578 (2016) 1–6

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Gene

journal homepage: www.elsevier.com/locate/gene

Review Pros and cons of pDNA and mRNA to study mRNA in mammalian cells

Dmitry E. Andreev a,⁎,IlyaM.Terenina,b, Sergey E. Dmitriev a,b, Ivan N. Shatsky a,⁎ a Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia b Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia article info abstract

Article history: synthesis in is subject to stringent control. The misregulation of translation of certain mRNAs Received 25 October 2015 is often a hallmark of many diseases, including malignancies and autoimmune disorders. To understand why and Accepted 7 December 2015 how it happens, it is important to investigate the translational control of specificmRNAs.Inthiscase,onecould Available online 8 December 2015 use reporter mRNAs in order to identify cis-acting elements responsible for regulation. Here we overview DNA (pDNA) and mRNA , their pitfalls and limitations, as well as some emerging applications for Keywords: Plasmid DNA mRNA transfection. Reporter gene © 2015 Elsevier B.V. All rights reserved. mRNA Transfection Translation Gene therapy

Contents

1. Introduction...... 1 2. DNAreporters...... 2 3. mRNAtransfection...... 2 4. Conclusions...... 5 Acknowledgements...... 5 References...... 5

1. Introduction how translation is regulated in living cells, but the main limitation of this technique is that it cannot explain how particular mRNA species During the last decade, we rapidly entered the “omics” era. It are regulated under specific conditions. In other words, we now know changed many areas of life sciences and, for instance, studies related (or could know if we wish) what is regulated and when it is regulated, to regulation of gene expression. In particular, a breakthrough in next but we do not understand how this is achieved. generation sequencing technology allowed scientists to develop the In order to investigate mechanisms of translational control of specif- profiling technique (Ingolia et al., 2009), which for the first ic mRNAs in detail, the initial step is to address the translation behavior time addressed translation at the whole transcriptome scale. Without of a particular mRNA and then to find and dissect cis-acting elements doubt, this powerful approach has already changed our understanding responsible for such behavior. For this, the use of reporter constructs is essential. It is thought that most of the features that affect the efficiency of Abbreviations: pDNA, plasmid DNA; UTR, untranslated region; DRB, 5,6-dichloro-1-β- translation of mammalian mRNAs are located in 5′ and 3′ untranslated D-ribobenzimidazole; TLR, Toll-like receptor; ARCA, anti-reverse cap analogue; MATra, regions. The 5′-UTR or 5′ leader needs to be traversed by the 43S magnet-assisted transfection; MEF, mouse embryonic fibroblasts; ES, embryonic stem preinitiation complex on its way to the translation initiation site, and cells; Ψ, pseudouridine; IRES, internal ribosome entry site. hence different features within the 5′ leader may facilitate or inhibit ⁎ Corresponding authors. ′ E-mail addresses: [email protected] (D.E. Andreev), [email protected] translation initiation. 3 -UTRs are believed to serve as regulatory plat- (I.N. Shatsky). forms for various effectors such as RNA binding and miRNAs,

http://dx.doi.org/10.1016/j.gene.2015.12.008 0378-1119/© 2015 Elsevier B.V. All rights reserved. 2 D.E. Andreev et al. / Gene 578 (2016) 1–6 which in turn may affect mRNA stability, localization, and translation Another important issue for pDNA transfection is that multiple aber- efficiency (Sonenberg and Hinnebusch, 2009; Jackson et al., 2010; rant mRNA isoforms may be produced from the expression cassette. Hinnebusch, 2014). To address the relative impact of either 5′-UTR, or This might be either because of alternative promoters located in the 3′-UTR, or both on translation, reporter constructs are usually generated 5′-UTR, alternative or cryptic splicing, or a combination of both (for a re- where UTRs of interest flank a particular reporter gene. A huge selection view, see Shatsky et al., 2010). Importantly, cryptic promoters may be of reporter are available, including those expressing fluorescent present not only in the inserted fragment but also in reporter gene itself reporter proteins, which can be directly measured in living cells, secret- (Vopalensky et al., 2008), or in the vector backbone (Lemp et al., 2012). ed reporters for measurement in the culture media, and enzymatic re- A good example of the latter is the presence of a cryptic mammalian porters, which can be measured after . Important properties promoter in the pGEM-4Z cloning vector that was not designed for ex- of reporter proteins that should be considered are their half-life and pression in mammalian cells but nevertheless is able to efficiently drive time required for maturation. The question addressed in this review is reporter expression (Chauhan et al., 2009). Importantly, cryptic pro- whether to use a pDNA transfection or mRNA transfection in cultured moters and/or splice sites may result in the production of reporter cells? We will share our experience regarding these techniques and mRNAs with unexpected 5′-UTRs, which either lack regulatory features highlight artifacts and caveats as well as advantages for each of them. of the original 5′-UTR or acquire some from vector backbone. Even low amounts of aberrant transcripts which are hard to detect by convention- al methods, such as Northern-blot, can significantly affect the results of 2. DNA reporters very sensitive reporter assays (Van Eden et al., 2004; Kozak, 2005, 2006; Gendra et al., 2007; Belancio, 2011). Searching for such aberrant tran- DNA reporters are (pDNA) where the expression of a scripts significantly increases the number of control experiments re- reporter gene, flanked by UTRs of interest, is driven by either a constitu- quired to perform. tively active (such as SV40, CMV etc), cell/tissue specific, or inducible pro- Moreover, various vector backbones can produce multiple cryptic moter. It is generally accepted that pDNA are translocated via endosomal non-coding transcripts that can directly influence reporter activity. uptake, cytosolic release, and nuclear entry. Because of the latter, a major Nejepinska et al. (2012) analyzed transcriptomes derived from various drawback of pDNA transfection is that it is usually ineffective for non- transiently transfected plasmids by means of high-throughput sequenc- dividing cells. ing. They detected unexpectedly complex spurious transcripts derived A combination of pDNAs bearing various reporters may be delivered from many parts of defined vector backbones, for example, a unique into cultured cells, and after a defined period of post-transfection, the population of edited sense and antisense small derived from the reporter activities may be estimated. Importantly, using single-cell Kan/Neo resistance cassette. Importantly, some vectors, when co- expression measurements from a pDNA expressing EGFP, the onset transfected, can inhibit the expression of luciferase reporters in a of fluorescence is detected 2 hours to 20 hours post-transfection dose-dependent manner. One possible explanation is that spurious (Leonhardt et al., 2014).This dispersion is probably a result of pDNA transcripts generated from the vector backbone may form dsRNAs and entry into the nucleus during in unsynchronized cells then activate PKR, which in turn phosphorylates eIF2, thus leading to (Wilke et al., 1996; Mortimer et al., 1999; James and Giorgio, 2000; translation repression (Garcia et al., 2007; Sadler and Williams, 2007). Akita et al., 2007). Another explanation for such kinetics is stochasticity Finally, it should be considered that pDNA may be immunogenic be- of mRNA synthesis in mammalian cells, also known as intrinsically ran- cause the presence of DNA in the cytoplasm is not normal for eukaryotic dom “ bursts” (Raj et al., 2006). cells. Indeed, it was noticed some time ago that bacterial DNA with non- There is a number of disadvantages of pDNA transfection, and methylated CpG motifs induce a strong immune response through Toll- because of that, the results of experiments must be considered with like receptor 9 (TLR9) (Hemmi et al., 2000). However, there are certain some caution. First, since pDNA transfection depends on mitotic activity lines of evidence indicating that the methylation status of CpG is not a of cells, conditions that affect progression can also affect primary determinant of an immune response, as self DNA also has the reporter production. Indeed, cells arrested in the G1 phase of the cell ability to induce TLR9 activation if it enters the endosomal compartment cycle by treatment with aphidicolin exhibited 20-fold lower reporter in which the recognition takes place (Brencicova and Diebold, 2013). gene activity than asynchronous control cells upon pDNA transfection This immune response leads to the activation of nuclear factor κB and (Mortimer et al., 1999). The second and logical caveat is transcription mitogen-activated protein kinase pathways (Krieg, 2002). Apart from of the reporter gene. From this ultimately arises the question whether Toll-like receptors, there seems to be many more players involved the effects on reporters observed in the experiment are because of in cytoplasmic DNA sensing and activation of an immune response altered translation or transcription. 5′-UTRs of mRNAs may serve as (reviewed in Paludan and Bowie, 2013) transcription modulators. They may bind various transcriptional factors Overall, it is clear that the results of pDNA transfection should be an- and hence may influence transcription itself in an unpredictable way. alyzed with caution. Nevertheless, in many studies on translation con- For instance, widespread transcription factor binding in genomic trol, DNA transfection is the only method of choice. At least, if other regions that are transcribed as 5′-UTRs can be easily visualized in the approaches are not applicable for some reasons, some control experi- UCSC genome browser in the “Txn Factor ChIP track,” which shows ments may be important. First, the reporter gene with corresponding DNA regions where transcription factors bind (Consortium, 2012). UTRs may be transferred to alternative vectors bearing different pro- Moreover, cryptic transcription enhancers may be present in the vector moters to rule out a direct influence of vector context on the results. backbone, which could modulate reporter gene expression in cell type- Second, the entire population of mRNAs produced from the pDNA dependent manner (Thirunavukkarasu et al., 2000). should be analyzed and quantified. Importantly, effects of mutations in- One possible way to discriminate between transcriptional and trans- troduced in pDNA may result in altered mRNA expression, and hence lational effects may be to block transcription before measuring reporter the levels of reporter should then be normalized to that for mRNA. activity. However, treatment with transcriptional inhibitors itself may repress translation. For instance, the inhibition of transcription 3. mRNA transfection with the widely used inhibitors actinomycin D or 5,6-dichloro-1-β-D- ribobenzimidazole (DRB) was found to induce phosphorylation of eIF2 The method of transferring in vitro transcribed mRNA into mamma- (Jiang et al., 2003; Dai et al., 2013), which significantly impairs transla- lian cells was first suggested in late the 1980s (Malone et al., 1989). tion. Alternatively, inducible promoters may be used to switch on or off Since this first study and until recently, surprisingly little attention has transcription of a particular reporter at desired time points without been paid to the use of messenger RNA for the transfection of cells, affecting global transcription and translation. which may be partially explained by the widely held belief that mRNA D.E. Andreev et al. / Gene 578 (2016) 1–6 3 is too unstable. However, it is clear that the transfection of cultured cells molecules. However, not every endosome releases its lipoplex cargo with reporter mRNAs lacks many of the disadvantages associated with into the cytosol, and the lysis rate is estimated to be 25–50%, which the use of DNA reporters. First, there is no de novo reporter mRNA tran- might lead to cytoplasmic delivery of 350–1400 mRNA molecules per scription in cells, and once mRNA is transfected, the yield of reporter is cell (Leonhardt et al., 2014). It turns out that transfected mRNA determined only by mRNA translation and turnover as well as the stabil- may represent a significant proportion of total cellular mRNA content, ity of the reporter protein. Second, in vitro transcribed and purified especially in certain cell types. While the average number of mRNA mol- mRNA represents only one desired isoform. Third, the same reporter ecules per cell was found to be 505,000 for mouse embryonic fibroblasts mRNA can also be directly assayed in cell-free translation systems. (MEF), this number was just 22,000 molecules in embryonic stem cells Technically, it is relatively easy to prepare m7G-capped and (ES) (Islam et al., 2011). Apart from some housekeeping genes, the vast polyadenylated reporter mRNAs from DNA constructs. PCR amplifica- majority of mRNA species are present at just in several copies per cell, tion could be carried out with the forward primer bearing a T7 or SP6 and the numbers depend on the cell type. For example, the top one promoter and reverse primer bearing an oligo-dT track of desired length thousand genes in ES cells ranged from 1 to 2709 molecules per cell, (which would be converted into a poly-A tail during transcription) whereas for MEFs the range was from 77 to 7044 molecules per cell (Dmitriev et al., 2007). In this case, there is no need for re-cloning of (Islam et al., 2011). This indicates that the number of molecules of re- DNA reporters previously studied in DNA transfection experiments porter mRNA, which enter the cell is comparable to that for most highly since all elements required for in vitro transcription are introduced expressed cellular mRNAs. It is reasonable to propose that the transla- upon PCR. The resulting purified PCR product is a suitable template for tion of some rare and non-abundant mRNAs may rely on low amounts an in vitro transcription reaction. PCR amplification with various combi- of specific translation regulators (i.e., RNA binding proteins, non- nations of primers allows one to easily investigate the translational be- canonical translation factors, miRNAs) present in cells, and then havior of 5′ and/or 3′ terminally truncated mRNAs without additional overloading of reporter mRNA may be misleading. However, the same cloning. Size selection and gel purification of PCR templates may be situation may happen during pDNA transfection, given the fact that important to avoid aberrant PCR products (which may still contain the each cell may acquire between 75 and 50,000 plasmids per nucleus de- T7 or SP6 promoter and thus may serve as templates for transcription). pending on transfection protocol applied (Cohen et al., 2009). Indeed, it The limitation here is that commercially available RNA polymerases can was demonstrated that there is poor correlation between mRNA ex- initiate transcription only with purines, which makes it impossible to pression and the corresponding protein production upon pDNA trans- obtain reporters with a 5′ terminal pyrimidine . fection in single-cell experiments (Akita et al., 2011), which may be To prepare m7G capped mRNA, there are two alternative ap- explained by sequestration of translation machinery by high amounts proaches, enzymatic and co-transcriptional capping (Rosenberg of reporter mRNA. The development of methods that allow controllable and Paterson, 1979; Contreras et al., 1982). First, uncapped mRNA delivery of precise amounts of mRNA into the cell would be very useful may be capped using a commercially available vaccinia capping in this regard. system (post-transcriptional capping). Second, mRNA may be capped The second technical disadvantage of RNA transfection is that it is co-transcriptionally by means of m7G-cap analogues, which can be hard to measure the stability of the transfected reporter mRNA directly. incorporated only at 5′ termini (for example, ARCA, anti-reverse cap As it was mentioned above, when mRNA is transfected by lipofection, a analogue). During co-transcriptional capping, it is possible to introduce significant portion of mRNA is not released from lyposomes, and be- other cap analogues, such as “non-functional cap,” ApppN, which is cause of that, a direct mRNA quantification is compromised. Indeed, however still able to prevent mRNA degradation (Dehlin et al., 2000). the quantification of deliberately unstable mRNAs transfected into the Capped and polyadenylated mRNAs can then be transfected into cells by lipofection showed no decrease in mRNA abundance (Barreau cells. Certain cell lines can be easily transfected by lipofection (in our et al., 2006). In our hands, this is also the case when magnet-assisted hands, HEK293T, HeLa, Huh7, NTera2/D1, HepG2, RKO, BHK21, COS-1, transfection (MATra; Bertram, 2006; Andreev et al., 2013) or cationic and Vero cell lines are good for mRNA lipofection). Alternatively, phys- polymer-based transfection was applied. To address the stability issue, ical methods such as may be used. However, it was re- an indirect approach that addresses the kinetics of reporter accumula- ported that, unlike lipofection, electroporation induces a strong stress tion after transfection was suggested (Dmitriev et al., 2007; Andreev response (Anderson et al., 2013). et al., 2013). The idea is that unstable mRNA would show different Another advantage of mRNA transfection is the very rapid reporter kinetics of reporter accumulation and would plateau shortly after trans- translation, which, in the case of luciferases, can be detected as early fection because all mRNAs that reach the cytoplasm would be degraded. as 30-60 minutes post-transfection (Rejman et al., 2010; Andreev Indeed, this was the case with uncapped reporter mRNA (Andreev et al., et al., 2013). In single-cell experiments with mRNA encoding EGFP, 2009). mRNA-transfected cells show an early and steady rise to a maximum Next, as for pDNA, in vitro transcribed mRNA may induce an im- with a subsequent decrease later (Leonhardt et al., 2014). This allows mune response. Similar to pDNA transfection, mRNA can activate cer- the investigation of the immediate effects of various stress responses tain Toll-like receptors (TLRs). For instance, TLR3 recognizes dsRNA in on translation, which are usually rapid due to the post-translational a sequence-independent manner (Alexopoulou et al., 2001), whereas modifications of components of the translation machinery. TLR7, TLR8, and TLR9 seem unable to distinguish between viral and Finally, another big advantage of reporter mRNAs is that the same self-nucleic acids on the grounds of distinct molecular patterns, but mRNA can be translated in various mammalian cell-free systems their activation relies solely on the mode of access to the endolysosomal (Dmitriev et al., 2009). Here, it is possible to address the tight kinetic compartment in which the recognition takes place (Brencicova and parameters of translation, the effect of various translation factors and Diebold, 2013). A strong argument that there is no distinguishable pat- mRNA binding proteins on reporter production, as well as an effect tern in pathogens RNAs, which are sensed by TLRs, is the observation of small molecules and compounds, which do not penetrate into cells that TLR7 can be activated by transfection of purified mammalian (Andreev et al., 2009; Vassilenko et al., 2011; Shagam et al., 2012; mRNA (Diebold et al., 2006). Terenin et al., 2013). Although the expression of TLRs is restricted in many cell types There are certain pitfalls of mRNA transfection into living cells. First, (they are expressed mostly in leukocytes), there are a cohort of foreign it is difficult to control how much mRNA is transfected in a single cell, in RNA sensors that are present in almost all cells. For instance, the agonist other words, what might be the concentration of transfected mRNA in for RIG-I has been defined as an RNA with a triphosphate (PPP) moiety the cell cytoplasm. According to one estimate (Leonhardt et al., 2014), and a base-paired stretch (reviewed in Goubau et al., 2013), as well during lipofection (with Lipofectamine 2000 reagent), each cell uptakes as the absence of 2′O-methylation at the 5′-terminal nucleotide N1 4–8 lipoplexes, and each lipoplex contains approximately 350 mRNA (Schuberth-Wagner et al., 2015). MDA5 seems to act as a sensor for 4 D.E. Andreev et al. / Gene 578 (2016) 1–6 long dsRNA (Pichlmair et al., 2009), and for the absence of 2′O-methyl- 2010). Although this approach definitely revolutionizes the field of ation of the 5′ cap structure (Zust et al., 2011). Both RIG-I and MDA5 gene therapy, we believe that it is hardly applicable for studying basic then activate the expression of the genes encoding IFN-α and IFN-β, mechanisms of translation control in cells. First, chemically modified as well as other antiviral genes (Goubau et al., 2013). Another sensor nucleosides may inactivate regulatory elements in mRNA, for example, is PKR, which is activated by a relatively long dsRNA and then inhibits by preventing the formation of secondary structures, and hence affect global protein synthesis (Nallagatla et al., 2011; Dabo and Meurs, translation control. Indeed, the incorporation of Ψ impairs the internal 2012). In some cases, PKR may be activated by the 5′PPP as well ribosome entry site (IRES) of the encephalomyocarditis virus (Thess (Nallagatla et al., 2007). et al., 2015). Some modifications such as Ψ also influence decoding In regard to mRNA transfection, it may be important to know that (Fernandez et al., 2013). UAA, UAG, and UGA are normally decoded as RIG-I signaling is sensitive to very small amounts of viral RNA and can stop-codons, whereas ΨAA, and ΨAG code for serine and threonine, be activated by less than 20 molecules of 5′ PPP viral RNA per cell and ΨGA codes for tyrosine and phenylalanine in yeast (Karijolich and (Zeng et al., 2010). Obviously, capping of in vitro transcribed mRNAs Yu, 2011). Notably, miscoding of mRNA may result in the accumulation is not 100% efficient, so even capped mRNA preparations may induce of protein aggregates and in the development of the unfolded protein an immune response. To reduce the risk of immune response activation, response, which may limit the application of modified mRNAs for capped mRNA preparation may be treated with phosphatase to remove gene therapy. residual triphosphates from 5′ ends. Finally, there is reasonable concern that transfected mRNA never Also, some endogenous mammalian mRNAs can directly activate pass through “nuclear events,” since it was not transcribed in and PKR. It was shown that IFN-gamma mRNA activates PKR through a exported from the nucleus-like endogenous mRNAs. Initially, we con- pseudoknot in its 5′-UTR, which is required for the autoregulation of sidered that this argument was used purely to describe the discrepan- translation (Ben-Asouli et al., 2002). PKR can be also activated by 3′- cies between the results of RNA and DNA transfections (Cammas et al., UTRs of some cytoskeletal proteins (Nussbaum et al., 2002) and TNF- 2007). However, now there is strong evidence that mRNAs transcribed α (Osman et al., 1999). Thus, an immune response induced by mRNA in the nucleus from specific promoters indeed may have a different transfection is not necessarily an artifact of transfection itself but may translational fate. It was shown that the identity of the promoter, name- be indicative of real translation control. ly, its responsiveness to heat-shock factor 1 (Hsf1), determines whether To overcome the immune response mediated by mRNA, several de novo transcribed mRNA would be translated or silenced upon glu- groups proposed to use of in vitro produced mRNA with incorporated cose starvation in yeast (Zid and O'Shea, 2014). The possible explana- modified nucleosides, such as m5C, m6A, m5U, s2U, pseudouridine (Ψ), tion for this phenomenon is that some features within promoters and their combinations (Kariko et al., 2005, 2008; Warren et al., determine the composition of specific RNPs assembled on the mRNA

Fig. 1. Schematic representation of pDNA transfection (A) and mRNA transfection (B). Possible sources of artifacts and misinterpretations are highlighted in red. For pDNA transfection, aberrant mRNA is represented by one with shortened 5′-UTR, which may drive more efficient reporter production. Stars represent reporter proteins. In right parts of A and B, represen- tative expected kinetic curves of reporter production in single cells are shown (adapted from Leonhardt et al., 2014). At the left part of B, the preparation of reporter mRNA from pDNA is proposed in order to emphasize simplicity of mRNA preparation. D.E. Andreev et al. / Gene 578 (2016) 1–6 5 in the nucleus, which is subsequently exported into the cytoplasm. A References combination of mRNA transfection and corresponding pDNA with its native promoter may be helpful to detect and investigate new cases of Akita, H., Ito, R., Kamiya, H., Kogure, K., Harashima, H., 2007. Cell cycle dependent tran- – scription, a determinant factor of heterogeneity in cationic lipid-mediated transgene such transcription translation coupling. expression. J. Gene Med. 9, 197–207. Akita, H., Umetsu, Y., Kurihara, D., Harashima, H., 2011. Dual imaging of mRNA and pro- tein production: an investigation of the mechanism of heterogeneity in cationic 4. Conclusions lipid-mediated transgene expression. Int. J. Pharm. 415, 218–220. Alexopoulou, L., Holt, A.C., Medzhitov, R., Flavell, R.A., 2001. Recognition of double- Both pDNA and mRNA transfection have common and individual stranded RNA and activation of NF-kappaB by Toll-like receptor 3. 413, 732–738. disadvantages (Fig. 1). Due to the immunogenicity of both pDNA and Anderson, B.R., Kariko, K., Weissman, D., 2013. Nucleofection induces transient eIF2alpha mRNA, its application may be compromised in certain cell types such phosphorylation by GCN2 and PERK. Gene Ther. 20, 136–142. as immune cells. Also, it is hard to control the concentration of reporter Andreev, D.E., Dmitriev, S.E., Terenin, I.M., Prassolov, V.S., Merrick, W.C., Shatsky, I.N., 2009. Differential contribution of the m7G-cap to the 5′ end-dependent translation mRNA, either directly delivered into the cell, or transcribed from pDNA. initiation of mammalian mRNAs. Nucleic Acids Res. 37, 6135–6147. Too high a concentration of reporter mRNA may be critical for cells with Andreev, D.E., Dmitriev, S.E., Terenin, I.M., Shatsky, I.N., 2013. Cap-independent transla- tion initiation of apaf-1 mRNA based on a scanning mechanism is determined by low mRNA content resulting in drawing the wrong conclusions about ′ fi some features of the secondary structure of its 5 untranslated region. Biochemistry cell-speci c translation control mechanisms. However, in general, (Mosc) 78, 157–165. pDNA transfection seems to be more prone of artifacts. 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Release 135, 166–174. vantage of mRNA transfection for such screenings is the very rapid accu- Consortium, E.P., 2012. An integrated encyclopedia of DNA elements in the human mulation of the reporter which can save time. genome. Nature 489, 57–74. A new and rapidly evolving field in RNA biology is “epitranscriptomic” Contreras, R., Cheroutre, H., Degrave, W., Fiers, W., 1982. Simple, efficient in vitro synthe- fi sis of capped RNA useful for direct expression of cloned eukaryotic genes. Nucleic modi cations of RNA (Lee et al., 2014; Wang and He, 2014). Non-coding Acids Res. 10, 6353–6362. RNAs such as rRNAs and tRNAs are extensively modified, whereas mRNA Dabo, S., Meurs, E.F., 2012. dsRNA-dependent protein kinase PKR and its role in stress, modifications are thought to be relatively low in frequency apart from the signaling and HCV . 4, 2598–2635. common terminal modifications, m7G cap, and poly(A) tail. 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