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processing of 5’ termini of RNAs produced by phage polymerases when such RNAs are used in mammalian cells. It is not clear how PKR and RIG-I pathways are integrated. RIG-I binds siRNAs (with or without 2-nt 3’ overhangs) in vitro and it shows greater unwinding of blunt-ended siRNAs. Unwinding is then translated into the interferon activation mediated via IRF-3. Toll-like Receptor 3 (TLR3) TLR3 is a member of the Toll-like receptor (TLR) family and functions as a sensor of extracellular, intracellular and viral dsRNAs (Amarante et al., 2011; Seo et al., 2013; Wang et al., 2015b; Wu et al., 2015b; Yang et al., 2006b). TLR3 has distinct or complementary roles to RIG-I and related helicases in sensing foreign molecules and activating downstream responses (Livengood et al., 2007; McCartney et al., 2009; Slater et al., 2010; Wu et al., 2015b). Oligoadenylate Synthetase (OAS) Interferon and dsRNA also activate 2’,5’-oligoadenylate synthetase (2’,5’-OAS) that produces 2’,5’ oligoadenylates with 5’-terminal triphosphate residues that subsequently induce activation of RNAse L; a protein responsible for general RNA degradation (de Veer et al., 2005). TARBP2 and PACT Interactions between RNAi, miRNA, and interferon response are poorly understood. There are two clear mechanistic connections between these two pathways. First, TARBP2 and PACT, two dsRNA binding proteins, which were mentioned earlier as Dicer-interacting proteins, interact also with PKR. Notably, while TARBP2 inhibits PKR (Cosentino et al., 1995; Park et al., 1994), PACT has the opposite role (Patel and Sen, 1998). While cytoplasmic long dsRNA in somatic cells apparently triggers the interferon response, it is not clear if the same dsRNA is also routed into the RNAi pathways. Experiments in oocytes and undifferentiated embryonic stem cells (Stein et al., 2005; Yang et al., 2001) suggest that RNAi dominates response to cytoplasmic long dsRNA in the absence of a strong interferon response and that the interferon pathway dominates when its relevant components are present. On the other hand, this view may be too simplistic as it does not explain the lack of both, RNAi and interferon response, in somatic cells expressing long dsRNA (Nejepinska et al., 2012; Nejepinska et al., 2014). In any case, understanding the role of TARBP2 and PACT isoforms in routing long dsRNA into RNAi and interferon pathways requires further studies. There is a clear evolutionary connection between RNAi and interferon response. The above-mentioned mammalian RNA helicases RIG-I, LGP2 and MDA5 are the closest homologs of helicases involved in processing of long dsRNA during RNAi in C. elegans. Notably, RIG-I is an established component of the interferon response to long dsRNA (Yoneyama et al., 2004). This suggests that the interferon response, which has a common
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trigger and evolved after the RNAi pathway, adopted several components from the latter pathway. It remains to be determined whether these and other components of RNAi lost their function in RNAi entirely or mediate some form of a cross-talk between RNAi and interferon response. Finally, there is also a complex relationship between miRNA and interferon pathways (Ingle et al., 2015; Ostermann et al., 2012; Shapiro et al., 2014; Xu et al., 2011). One connection is exemplified by viral miRNAs, which viruses use to regulate the host response, in particular factors of the interferon pathway (Ostermann et al., 2012) or other cellular signalling (Xu et al., 2011). However endogenous cellular miRNAs may also act to suppress the interferon response factors, such as the case mir-485, which has a dual role in targeting RIG-I as well as the influenza virus H5N1 (Ingle et al., 2015). Other dsRNA-associated mechanisms II – Adenosine deamination A-to-I editing is a covalent RNA modification system of broad significance (reviewed in Nishikura, 2016). It is mediated by adenosine deaminases acting on RNA (ADARs), enzymes that carry two or three dsRBD and recognize both inter- and intramolecular dsRNAs longer than 20–30 bp (Nishikura et al., 1991). ADARs convert adenosines to inosines, which base pair with cytosines, which are interpreted as guanosines during translation. Thus, RNA editing affects coding potential, fidelity of RNA replication reverse transcription, or formation/stability of RNA secondary structures where a change of a single base in a sequence may result either in dsRNA destabilization (inosine-uridine pair) or stabilization (inosine-cytidine pair) (Nishikura, 2010). Such transition in the local and global stability of dsRNA structure can influence further processing of dsRNA, such as the selection of the effective miRNA strand (Bartel, 2004; Meister and Tuschl, 2004). Mammals (and vertebrates in general) have three ADAR genes (reviewed in Nishikura, 2016) (Fig. 3). Two encode proteins carrying deaminase activity: ADAR1, which is interferon-inducible, and ADAR2, which is constitutively expressed. ADAR3 is mostly expressed in the brain but its editing activity has not been shown yet. The specificity of the ADAR1 and ADAR2 deaminases ranges from highly site-selective to non-selective, dependent on the duplex structure of the substrate RNA. The complete ADAR structure has not been solved yet but structure of several domains is known – the Z alpha domain of the human editing enzyme ADAR1 (Schwartz et al., 1999) and dsRBDs of ADAR2 (Stefl et al., 2010). The analysis of dsRBDs provided an insight into editing of a specific substrate and revealed that dsRBDs of ADAR not only recognize the shape but also the sequence of the dsRNA (Stefl et al., 2010). The unexpected direct readout of the RNA primary sequence by dsRBDs is achieved via the minor groove of the dsRNA and this recognition is critical for both editing and binding affinity of edited RNA (Stefl et al., 2010). It was also shown that ADAR2 forms dimers in vivo and that dsRBDs are necessary and sufficient for dimerization of the enzyme (Poulsen et al., 2006). ADARs exhibit complex regulation of localization. For example, it was shown that mouse ADAR1 isoforms are differentially localized in cellular compartments and that their
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Figure 3 Domain composition of mammalian ADAR proteins NES, nuclear export signal, NLS, nuclear localization signal; dsRBD, dsRNA binding domain.
localization is controlled by several independent signals, which include a nuclear localization signal (NLS), the nucleolar localization signal (NoLS), the nuclear exporter signal (NES) near the N terminus (Nie et al., 2004). ADAR1 interacts with TuDOR-SN nuclease (Nishikura, 2010; Scadden, 2005; Weissbach and Scadden, 2012; Yang et al., 2006a) and localizes to stress granules upon stress induction (Weissbach and Scadden, 2012) while tudor-SN degrades hyperedited dsRNA (Scadden, 2005). RNA editing concerns a broad range of RNAs including viral and cellular RNAs. Many long perfect dsRNAs (>100 bp) undergo extensive editing with a conversion of approximately 50 % of adenosines to inosines (Nishikura et al., 1991; Polson and Bass, 1994). Extensive editing (hyperediting) is linked with nuclear retention (reviewed in DeCerbo and Carmichael, 2005). On the other hand, short RNAs (~20–30 bp) or imperfect long dsRNAs are edited selectively; usually only a few adenines at specifi c sites are deaminated (Lehmann and Bass, 1999). High throughput analyses revealed the extent of RNA editing of mammalian RNAs in terms of substrate diversity and frequency of editing in the transcriptome (Carmi et al., 2011; Peng et al., 2012). Edited endogenous RNAs (Dawson et al., 2004; Hundley et al., 2008; Morse et al., 2002; Salameh et al., 2015) include mRNAs, repetitive sequences (mainly Alu (Athanasiadis et al., 2004)), and miRNAs. It was predicted that more than 85% of pre-mRNAs may be edited, predominantly in the non-coding regions (Athanasiadis et al., 2004). Several pri-miRNAs (e.g. miR-142) are known to undergo editing, which inhibits Drosha cleavage or even causes degradation of pri-miRNA by Tudor SN (Nishikura, 2010; Scadden, 2005; Yang et al., 2006a). In other cases, pri-miRNA editing does not infl uence Drosha activity but inhibits processing of pre-miRNA by Dicer (e.g. miR-151) (Kawahara et al., 2007a). Last but not least, RNA editing might also inhibit export of miRNAs from the nucleus (Nishikura, 2010). A systematic analysis of edited miRNAs in the human brain showed that editing of miRNAs affects several miRNAs but it is not widespread (Alon et al., 2012). A similar picture was obtained from analysis of embryonic miRNAs (Garcia-Lopez et al., 2013; Vesely et al., 2012) One of the roles of ADARs in immunity is to prevent innate immune sensing of self-RNA (Heraud-Farlow and Walkley, 2016). ADARs also affect viral RNAs in various ways – ADARs are both antiviral and proviral; the effect on virus growth and persistence depends
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