-5′ exonuclease, Nibbler (Nbr) (Han et al. 2011; Liu et al. 2011), highlighting the potential importance of regulation of miRNA length in age-associated processes. In addition to length and sequence heterogeneity, the 2′-OH of the 3′ terminal ribose of miRNAs can be modified by 2′-O-methylation (Zhao et al. 2012a). In plants, this modification occurs on nearly all miRNAs and siRNAs (Yu et al. 2005; Zhao et al. 2012a,b) and protects the small RNAs from HESO1-mediated uridylation and degradation (Li et al. 2005; Yu et al. 2005; Ren et al. 2012; Zhao et al. 2012b). In Drosophila, most miRNAs are loaded into Ago1 and remain unmodified (Hutvagner et al. 2001; Okamura et al. 2004; Vagin et al. 2006). However, a subset of miRNAs are found to be 2′-O-methylated (Forstemann et al. 2007; Horwich et al. 2007; Ghildiyal et al. 2008; Czech et al. 2009; Okamura et al. 2009). Importantly, this modification is also protective in animals: Loss of 2′-O-methylation of small RNAs leads to destabilization as well as tailing and trimming of the small RNAs (Kurth and Mochizuki 2009; Ameres et al. 2010; Kamminga et al. 2010). In Drosophila, 2′-O-methylation of small RNAs is associated with loading of the miRNAs into different Ago complexes: While most miRNAs are loaded into Ago1 (Ago1-RISC) and remain unmodified, those loaded into Ago2 (Ago2-RISC) are 2′-O-methylated (Czech et al. 2009; Okamura et al. 2009; Ghildiyal et al. 2010). The miRNA/miRNA* duplex structure and 5′ nucleotide preference are two suggested mechanisms by which miRNAs are directed into the two distinct Ago complexes (Forstemann et al. 2007; Tomari et al. 2007; Okamura et al. 2009; Ghildiyal et al. 2010). miRNAs loaded into Ago2 are also active in translational silencing of the target genes, like miRNAs loaded into Ago1, but by a potentially different mechanism and with different silencing efficiency (Czech et al. 2009; Iwasaki et al. 2009; Okamura et al. 2009). However, despite the discovery of functional Ago2-loaded and 2′-O-methylated miRNAs in Drosophila, whether such loading of miRNAs to Ago2 is biologically regulated or biologically important in vivo is not yet clear. Here, we pursued the study of several Nbr-dependent miRNAs that, like miR-34, show multiple isoforms. Examination of changes in their isoform patterns with age unexpectedly revealed a diversity of patterns. The age-associated increase of some Drosophila miRNAs reflected increased 2′-O-methylation of select isoforms. Associated with this increase, we found increased loading of specific miRNA isoforms into Ago2, but not Ago1, with age. Furthermore, the lack of 2′-O-methylation by Hen1 and Ago2 mutations resulted in reduced life span and brain degeneration, raising the possibility that the increased protection of small RNAs with age may impact age-associated events.