The increasing prevalence of worldwide obesity has emerged as a major risk factor for type 2 diabetes (T2D), hepatosteatosis, and cardiovascular disease. Accumulating evidence indicates that obesity has strong inflammatory underpinnings tightly linked to the development of metabolic diseases. However, the molecular mechanisms by which obesity induces aberrant inflammation associated with metabolic diseases are not yet clearly defined. Recently, RNAs have emerged as important regulators of stress responses and metabolism. RNAs are subject to changes in modification status, higher-order structure, and cellular localization; all of which could affect the affinity for RNA-binding proteins (RBPs) and thereby modify the RNA-RBP networks. Proper regulation and management of RNA characteristics are fundamental to cellular and organismal homeostasis, as well as paramount to health. Identification of multiple single nucleotide polymorphisms (SNPs) within loci of fat mass- and obesity-associated protein (FTO) gene, an RNA demethylase, through genome-wide association studies (GWAS) of T2D, and functional assessments of FTO in mice, support the concept that disruption in RNA modifications leads to the development of human diseases including obesity and metabolic disorder. In obesity, dynamic alterations in modification and localization of RNAs appear to modulate the RNA-RBP networks and activate proinflammatory RBPs, such as double-stranded RNA (dsRNA)-dependent protein kinase (PKR), Toll-like receptor (TLR) 3 and TLR7, and RNA silencing machinery. These changes induce aberrant inflammation and the development of metabolic diseases. This review will describe the current understanding of the underlying causes of these common and altered characteristics of RNA-RBP networks which will pave the way for developing novel approaches to tackle the pandemic issue of obesity. In the past several decades, dramatic and rapid changes in lifestyle and dietary trends have led to obesity becoming a worldwide problem, bringing with it a host of chronic metabolic diseases, including type 2 diabetes (T2D), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and cardiovascular disease (CVD). The global prevalence has reached epidemic proportions. In 2015, ~603.7 million adults and 107.7 million children were considered obese, and yet these figures are predicted to continue to increase worldwide (N6-methyladenosine (m6A) demethylase of eukaryotic messenger RNA (mRNA) , N1-methyladenosine (m1A), and regulate RNA stability  evaluating patients with T2D revealed fat mass- and obesity-associated protein (FTO) as the first GWAS-identified diabetes susceptibility locus . InitialA (mRNA) , 11, andA (mRNA) , 13. Subtability , 15, witThe biological significance of RNA modification remains largely unknown in metabolic disorders, yet accumulated evidence suggests that the dynamic regulation of RNA modifications could have profound impacts on mRNA splicing, translation, maturation of non-coding microRNAs (miRNAs), and interactions with RNA-binding proteins , 17. RecFTO genetic locus are also associated with onset and development of insulin resistance, metabolic syndrome, atherosclerosis, systemic hypertension, and alteration in C-reactive protein levels , and obesity . Approxin levels  56–60).–60.FTO gn levels .In a study investigating the link between the obesity-associated FTO SNPs and FTO expression, expression quantitative trait locus (eQTL) analysis failed to demonstrate that SNPs affected FTO expression in human tissues , 62, 63.These findings suggest the obesity and obesity-associated phenotypes observed in individuals who have FTO SNPs in intron 1 may be attributed to their potential to influence expression of IRX3 and IRX5, instead of FTO itself , 67. Non2+ binding motif and a 2-oxyglutarate (2OG)-dependent oxygenase (6A) demethylase of mRNA (FTO contains within its sequence a Fexygenase . Structuxygenase –77. A su of mRNA  78). In. In2+ bi of mRNA , while m of mRNA , 81. Rec of mRNA . Genome- of mRNA . Further of mRNA , 84.Of note, a recent study identified a novel role of FTO for RNA modification of the first encoded nucleotide next to the 7-methylguanosine cap, N6,2-prime-O-dimethyladenosine (m6Am), in controlling mRNA stability . This RNBeyond simply the importance of FTO-mediated RNA methylation lies the presence of effective RBPs capable of recognizing and binding these methylated RNAs. One such RBP, YTH N6-Methyladenosine RNA Binding Protein 2 (YTHDF2), is an RNA-binding protein that belongs to the YTH domain-containing family. It regulates decay, destabilization, and degradation of methylated genes by selectively binding to previously described m6A-methylated mRNA within the G(m6A)CU/A consensus, or by recruiting CCR4-NOT deadenylase complex (see the CCR4-NOT section below) within P-bodies found in the cytoplasm –92. YTHDTechnical advances have enhanced our ability to define the distribution profile and dynamic properties of m6A methylation by using sensitive and specific approaches. While chemical and antibody-based approaches have been developed and provided us important findings, there appear to be some limitations in these approaches. For instance, methylated RNA immunoprecipitation with anti-methylated RNA-specific antibodies and sequencing (MeRIP-seq) has been used to detect global m6A levels within 100 nucleotides length , 96. HowO-methylation close to the cap are marked as “non-self” and trigger the release of type I IFNs. Cap-specific mRNA (nucleoside-2′-O-)-methyltransferase 1 (CMTR1) dictates mRNA cap1 2′-O-ribose methylation of the mRNA 5′-cap structure. Importantly, dysregulation in CMTR1 expression is associated with several pathologies related to self-recognition immune mechanism from non-self RNAs. Consistently, it has been shown that uncapped viral RNAs can trigger an interferon-mediated antiviral response from the host. Mechanistically, several pro-inflammatory RBPs, including RIG-I and MDA5, are potent regulators that bind and discriminate between “self” and “non-self” RNA  through TLR7 signaling in a methylation status-dependent manner  production, ER stress, and subsequent cell death, independent of 2′-O-methylation of rRNA targets. A subsequent genome-wide shRNA-based screen recently identified nuclear export factor 3 (NXF3) as a transporter that can change the nucleo-cytoplasmic distribution pattern of these box C/D snoRNAs. Of note, these snoRNAs are involved in glucose intolerance and cholesterol trafficking mechanisms after treatment with fatty acids , a syndrome in which affected individuals displaying profound food-seeking behavior and are, consequently, severely obese with or without diabetes  are small non-coding RNAs that manage chemical modifications of other RNAs including ribosomal RNA (rRNA) and tRNA . SnoRNAsty acids . For insolerance , whereasolerance . Interes tissues  126), a, aRpl13adiabetes . A recena of PWS . AlthougSeveral RBPs, including TLR7, function as receptors for pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) capable of inducing atypical inflammation under obese conditions , 129–131in vivo when overexpressed in cells  stress, in the absence of pathogenic infection. The PKR activation induced by these cellular stresses, when compared to the robust activation by dsRNA or analogs like polyinosinic-polycytidylic acid (Poly(I:C)), are generally not drastically increased, but are sufficient to induce JNK activation and eIF2α phosphorylation . These fin cells .in vivo and in vitro , mitochondrial tRNAs, and mRNAs, in mouse and human cells , 141. Foin vitro . Intriguin vitro . These fAlthough PKR appears to play a critical role in immuno-metabolic regulation and, indeed, several studies have shown that PKR-deficiency in mice exhibits beneficial immuno-metabolic phenotypes, contradicting observations have been reported , 146–149ob/ob) to generate C-PKR KO-ob/ob mice. The beneficial metabolic phenotypes, including improved systemic glucose tolerance and insulin sensitivity, were also observed in C-PKR KO-ob/ob mice, while body weight was not affected -induced colitis , whereasIn the C-PKR KO mice, maintaining expression of dsRBDs, in addition to deletion of the kinase domain, of PKR may be associated with beneficial effects during the pathogenesis of obesity. The dsRBDs can still interact with endogenous RNAs, including snoRNAs, particularly under metabolically-driven stress conditions , 151. InHypothetically, if nutrient and pathogen response systems are integrated, then involvement of pathogen sensors would be anticipated during metabolic regulation and stimulate the pursuit toward understanding the potential role of PKR in metabolism . Other tob/ob obese mice results in a significant reduction of JNK activity and eIF2α phosphorylation, which coincides with the alleviation of systemic glucose intolerance, insulin resistance, and hepatic steatosis  and insulin-like growth factor binding protein 1 (Igfbp1) mRNAs were higher with longer poly(A) tails when compared to controls due to reduced deadenylation activity  sequence at the 3′ end of mRNA tail contributes to post-transcriptional gene regulation by acting on mRNA metabolic stability and frequency of translation . Shortensitivity . Mechaniactivity . Liver-sactivity . Inhibitenditure . These fenditure .Ucp1), the main driver of non-shivering thermogenesis, in inguinal white adipose tissue (iWAT)  . The Tobe (iWAT) –197. Tobficiency . Convers elusive . These fin vitro, ex vivo, and in vivo experimental data highlighting potential underlying mechanisms of RNA-RBPs that might be crucial to answering important questions during the study of immune-metabolic dysregulation. As previously described, RNA-RBPs are considered novel players essential for the association between abnormal immune system response, posttranscriptional gene regulation, and metabolic dysfunction both at the tissue-specific and systemic scale. Clearly, it could be hypothesized that specific pharmacological inhibitors against particular RNA-RBPs would restore normal insulin signaling, glucose and lipid metabolism, and ameliorate inflammation and fibrosis of metabolic tissues, such as is observed in obesity-induced steatohepatitis. As a proof of principle, small chemical inhibitors were tested to block the activity of PKR, such as imoxin and 2-aminopurine  have been designed that bind to target miRNA and inhibit the function of miRNA by preventing its binding to target mRNA, followed by sequestration and/or RNase-mediated degradation. Currently, pharmaceutical laboratories have produced several generations of AMOs in order to improve the pharmacokinetic parameters with less toxicity by engineering AMOs containing other modifications, such as 2′-fluoro and/or 2′Omethoxyethyl, as well as 2′-O-methyl modifications, phosphorothioate bonds, and a 3′ end cholesterol tail seen in the first AMO generation –208. By Even in the presence of potentially effective, though limited, therapeutics targeting specific RNAs, further studies are required to elucidate the molecular mechanisms underlying the development of immune-metabolic disorders. Nonetheless, there is clear evidence that RNA-RBP networks play a critical role in the regulation of tissue-specific and/or whole-body glucose metabolism and inflammation, and that novel approaches targeting RNA-RBPs network in the setting of metabolic disorders will facilitate clinical research toward the identification of effective medical therapies for treating obesity and its associated metabolic diseases.Knowledge of the role of RNA networks associated with RNA modifications and RNA-RBP interactions in inflammatory and metabolic regulation in obesity is limited. However, accumulating evidence has shed light on how RNA networks dynamically affect metabolic, developmental, and inflammatory mechanisms in the pathogenesis of obesity-associated disorders. Indeed, functional changes of additional RBPs, including but not limited to LIN28A, IGF2BP2/IMP2, and HuR, are linked to the development of metabolic diseases –215. RNAWhile RNA modifications are generally considered a fine-tuning process of cellular homeostasis, prolonged external cues, including chronic HFD-feeding, may gradually accumulate erroneous RNA modifications that drive the cell fate toward chronic and low-grade inflammatory conditions which are well-observed cellular features in obesity. This concept is supported by evidence that several inflammatory RBPs, including TLR3, TLR7, and PKR, are involved in the induction of obesity-associated chronic inflammation. As mRNA methylation modification could influence post-transcriptional gene silencing and control targeted gene expression, e.g., through affecting interactions with mRNA/miRNA/Ago proteins complex, there may be specific types and modifications of RNAs that activate metabolic and inflammatory programs in the pathogenesis of obesity.in vivo RNA targets of RBPs (Current technical advances have allowed us to investigate quantitative changes in many types of RNA species; either coding or non-coding RNA levels, and methylation status –221. How of RBPs . UtilizaAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite advances in the development of targeted therapy, prognosis of MCL is poor, with a median survival of 5–7 years2. The pathogenesis of MCL is complex due to molecular alterations at several regulatory hubs. Regulatory control of gene transcription is well studied in most of the cancers, including MCL; however, the regulatory mechanisms of protein translation in cancers including MCL are less understood. Translation machinery, also referred as translation initiation complex (eIF4F), regulates the translation of mRNAs mainly via translation initiation factor-4E (eIF4E). We and others have previously shown that eIF4E is overexpressed in MCL tissue, and is correlated with poor prognosis4.Mantle cell lymphoma (MCL) is a type of aggressive non-Hodgkin B cell lymphoma derived from naive pre-germinal center cells of mantle zones5, the role of lncRNAs in translation regulation has been revealed only in recent years. We have recently reported that lncRNA ROR-AS1 is highly upregulated in MCL tissue and regulates gene transcription via the epigenetic repressive complex6. Recent ribosome profiling as well as polysome profiling approaches have presented evidence suggesting that some lncRNAs can be found associated with ribosomal and poly-ribosomal fractions7. This raises the possibility that ribosome-bound lncRNAs can be involved in fine-tuning the speed or specificity of the translation machinery.Long non-coding RNAs (lncRNAs) are vaguely defined by the length range of >200 nucleotides to several kilobases of RNA, and they lack coding potential. Due to their important role in the epigenetic process of gene expression lncRNAs have recently received considerable attention. While transcriptional role of lncRNAs in cancers is well studiedn = 4), MCL cell line (Jeko), and normal controls (n = 3) were pulled down with anti-eIF4E antibody, followed by RNA sequencing and secondary analysis to detect lncRNAs. Specific binding of lncRNAs with eIF4E was evaluated by enrichment analysis, in which the reading counts mapped to the genes of the RNA sample immunoprecipitated with eIF4E (IP) were compared with those of the total RNA sample. Data analysis demonstrated hundreds of lncRNAs associated with eIF4E. Based on twofold changes, we selected the top eight eIF4E-enriched lncRNAs in the MCL patient samples, including novel (NBPF8 and RP4-550H1.6) and known-lncRNAs  as compared to normal controls . To further confirm direct association of SNHG12 lncRNA with eIF4E, we performed RNA-IP in three MCL lines  and in normal controls (n = 3) with IgG and eIF4E antibodies, followed by targeted reverse transcriptase (RT)-PCR. RT-PCR data showed that SNHG12 was highly enriched after RNA-IP with anti-eIF4E antibody as compared to IgG control in all three MCL cell lines tested, while normal B cells had no demonstrable SNHG12 enrichment with anti-eIF4E IP -sequencing. RNA extracted from MCL patients . To examine the relative importance of RNA-binding and cap binding motifs of eIF4E for the interaction between eIF4E and lncRNAs, various plasmid constructs: (i) eIF4E (HA-eIF4EWT), (ii) eIF4E with RNA-binding motifs deleted (HA-eIF4EDel1&2), and (iii) cap mutant (HA-eIF4Ecap mutant), were transfected into HEK-293T cells and RNA-IP was performed for the lncRNAs FTX and SNHG12. There was a net 3–5-fold increase in binding of SNHG12 or FTX lncRNAs in cells overexpressing HA-eIF4EWT as compared to those transfected with an empty vector. However, overexpression of HA-eIF4EDel1&2 but not the HA-eIF4Ecap mutant decreased the binding between eIF4E and SNHG12 or FTX lncRNAs  reduced the 20–25% proliferation as assessed by the MTT assay Fig. ; S2A–D. NAs Fig. . We thenIn summary, to the best of our knowledge, we report for the first time that several lncRNAs including SNHG12, FTX, SNHG5, and ZNFX1-AS1 are found to be associated with translation machinery via eIF4E in MCL tumor cells. We also demonstrate that RNA-binding motifs (but not cap-binding domain) of eIF4E are responsible for binding of lncRNAs with eIF4E. Among lncRNAs that bound to eIF4E, at least SNHG5 and SNHG12 can modulate c-Myc translation in MCL cells. Future work will need to clarify whether eIF4E-bound or ribosome-bound lncRNAs are capable of eliciting additional regulatory roles to the translation machinery. Our results expand the known repertoire of translation regulation and lncRNA biology for potential implications in cancer therapies. In future studies may clarify if eIF4E- or ribosome-bound lncRNAs elicite additional regulatory roles on translation machinery.S1S2S3


Soft X-ray microscopy was applied to study the quantitative distribution of DNA, RNA, histone, and proteins other than histone (represented by BSA) in mammalian cells, apoptotic nuclei, and a chromosome at spatial resolutions of 100 to 400 nm. The relative distribution of closely related molecules, such as DNA and RNA, was discriminated by the singular value decomposition (SVD) method using aXis2000 software. Quantities of nucleic acids and proteins were evaluated using characteristic absorption properties due to the 1s–π * transition of N=C in nucleic acids and amide in proteins, respectively, in the absorption spectra at the nitrogen K absorption edge. The results showed that DNA and histone were located in the nucleus. By contrast, RNA was clearly discriminated and found mainly in the cytoplasm. Interestingly, in a chromosome image, DNA and histone were found in the center, surrounded by RNA and proteins other than histone. The amount of DNA in the chromosome was estimated to be 0.73 pg, and the content of RNA, histone, and proteins other than histone, relative to DNA, was 0.48, 0.28, and 4.04, respectively. The method we present in this study could be a powerful approach for the quantitative molecular mapping of biological samples at high resolution. In principle, X-ray microscopy has the following advantages for the observation of biological samples over other microscopic methods: higher resolution than optical microscopy with respect to the diffraction limit; good absorption contrast in hydrated conditions with soft X-rays in an energy range, the so-called water window; better transmittance than electron microscopy; and the discrimination of biological molecules by spectromicroscopy, the combination of microscopy and spectroscopy using absorption of fine structures in biomolecules according to the energies of carbon, nitrogen, and oxygen absorption edges. These advantages have been demonstrated by the development of X-ray microscopes  with absThe characteristic nature of the X-ray absorption spectrum near the absorption edge of each element—called near-edge X-ray absorption fine structure (NEXAFS)—is useful for the separation of constituent molecules in biological specimens because NEXAFS depends on the chemical structures of the molecules. NEXAFS at the nitrogen K absorption edge shows clear separation of the absorption spectra of nucleic acids and proteins, and it has been applied to identify these molecules in images of a mammalian cell and a chromosome observed with a scanning transmission soft X-ray microscope (STXM) ,24, a mi2 atmosphere. For the study of apoptotic nuclei, HeLa S3 nuclei were prepared as described previously [CHO cells at interphase or mitotic (M) phase were prepared as described previously ,24. HeLaeviously . 3N4) membrane  and processed using a previously described method [® Cell-Tak Cell Tissue Adhesive, washed with an increasing concentration of ethanol, and dried. Molecules including calf thymus histone  were obtained from Sigma-Aldrich Co. , and they were prepared according to previously described methods [For the observation of CHO and HeLa S3 cells, these cells were plated and grown on a silicon nitride  images at 398 eV in DNAspect, RNAspect, histone-spect, or BSAspect were converted to mass thickness, defined as mass per unit area, by using the mass absorption coefficients of DNA, RNA, histone, and BSA at 398 eV, calculated from the database for mass absorption coefficients of constituent elements . For comObserved absorption images (spect1) and residual images after subtraction of nucleic acids and proteins (spect3) are shown in 2), by the method described in Materials and Methods using aXis2000 software , RNA (green), and histone (blue) in the CHO cell are shown in We obtained local absorption of spect1 at three positions in the CHO cell , four poQuantities of DNA, RNA, histone, and other proteins were obtained from We have demonstrated, here, a method for quantifying the distribution of DNA, RNA, histone, and proteins other than histone in mammalian cells at different spatial scales using soft X-ray microscopy. Our results showed that DNA and histone were located in the nucleus, whereas RNA was clearly discriminated and found mainly in the cytoplasm. The images of DNA and RNA in the absorption images of the energy stack file (spect1) of the sample were extracted by the SVD method on aXis2000 using the spectra of DNA and RNA, each of which was measured at the photon energy regions of carbon, nitrogen, and oxygen K absorption edges and combined. The fraction of DNA in DNA plus RNA was applied to the quantitative image of nucleic acids in spect1 for the extraction of a quantitative DNA image. The energy stack file of DNA was then constructed and subtracted from spect1. The RNA image was obtained from the resulting stack file, spect2D. In the same manner, histone and BSA (as a representative of proteins other than histone) images were obtained. The residual stack file (spect3) contains spectra of molecules other than nucleic acids and proteins. In these processes, clear separation of the 1s–π * transition between N=C bond in nucleic acid at 399.6 eV and amide bond in protein at 401.4–401.5 eV  at the nTwo-dimensional distributions of DNA, RNA, histone, and proteins other than histone were estimated by the present method in all samples , showingInterestingly, we found that DNA and RNA were separated within the chromosome. Distribution of mass thickness  and RGB We observed highly different structures in the apoptotic nucleus e compareIn measuring local spectra in the absorbed image (spect1), we found similar spectral profiles in the CHO cell  and the In principle, X-ray microscopy is an imaging method complementary to optical microscopy and electron microscopy, with respect to the resolution defined by diffraction limit and transmittance. Therefore, it has been expected to be developed as a good candidate method for investigating biological samples. To date, the most successful biological application of X-ray microscopy seems to be the 3D observation of frozen hydrated samples. However, there is another approach: spectromicroscopy, which allows for direct observation of the quantitative distribution of biological molecules ,24.Our findings showing that DNA and histone were located in the nucleus, whereas RNA was clearly discriminated and found mainly in the cytoplasm, suggesting that the method reported here has enormous potential for examining a wide range of component molecules. We have laid out a simple process for analyzing the distribution of DNA and RNA for the observed absorption data (spect1) by the SVD method and subtraction of the energy stack-files of these molecules from spect1; then, analysis of distributions of proteins as many kinds of proteins as the absorption spectra observed for the subtracted energy stack file by the SVD method and subtraction of the energy stack-files for these molecules from the subtracted data; and so on. This process could be continued for all the remaining molecules, even minor components in the sample, so far as the spectra for them could be prepared. However, limitations should be considered for the fidelity of the results.To observe thick samples, such as the nucleus, it is not easy to supply a sufficient flux of the probing X-rays, as was shown in For imaging minor molecules, there is the same problem as discussed above. Higher flux is required for the identification of minor fraction. The possibility of success for imaging is a matter of trade-off. Therefore, there is a limitation to observing and analyzing minor fractions of molecules in the sample. Observation may be successful in the case where thin-sliced samples are able to be prepared, although the preparation solely depends on the purpose of the observation.Fixative may be a factor to modifying the X-ray image. In the present study, glutaraldehyde or a mixture of ethanol and acetic acid was used to fix the samples. These fixatives would not influence our results because they contain no nitrogen, and absorption peaks at the nitrogen K absorption edge region were applied for our analysis of quantitative distribution.We have established a method for the analysis of biological data obtained with X-ray spectromicroscopy and estimated the quantitative distribution of DNA, RNA, histone, and proteins other than histone in a CHO cell, the cytoplasmic area of a HeLa cell, an apoptotic nucleus, and a chromosome for the first time. In particular, the results revealed the distinctive distribution of RNA in an apoptotic nucleus and a chromosome. The results demonstrated that we could determine the spatial distribution of molecules in cells by the quantitative treatment of X-ray absorption and suggested that the present method, in combination with X-ray spectromicroscopy, has an infinite potential for extending the analysis to the quantitative distribution of various molecules of interest in accordance with the technical improvement of an imaging system. In other words, the present method will shed light on novel cellular localization of biological molecules, which may lead to unique studies, e.g., elucidation of functional roles of microRNAs in chromosomal architecture or apoptotic structures.


Thionucleotides, especially 4-thiouridine and 6-thioguanosine, are photosensitive molecules that photocrosslink to both proteins and nucleic acids, and this feature is a major reason for their application in various investigations. To get insight into the thermodynamic and structural contributions of 6-thioguanosine to the properties of RNA duplexes a systematic study was performed. In a series of RNA duplexes, selected guanosine residues located in G-C base pairs, mismatches , or 5′ and 3′-dangling ends were replaced with 6-thioguanosine. Generally, the presence of 6-thioguanosine diminishes the thermodynamic stability of RNA duplexes. This effect depends on its position within duplexes and the sequence of adjacent base pairs. However, when placed at a dangling end a 6-thioguanosine residue actually exerts a weak stabilizing effect. Furthermore, the structural effect of 6-thioguanosine substitution appears to be minimal based on NMR and Circular Dichroism (CD) data. In addition to the thionucleotides listed above, there are also five 2-methylthio-N6-substituted derivatives of adenosine. The biological functions of some of these thionucleotides are discussed in several reviews2.RNA contains many modified nucleotides performing various biological functions, and thionucleotides constitute one group of these modified nucleotides. The following thionucleotides can be found among the 112 entries of the RNA modification database  and 6-thioguanosine (s6G), are photosensitive molecules that photocrosslink to both proteins and nucleic acids, and this feature is a major reason for their application in various investigations19. In contrast, for the same set of oligonucleotides modified with 2-thiouridine (s2U), it has been shown that s2U increases the stability of base pairing with adenosine more than that with guanosine. This correlation indicated that s2U preferentially pairs with adenosine over guanosine. To the best of our knowledge, there are no extensive studies concerning the contribution of s6G to the thermodynamic stabilities of RNA duplexes. However, it was previously reported that the incorporation of 2′-deoxy-6-thioguanosine diminishes the thermodynamic stability of DNA duplexes20. Extending these studies to RNA duplexes is important because there are several reports on the application of 6-thioguanosine residues for inter- and intramolecular photocrosslinking of RNAs and photocrosslinking of RNAs and proteins12. For example, derivatives of 6-thioguanosine have been incorporated into RNA via transcription with the 5′-O-triphosphate of 6-thioguanosine (s6GTP)21. A mixture of GTP and s6GTP was used for transcription, which resulted in the incorporation of a single s6G into target RNA. Incorporation of a single s6G into RNA did not change the overall structure of large RNAs; however, a local structural rearrangement is possible.It is important to evaluate whether the substitution of uridine with 4-thiouridine and guanosine with 6-thioguanosine influences the structure and stability of RNA. Studies of RNA duplex formation by oligonucleotides with either an internal or a terminal 4-thiouridine showed an increase in their thermodynamic stability and indicated that the presence of s4U increases the stability of base pairing with guanosine more than that with adenosineIn this report, we performed extensive studies to obtain detailed information on the influence of s6G on the thermodynamic stabilities of RNA duplexes and selected guanosines located in G-C base pairs, mismatches , and 5′ and 3′-dangling ends were replaced with 6-thioguanosine. Together, eleven 6-thioguanosine containing RNA duplexes were investigated. The influence of s6G on the thermodynamic stability of RNA was obtained by comparison with unmodified duplexes with guanosine at the same position. Generally, the presence of 6-thioguanosine diminished the thermodynamic stability of RNA duplexes, however, this effect depended on its position within duplexes and the sequence of adjacent base pairs. Furthermore, the structural effect of 6-thioguanosine substitution appeared to be minimal based on NMR and Circular Dichroism (CD) data.Guanosine that was used for synthesis and most other reagents  were purchased from Sigma-Aldrich. Dimethoxytrityl chloride and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite were purchased from ChemGenes. Solvents were used at the highest available purity and dried with molecular sieves, if necessary. The chemical stepwise synthesis of 3′-O-phosphoramidite of the protected 6-thioguanosine is described in detail in Supplementary Materials.22 and commercially available phosphoramidites . The synthesis was conducted using a standard RNA protocol, except that milder conditions were used for oxidation . For deprotection, 6-thioguanosine oligoribonucleotides were treated with a mixture of isoamylamine/pyridine (2/1 v/v) for 24 hours at room temperature24. Silyl-protecting groups were cleaved by treatment with triethylamine  trihydrofluoride. Deprotected oligonucleotides were purified by silica gel thin layer chromatography (TLC) in 1-propanol/aqueous ammonia/water (55/35/10 v/v/v), as described in detail previously23.Oligonucleotides were synthesized on a BioAutomation MerMade12 DNA/RNA synthesizer using β-cyanoethyl phosphoramidite chemistry−3–10−6 M on a JASCO V-650 UV/Vis spectrophotometer in buffer containing 1 M sodium chloride, 20 mM sodium cacodylate, and 0.5 mM Na2EDTA (pH 7)25. Oligonucleotide single strand concentrations were calculated from the absorbance above 80 °C, and single strand extinction coefficients were approximated by a nearest-neighbor model. The extinction coefficient of 6-thioguanosine was considered the same as for guanosine26. Absorbance vs. temperature melting curves were measured at a wavelength of 260 nm with a heating rate of 1 °C/min from 0 to 90 °C on a JASCO V-650 spectrophotometer with a thermoprogrammer. The melting curves were analyzed, and the thermodynamic parameters were calculated from a two-state model with MeltWin 3.5 software27. For most sequences, ∆H° derived from TM−1 vs. ln(CT/4) plots was within 15% of that derived from averaging the fits to individual melting curves, which was expected if the two-state model is reasonable.Thermodynamic measurements were performed for nine different concentrations of RNA duplex in the range of 102EDTA (pH 7.0) to achieve a 20 µM sample concentration. All samples were denatured for 3 min at 90 °C and then slowly cooled to room temperature overnight before data collection. The measurements were taken at 5 °C in the 205–350 nm wavelength range with a 1 nm data point interval. CD curves were established as an average of three CD measurements. The buffer spectrum was subtracted from the sample spectra.CD spectra were recorded on a JASCO J-815 spectropolarimeter using 1.5 mL quartz cuvettes with a 5 mm path length. Oligonucleotides were dissolved in a buffer containing 1 M sodium chloride, 20 mM sodium cacodylate and 0.5 mM Na2EDTA. To ensure that the RNA was uniquely present in the duplex form, samples were further washed with the same buffer on an Amicon centrifugal filter with a 3 kDa molecular weight cutoff (any excess single-stranded RNA would pass through the pores of the Amicon membrane). NMR data were collected on a Bruker AVANCE III 700 MHz spectrometer, equipped with a QCI CryoProbe. 1D proton NMR spectra as well as 2D 1H-1H NOESY spectra were measured in H2O/D2O  at 25 °C and 5 °C. An additional set of NOESY spectra was acquired in 99.990% D2O (Sigma Aldrich) at 25 °C to facilitate the assignment of nonexchangeable protons. All the observable imino and amino protons as well as the nonexchangeable aromatic and anomeric protons were assigned using standard methodologies28, as described in the Supplementary Materials and exemplified in Figs For the acquisition of NMR data, all RNA duplexes were dissolved in a 10 mM sodium phosphate buffer (pH 6.8) containing 150 mM sodium chloride and 0.1 mM Na29, 2-cyanoethyl30 and 2-[(2-ethylhexyl) oxycarbonyl] ethyl31 were all applied previously for the protection of the sulfur atom; in this study, the 2,4-dinitrophenyl group was used  of the reference duplexes M9, M11, M13 and M15 were 13.74, 13.37, 10.28 and 10.19 kcal/mol, respectively. The replacement of guanosine with 6-thioguanosine at the central position in duplexes M10, M12, M14 and M16 significantly diminished their thermodynamic stability to 11.10, 10.89, 8.53 and 7.61 kcal/mol, respectively. The changes in the thermodynamic stability (ΔΔG°37) of duplexes for 5′UGA/3′ACU, 5′AGU/3′UCA, 5′CGG/3′GCC, and 5′GGC/3′CCG (where G corresponds to 6-thioguanosine) 6-thioguanosine arrangements were 2.58, 1.75, 2.48 and 2.64 kcal/mol, respectively. Thus, for three among the tested arrangements, the destabilizing effect of 6-thioguanosine was consistently around 2.5–2.6 kcal/mol, while for the fourth one, containing an adenosine residue 5′-adjacent to 6-thioguanosine, the destabilizing effect was around 30% weaker.We found that replacing G with s6G in a base pair with C always destabilized RNA duplexes and the effectiveness of the destabilization was dependent on the sequence and arrangement of adjacent base pairs. The thermodynamic stability  of 10.41 and 10.98 kcal/mol, respectively. This finding indicates that due to the presence of 5′- or 3′-dangling ends, the stability of M1 and M3 duplexes increased by 0.15 and 1.02 kcal/mol, respectively. Alternatively, placing 6-thioguanosine at the 5′- and 3′-dangling ends led to the formation of M2 and M4 duplexes with thermodynamic stabilities of 10.96 and 11.70 kcal/mol, respectively . Therefore, it can be concluded that replacing guanosine with 6-thioguanosine contributes to the stability of RNA duplexes by 0.55 and 0.72 kcal/mol for 5′- and 3′-dangling ends, respectively.In general, the presence of 5′- and 3′-dangling ends stabilizes RNA duplexes38. Stacking interactions of a terminal base pair (considered as 5′- and 3′-dangling ends) and hydrogen bonding of terminal base pairs both contribute to the thermodynamic stability of a terminal base pair. Comparisons of the stabilization effects of 6-thioguanosine on 5′- and 3′-dangling ends with the destabilization effect of a terminal s6G-C base pair suggest that weakened hydrogen bonding is responsible for the overall destabilization effect of the terminal s6G-C base pair39. A compensation between the effects of stacking and hydrogen bonding is very often observed in RNA39.It is postulated that the thermodynamic effects of dangling ends are related to their stacking ability43. In comparison to canonical G-C base pair mismatches, G-G, G-A and G-U stabilize RNA duplexes much less efficiently. Indeed, the thermodynamic stability of duplex M9 with a canonical G-C base pair and duplexes with G-G (M17), G-U (M19) and G-A (M21) mismatches were 13.74, 10.30, 11.53 and 8.45 kcal/mol, respectively. These results show that the presence of G-G, G-U and G-A mismatches destabilizes the canonical duplex M9 by 3.44, 2.21 and 5.29 kcal/mol, respectively. Replacing guanosine with 6-thioguanosine results in the formation of either the fully complementary duplex (M10) or duplexes containing s6G-G (M18), s6G-U (M20) and s6G-A (M22) mismatches with thermodynamic stabilities of 11.10, 7.74, 9.56 and 6.63 kcal/mol, respectively. These results indicate that the presence s6G in duplexes containing s6G-G (M18), s6G-U (M20) and s6G-A (M22) mismatches diminished destabilization by 3.36, 1.54 and 4.27 kcal/mol, respectively. A comparison of the destabilization effect resulting from the presence of mismatches with guanosine and 6-thioguanosine indicated that the mismatches formed by s6G were further destabilized (ΔΔG°37) by 0.29, 0.30 and 0.65 kcal/mol when s6G formed mismatches with G, U and A, respectively.Guanosine is a unique nucleotide that, in addition to its canonical interactions with C, also pairs with A, G and U44. The replacement of guanosine with 6-thioguanosine within the tested RNA duplexes containing base-paired and mismatched s6G results in red shift of the maximum ca. 260 nm by 5–10 nm, without altering the overall shape of the spectrum . The line broadening was severe enough that for M14, the G5 imino proton was barely visible at 25 °C, while for M12, it only became observable at 5 °C , as well as their unmodified counterparts , were investigated by NMR spectroscopy. The chemical shift changes (ΔCS) induced by the G to s6G modification were rather limited and qualitatively very similar for all four studied pairs of duplexes. The ΔCS observed for the M9-M10 and M11-M12 pairs of duplexes are shown in Tables  °C Fig. . This brdownfield with respect to the nonmodified duplex  or its s6G-modified counterpart (M20) at an internal position, was also investigated by NMR. The ΔCS measured for nonexchangeable protons were comparable to those observed for the duplexes with modified G-C pairs and, once again, localized in direct proximity of the modified base pair. This finding suggests that also for the G-U base pair, the G to s6G substitution results in very minimal structural rearrangements. The imino protons of G and U residues involved in the s6G-U wobble pair were both shifted by ca. 0.2 ppm lex Fig. . In contlex Fig. . This NM46 and thermodynamic studies47 indicated that, for a s6dG-T wobble pair in a DNA duplex, the s6G modification actually slightly stabilized the structure. In order to formulate a plausible explanation for this difference, the effects of s6G introduction into G-C pairs in DNA and RNA were also compared. The DNA study by Bohon and de los Santos47 reports an approximately 0.9 kcal/mol destabilization at 37 °C accompanied by around 3 °C decrease in TM when s6G is introduced in the 5′TGA/3′ACT sequential context. On the other hand, our current data reveal a 2.6 kcal/mol destabilization and around 10 °C drop in TM when the same substitution is made in RNA. Thus, the destabilizing effect observed in RNA appears to be much more pronounced. Taken together, the results for s6G-C base pairs and for the s6G-U mismatches could indicate that the s6G modification is easier to accommodate in B-form helices (DNA) compared to A-form helices (RNA). However, much caution is required when directly comparing these data due to different polymer types and different overall sequences.Interestingly, contrary results were previously observed for DNA. Both NMR6-Thioguanosine residues were placed in duplexes at central and terminal positions with various sequences and arrangements of 5′- and 3′-adjacent base pairs, as well as, at 3′- and 5′-dangling ends. Placing the s6G residue at the center of RNA duplexes diminished their thermodynamic stability up to 2.64 kcal/mol, and this destabilizing effect did not substantially depend on the sequence and arrangement of 5′- and 3′-adjacent pairs. The presence of 6-thioguanosine in 5- or 3′-terminal base pairs of the duplexes reduced their stability by ca. 1 kcal/mol. However, when guanosine 5′- and 3′-dangling ends were replaced with the s6G residue, the thermodynamic stability of RNA duplexes was enhanced by 0.5–0.7 kcal/mol. Comparisons of the influence of s6G involved in base pair formation as well as its presence on 5′- and 3′-dangling ends suggest that replacing guanosine with s6G led to increased stacking interactions, while diminishing hydrogen bonding strength within RNA duplexes.The presence of the s6G modification within s6G-G, s6G-U and s6G-A mismatches also destabilized RNA duplexes; however, this effect was smaller than the destabilization due to the introduction of G-G, G-U and G-A mismatches themselves without s6G modification.NMR investigations indicated that the structures of RNA duplexes containing natural base pairs and base pairs with s6G modification were similar. This observation is consistent with the thermodynamic data and CD spectra reported herein, as well as, with the previously reported results for DNA duplexes containing 2′-deoxy-6-thioguanosine. Taken together, our results demonstrate that the presence of 6-thioguanosine in helical regions of RNA only negligibly affects their structure. This finding is reassuring for photocrosslinking studies in which a single 6-thioguanosine residue is usually present in the group of RNAs as its presence should not alter the formed secondary structures.48.On the other hand, in some special cases minor structural and electronic changes induced by the 6-thioguanosine substitution may significantly affect the properties of RNA molecules. For example, a 6-thioguanosine introduced at the active sites of ribozymes may be enough to affect their activity. Thus, in this context, a G to s6G substitution could provide valuable data regarding the mechanisms of action of these types of RNA moleculesSupplementary Info


Eukaryotic mRNA has a cap structure and a poly(A) tail at the 5′ and 3′ ends, respectively. The cap structure is recognized by eIF (eukaryotic translation initiation factor) 4 F, while the poly(A) tail is bound by poly(A)-binding protein (PABP). PABP has four RNA recognition motifs (RRM1–4), and RRM1-2 binds both the poly(A) tail and eIF4G component of eIF4F, resulting in enhancement of translation. Here, we show that PABP interacts with the 40S and 60S ribosomal subunits dynamically via RRM2-3 or RRM3-4. Using a reconstituted protein expression system, we demonstrate that wild-type PABP activates translation in a dose-dependent manner, while a PABP mutant that binds poly(A) RNA and eIF4G, but not the ribosome, fails to do so. From these results, functional significance of the interaction of PABP with the ribosome is discussed. These terminal structures enhance translation. The cap structure is bound by eukaryotic translation initiation factor (eIF) 4F, which comprises three proteins . eIF4E interacts directly with the cap, and eIF4A is an ATP-dependent RNA helicase thought to work with the RNA-binding initiation factor eIF4B to unwind the secondary structure of the 5′-untranslated region of the mRNA. eIF4G is a large modular scaffolding protein with binding sites for eIF4E, eIF4A and eIF3, a multi-subunit initiation factor that interacts directly with the small (40S) ribosomal subunit3. The N-terminal two RRMs (RRM1-2) bind poly(A) RNA as well as eIF4G6.The poly(A) tail is bound by poly(A)-binding protein (PABP), which consists of four RNA-recognition motifs  and a C-terminal portion containing an unstructured region and an α-helical peptide-binding domain (PABC)7. PABP binding to eIF4G activates translation initiation most probably by stabilizing the association of eIF4G with the mRNA9.Targeting of poly(A) RNA and eIF4G to RRM1-2  of PABP is cooperative, ensuring communication between the mRNA 5′ and 3′ ends11. Paip 2 also can bind PABC although less avidly, and a similar mode of binding to PABP is observed for Paip112. Eukaryotic release factor 3 (eRF3) and the microRNA-related protein GW182 bind to PABC to influence translational and post-translational events13.In addition, PABP harbors binding sites for other molecules important for translation and metabolism of RNA and proteins. Paip (PABP-interacting protein) 2 binds RRM2-3 of PABP in competition with the poly(A) RNA and eIF4G, resulting in translation inhibitionHere we report that PABP dynamically associates with the ribosomal RNAs, and this interaction might support translation.14. PABP-free ribosomes were prepared as follows: a crude ribosome preparation obtained from HeLa S3 cells (1.5 L) was treated with 1 mM puromycin at 37 °C for 10 min and then centrifuged at 15,000 × g for 10 min at 4 °C. The supernatant was again centrifuged at 15,000 × g for 10 min at 4 °C. The supernatant (2.5 ml) was incubated with GST-Paip215 (2 mg) at room temperature for 30 min and then resolved by the HiPrep 16/60 Sephacryl S-500 HR gel-filtration column , equilibrated with a buffer  sucrose, 1 mM DTT). Elution was carried out with the same buffer at a flow rate of 0.5 ml/min, and 1.5-ml fractions were collected with the ÄKTAprime plus system . The fractions that contained ribosomes (fraction numbers: from 30 to 51) were combined and concentrated using the Amicon Ultra-15  to approximately 5 ml.Ribosomes that were not depleted of PABP were purified as described16. The resulting mixture was resolved by 10–40% (wt/vol) sucrose-gradient centrifugation with a buffer  at 25,000 rpm in the SW28 rotor (Beckman Coulter) for 16 h at 4 °C. Two-milliliter fractions were successively taken from the top of the gradient, and an aliquot of each fraction was analyzed by SDS-PAGE followed by Coomassie brilliant blue staining.This ribosome-rich sample was incubated again with GST-Paip2 (2 mg) at room temperature for 30 min. After incubation, the sample was mixed with KCl  and a mixture of ATP and magnesium acetate  to disrupt contaminating chaperonin CCTThe fractions that mainly contained the 40S subunit were combined and diluted by four times with a sucrose-free buffer . The diluted 40S fraction was concentrated using Amicon Ultra-15  to approximately 5 ml. The fractions that mainly contained the 60S subunits were also diluted and concentrated in the same manner. These 40S and 60S fractions were incubated with GST-Paip2 (2 mg) at room temperature for 30 min and separately resolved by sucrose-gradient centrifugation in the same conditions as described above but without pre-treatment with ATP. The fractions that contained the 40S subunit only and the 60S subunit only were concentrated as described above and dialyzed against a 100-fold volume of a buffer  for 3 h at 4 °C using a dialysis membrane , and then against a new batch of the same buffer overnight.17.The dialyzed samples of the 40S and 60S subunits were applied to GST-Paip2-bound Glutathione Sepharose 4B resin . The unbound fraction was collected and then passed through Glutathione Sepharose 4B resin (0.2 ml) to remove possibly contaminating GST-Paip2. The samples were concentrated as described above and the concentration of 40S and 60S ribosomes was determined by measuring optical density (OD) at 260 nmg for 30 min at 4 °C. The supernatant was then loaded onto a Ni-NTA resin , and the resin was washed with the same buffer (10 ml) and further washed with another buffer . The proteins were eluted in a stepwise manner with increasing concentrations of imidazole  in a buffer . The eluates with 50, 100, and 250 mM imidazole were combined and passed through the Q Sepharose resin  to remove possibly contaminating RNA. The flow-through fraction was applied onto the Heparin Sepharose resin , and the resin was then washed with a buffer . Bound proteins were eluted in a step-wise manner that increased the concentration of KCl from 300 to 1000 mM in a buffer . The eluates with 300 and 400 mM KCl were combined and concentrated by Amicon Ultra-15  to approximately 2.5 ml and then loaded onto a PD-10 column  equilibrated with a buffer . After elution with 3.5 ml of the same buffer, the eluate was concentrated.For PABP-His, PABP- His-PA, and deletion mutants-His-PA, a bacterial strain BL-21 (DE-3) was transformed with pET28b-PABP-His, pET28b-PABP-His-PA, or deletion mutants-His-PA and grown in Luria broth (300 ml) at 37 °C until OD at 600 nm reached 0.4 to 0.6. Isopropyl-β-D-thiogalactopyranoside was added to 0.1 mM, and cells were cultured at 30 °C for 12 to 16 h. After washing with a buffer , the bacterial pellet was kept at −20 °C until use. The frozen pellet was suspended in a buffer ), lysed by sonication, and centrifuged at 15,000 × PABP- or 12C-FLAG: BL-21 (DE3) was transformed with pET28 PABP-FLAG or pET28 12C-FLAG. Proteins were expressed as described for PABP-His-PA and purified with anti-FLAG M2 agarose chromatography (Sigma).PABP-His (60 pmol) was mixed with the 40S subunit (30 pmol) or the 60S subunit (30 pmol) in a buffer  in the presence or absence of an RNA fragment . Then, the 40S subunit or the 60S subunit (4.8 pmol each) was added to the resin in the binding buffer (50 μl) and incubated on ice for 15 min. After incubation, the unbound fraction was removed and the resin was washed five times with the washing buffer (400 μl each). Bound materials were eluted by incubation with a buffer  for 10 min at room temperature. The eluate was analyzed by western blotting with antibodies against ribosomal protein S6, ribosomal protein L13a or the PA tag (WAKO).Full-length PABP-His-PA or truncated versions of the protein (3 μg each) were immobilized on TALON metal affinity resin  by incubation in a binding buffer  at room temperature for 30 min. After incubation, the unbound fraction was removed by centrifugation at 5000 × The following components were mixed in a test tube: eIF1 (61 ng), eIF1A (76 ng), eIF2 (547 ng), eIF2B (51 ng), eIF3 (100 ng), eIF4A (570 ng), eIF4B (144 ng), eIF4G/eIF4E (141 ng), eIF5 (76 ng), eIF5B (85 ng), DHX29 (67 ng), tRNA (5 μg), eEF1 (25 μg), eEF2 (500 ng), eRF1 (125 ng), eRF3 (125 ng), 40S ribosomal subunit (4.8 pmol), 60S ribosomal subunit (4.8 pmol), aminoacyl-tRNA synthetases (750 ng), amino acid mixture , and 10 × reaction buffer . This combination (4.5 μl) was then mixed with a template RNA (0.5 μl) and PABP or a truncated PABP protein , and incubated for 1.5–6 h at 32 °C.An aliquot (1 μl) from the translated sample was used for the luciferase reporter assay using the Dual-Luciferase Reporter Assay system (Promega). The remaining sample (4 μl) was subjected to western blotting with anti-HA tag  or anti-Myc tag  antibody. Protein bands were analyzed using the ImageQuant LAS 4000 mini (Fujifilm).in vitro dissociation experiment, CRAC analysis and other methods, see Supplementary Methods.For the During purification of ribosomes from HeLa cells according to the procedure outlined in Fig. A crude ribosomal preparation from HeLa cell lysates was treated with puromycin and resolved by gel-filtration chromatography Fig. . Each frThese fractions containing both the ribosomes and PABP were resolved by sucrose- gradient centrifugation Fig. . The 40S18, and recent proteomics studies included PABP in the list of the numerous ribosome-associated proteins of yeast19 and mammals20.To further purify 40S and 60S ribosomal subunits, these 40S and 60S fractions were individually subjected to the second sucrose-gradient centrifugation Fig. , and theThe purified 40S and 60S ribosomal subunits were estimated to contain PABP molecules roughly at a 10:1 molar ratio each, meaning that the molar ratio of PABP to the ribosome (40S + 60S subunits) would be 1:5 in the final preparation, as assayed by western blot Fig. . BecauseS-transferase (GST)-Paip215 at multiple purification steps, indicated by dotted arrows in Fig. 15. With this procedure, both the 40 and 60S subunits were depleted of PABP to undetectable levels  and resolved the mixture by 10–40% sucrose-gradient centrifugation. When PABP alone was resolved, it mainly remained near the top of the gradient Fig. . In addiBecause PABP binds poly(A) RNA, we next examined whether a ternary complex consisting of the ribosome, PABP and poly(A) RNA could be formed. When poly(A) RNA  RNA or the HA-Rluc-N RNA. The 40S and 60S subunits were dissociated from the resin in an RNA dose-dependent manner, leaving PABP-His on the resin Fig. . These rBecause PABP interacts with eIF4G, we then examined whether a ternary complex consisting of the ribosome, PABP, and eIF4G could be formed. When eIF4G/4E, PABP, and the 40S or 60S subunit were mixed and resolved by the sucrose-gradient centrifugation, eIF4G did not co-sediment with the ribosomes  domains and the C-terminal domain . Poly(A) RNA binds to RRM1-2, and eIF4G binds to RRM2in vitro binding assay using recombinant PABP fragments and the PABP-free 40S or 60S ribosomal subunits. The carboxy-terminal His-PA-tagged PABP protein (1-2-3-4-C-His-PA), various RRM-deletion mutants . The PABP-depleted 40S or 60S subunits and the recombinant PABP-His-PA protein were incubated and irradiated with UV light. The samples were then treated with guanidine-HCl to disrupt the ribosomal structure and incubated with a nickel resin. After extensive washes, the resin was treated with RNases, and protein–RNA complexes containing PABP-His-PA were eluted with imidazole. Complementary DNA for the UV-crosslinked RNA was cloned and sequenced -cross-linking and analysis of cDNAs) method 23 Fig. , probabl14. To establish an eIF-dependent translation system, we purified all the eIFs -supported protein synthesis system reconstituted with mammalian (human) factors. We previously constructed an eIF-independent translation system by assembling the 40S subunit, 60S subunit, eEF1, eEF2, eRF1/3, tRNAs and aminoacyl tRNA synthetases with an HCV (hepatitis C virus) IRES -driven templateWe then incubated this system with each of the four types of Rluc-based mRNA (Rluc: uncapped without poly(A); Rluc-A: uncapped with poly(A); cap-Rluc: capped without poly(A); and cap-Rluc-A: capped with poly(A); the open reading frame was tagged with HA and myc at the N- and C-termini, respectively) in the presence or absence of PABP. After incubation, translational activity was assessed by western blot with an anti-myc antibody and by Rluc activity. Both assays showed that the cap-structure and poly(A) tail stimulate translation, and the highest productivity was observed with the capped and poly(A)-tailed RNA in the presence of PABP Fig. .Figure 59, which cannot bind to eIF4G, was used in place of wild-type PABP, no activation was observed regardless of the presence of the poly(A) tail , stimulation by PABP was abolished  structures.When a PABP mutant (M161A)7, but not to the ribosome -supported translation system, we explored the functional significance of the association of PABP with the ribosome. For this aim, we used PABP (1-2-C), a PABP mutant that binds to poly(A) and eIF4Gome Fig. . Low dosome Fig. . A similome Fig. .Figure 627. The translation repression by Paip2 might in part trace to dissociation of PABP from the ribosome  in the presence of ABCE1These outcomes might mean that, although PABP in general binds directly to mRNA and may thus stimulate translation, some of the highly concentrated PABP molecules could be trapped by the ribosome for translation activation; a large part of the same concentration of PABP (1-2-C) molecules would remain unavailable to translation. However, the possibility is not rigorously excluded that RRM3-4 would have a function other than binding to the ribosome, and truncation of RRM3-4 would diminish the PABP function in translation.29, with an estimated concentration of 4 μM in HeLa cells17. Consequently, only 30% of PABP molecules are thought to bind to the poly(A) tail of mRNAs with the majority of PABP being associated with non-poly(A) RNAs29. Considering that the concentration of ribosomes is also high (approximately 1.5 μM in HeLa cells)29, the ribosome would be expected to be a primary site for free PABP to bind. Because the interaction between PABP and the ribosome is dynamic; PABP is readily dissociated with a non-specific RNA  signal33 and A-rich sequences of non-coding RNAs, probably via RRM1-2 and/or RRM3-434. The expansion segments of the ribosome, which are GC-rich23, likely interact with RRM2-3 and RRM 3-4 of PABP  tail, as its name indicates, but it also is reported to bind RNA sequences other than the poly(A) tails. PABP binds to AU-rich sequences in the 5′ and 3′ untranslated regions9 experiments, PABP also was shown to stimulate translation of poly(A)-minus mRNA to some extent. It is likely that PABP is recruited to the translation machinery in multiple ways, including through the ribosome. For example, RNA-binding proteins such as the herpes simplex virus-1 protein, ICP27 and a cellular mRNA-specific regulator, Deleted in Azoospemia-like  bind both PABP and a specific mRNA to enhance its translation37. Of note, the length of the poly(A) tail is not directly related to translational efficiency in somatic cells39.In the present Fig.  and prev23. The expansion segments might be used as hubs for PABP because the segments that protrude from the ribosomal body are unlikely to participate in protein synthesis directly; therefore, the binding to them should not interfere with translation. Thus, these segments might trap not only PABP but also other RNA-binding proteins to support translation.Finally, we demonstrated the PABP-binding sites included the expansion segments of the ribosome. The expansion segments of the mammalian ribosome consist mostly of duplexed RNA, and some of the segments protrude from the ribosome body like tentaclesSupplementary information


The structure of a 22-base-pair RNA helix with mismatched pyrimidine base pairs is reported. The pyrimidine mismatches are unusual and display sheared, cross-strand stacking geometries that locally constrict the helical width. The structure of a 22-base-pair RNA helix with mismatched pyrimidine base pairs is reported. The helix contains two symmetry-related CUG sequences: a triplet-repeat motif implicated in myotonic dystrophy type 1. The CUG repeat contains a U–U mismatch sandwiched between Watson–Crick pairs. Additionally, the center of the helix contains a dimerized UUCG motif with tandem pyrimidine (U–C/C–U) mismatches flanked by U–G wobble pairs. This region of the structure is significantly different from previously observed structures that share the same sequence and neighboring base pairs. The tandem pyrimidine mismatches are unusual and display sheared, cross-strand stacking geometries that locally constrict the helical width, a type of stacking previously associated with purines in internal loops. Thus, pyrimidine-rich regions of RNA have a high degree of structural diversity. However, only a few percent of the structural coordinates in the Worldwide Protein Data Bank correspond to RNA, and some of these are redundant. At the time of writing, there are 1213 unique RNA-containing structures at a resolution of 3 Å or higher in the RNA 3D Motif Atlas  is a heritable disease caused by the expansion of genomically encoded CUG repeats in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) mRNA ] to provide a platform for analyzing compounds designed to target the CUG repeat sequence  and immobilized on a 1 ml HiTrap Q column . The column was washed with ten volumes of buffer prior to step elution in anion-exchange buffer supplemented with 2 M NaCl. The resulting eluate was concentrated using centrifugal filters with a 3 kDa cutoff (Amicon) and then iteratively diluted tenfold and reconcentrated three times using a buffer containing only 20 mM deuterated bis-Tris pH 6.5. The RNA was then concentrated to 150 µM. A small aliquot of this RNA was resolved on an analytical nondenaturing polyacrylamide gel, which showed a trace amount (∼5%) of RNA migrating as a slower species that is presumed to be an intermolecular dimer (data not shown).A putative RNA hairpin (5′-GGGCUGCACUUCGGUGCUGCCC-3′) was purchased from Integrated DNA Technologies. The synthesized RNA was resuspended in anion-exchange buffer (300 mM (∼10 mg ml−1) prior to monitoring its association with the compound ‘JFA’ . For this process, the compound JFA was in 100% DMSO and was added stepwise to a final approximate twofold stoichiometric excess, resulting in a 300 µl sample containing approximately 800 µM RNA, 1600 µM JFA, 5% DMSO, 5% D2O and 20 mM deuterated bis-Tris pH 6.5%. The RNA with ‘JFA’ was finally concentrated using 3 kDa cutoff centrifugal filters (Amicon) to a volume of approximately 100 µl without additional treatment before crystallization screening.The RNA was subsequently concentrated to approximately 1.5 m2.2.M HEPES pH 7.4, 20% PEG 3350, 20% glycerol, 10% MPD. Crystals were harvested with 100 µm LD MicroLoops (MiTeGen) and vitrified via rapid immersion in liquid nitrogen.High-throughput crystallization screening was performed by sitting-drop vapor diffusion in 96-well plates at 4°C using 0.2 µl RNA solution, 0.2 µl crystallization reagent and a reservoir volume of 50 µl with a Mosquito crystallization robot (TTP Labtech). After a few weeks, several small crystals (∼10 × 50 µm) were obtained using a crystallization reagent consisting of 0.1 et al., 2013XDS . Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 6e7l; diffraction images are available from the SBGrid Data Bank at https://doi.org/10.15785/SBGRID/712.Initial phases were determined by molecular replacement using 3.a)]. The crystals diffracted X-rays to 2.59 Å resolution [Fig. 1b) and Table 1c)]. For the purposes of discussion, we give one strand in the duplex the numbering 1–22 and the other 1′–22′. The RNA adopts an A-form geometry for all nucleotides except the UUCG sequence regions, which are involved in crystal contacts [Figs. 1d) and 2a)].The 22-nucleotide RNA strand contains two CUG repeats and a UUCG sequence, and is capable of forming a hairpin or a duplex conformation . The U–C base pairs form a hydrogen bond between the uracil O2 and the cytosine N3 amino group. An additional inter-strand hydrogen bond is formed between the uracil N3 and the uracil ribose O2′. This conformation is significantly different from previous structures of the same sequence, which lacked cross-strand stacking ]. The cross-strand stacked U–C base pairs are flanked by U–G wobble pairs. The U–G wobble-pair region is involved in helical packing within the crystal lattice [Fig. 1d)], mediated by minor-groove interactions that are stabilized by inter-helical hydrogen bonds involving 2′ hydroxyl groups, similar to ‘ribose-zipper’ interactions  form helices with U–U mismatches that display heterogeneous base-pairing patterns et al., 2005et al., 1997et al., 1994et al., 2006et al., 1990et al., 2001Cross-strand stacking in RNA tertiary structure typically involves purines (Chen dimerized UUCG motif, 6e7lPDB reference: https://doi.org/10.15785/SBGRID/712Diffraction images.:


Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD NCIN-meRNAP II)  and eukaRNAP II) . Thus, w+ and NADH, respectively . The eukaryotic nuclear RNAPs--RNAP I, II, and III--are multi-subunit RNAPs closely related in sequence and structure to bacterial RNAP . Furthermore, NudC treatment of full-length products obtained with [32P]-NAD+ yielded products that were sensitive to alkaline phosphatase NCIN / (kcat/KM)ATP, for mtRNAPs NCIN / (kcat/KM)ATP of ~0.3 to ~0.4 for NCIN-mediated initiation with NAD+ and NADH by S. cerevisiae mtRNAP and ~0.2 to ~0.6 for NCIN-mediated initiation with NAD+ and NADH by human mtRNAP. These values imply that NCIN-mediated initiation with NAD+ or NADH is up to 40% as efficient as initiation with ATP for S. cerevisiae mtRNAP and up to 60% as efficient as initiation with ATP for human mtRNAP.We next determined the relative efficiencies of NCIN-mediated initiation vs. ATP-mediated initiation,  (+ and NADH by T7 RNAP were nearly as high as the efficiencies of NCIN-mediated initiation by mtRNAPs. We conclude that there is a quantitative difference in the efficiency of NCIN capping between members of the single-subunit RNAP family (T7 RNAP and mtRNAPs) and members of the multi-subunit RNAP family .We next used the same tailed template and reaction conditions as in assays performed with mtRNAPs to determine the efficiency of NCIN-mediated initiation with NADT7 RNAP) . The eff+ and NADH by bacterial RNAP is determined by promoter sequence, particularly at and immediately upstream of, the transcription start site (TSS)  . NCIN cate (TSS) .+ capping by S. cerevisiae mtRNAP using promoter derivatives having A:T or G:C at position +1  at position +1 , observed with bacterial RNAP, also is observed with mtRNAP, we assessed NADition +1 . We obseition +1 , indicatition +1 . We obse+ capping by S. cerevisiae mtRNAP using promoter derivatives having either R:Y (A:T or G:C) or Y:R (C:G or T:A) at position −1 . We further conclude that the sequence and strand preferences at positions +1 and −1 for NCIN capping with NAD+ by mtRNAP match the sequence and strand preferences observed for bacterial RNAP . Here, to overcome these limitations, we have combined boronate affinity electrophoresis with use of oligodeoxynucleotide-mediated RNA cleavage (‘DNAzyme’ cleavage)   and use leavage) . Use of leavage) . Use of leavage) .+ and NADH in mitochondrial RNA isolated from cells, we employed the following steps: (i) DNAzyme cleavage of target RNAs to generate 5'-end-containing subfragments < 80 nt in length , and show that S. cerevisiae mitochondrial RNAs also undergo NADH capping in cells.We selected for analysis two  and 21S . We isol+- and NADH-capping of S. cerevisiae mitochondrial RNAs are remarkably high. For COX2 RNA, NAD+-capped RNA comprises ~50% of the total COX2 RNA pool and NADH-capped RNA comprises ~10% of the total COX2 RNA pool   .+-capped species comprising ~15% of the total LSP-derived RNA pool .We performed analogous experiments analyzing RNAs produced by transcription from the human mitochondrial LSP promoter , top. WeRNA pool , top. Th+ to NADH and oxidative phosphorylation oxidizes NADH to NAD+. Our finding that mtRNAPs perform NCIN capping with NAD+ and NADH at efficiencies that vary in a simple mass-action-dependent fashion with [NAD+] / [ATP] and [NADH] / [ATP] ratios in vitro  cycle and oxidative phosphorylation. The TCA cycle reduces NADin vitro  and 2, a in vivo  and 5, r+] / [NADH] ratios results in changes in NAD+ and NADH capping of mitochondrial RNAs. We isolated total RNA from S. cerevisiae grown under conditions that result in either high or low [NAD+] / [NADH] ratios  and an anti-correlated increase in the level of NADH capping (from ~10% to ~40%). Notably, the total level of NAD+ and NADH capping, NAD(H) capping, remains constant under the two conditions (~60%), indicating that the relative levels of NCIN-mediated initiation and ATP-mediated initiation do not change , the level of NADH capping increases (from 0% to ~20%), and the total level of NAD+ and NADH capping remains constant (~30%). The results indicate that changing the [NAD+] / [NADH] ratio changes transcription outputs in vivo.As a first test of this hypothesis, we assessed whether changing intracellular [NAD] ratios : respira] ratios . We obset change . For 21St change : on chanAs a second test of this hypothesis, we assessed whether changing intracellular total NAD(H) levels results in changes in NCIN capping of mitochondrial RNAs . We isol+] / [NADH] ratios to relative levels of NAD+- and NADH-capped mitochondrial RNAs   , in conthondria) . Fourth, in vivo  and 5, w+ and NADH capping by mtRNAP correlate with changes in intracellular levels of NAD+ and NADH  was prepared from E. coli strain BL21(DE3) transformed with pJJ1399 (gift of Judith A. Jaehning) using culture and induction procedures, polyethyleneimine treatment, ammonium sulfate precipitation, Ni-sepharose, DEAE sepharose and Heparin-sepharose chromatography as in (S. cerevisiae Mtf1 was prepared from E. coli strain BL21(DE3) transformed with pTrcHisC-Mtf1 (hy as in . S. cereisC-Mtf1  and puriisC-Mtf1 .E. coli strain BL21(DE3) transformed with pPROEXHTb-POLRMT(43–1230)−6xHis (E. coli strain ArcticExpress (DE3) (Stratagene) transformed with pT7TEV-HMBP4 (Human mtRNAP (POLRMT) and TFAM were purified from 0)−6xHis  or pPROE0)−6xHis , respect0)−6xHis . Human TEV-HMBP4 , using cEV-HMBP4 .E. coli strain BL21 transformed with pAR1219 using culture and inductions procedures, SP-Sephadex, CM-Sephadex and DEAE-Sephacel chromatography as described in (T7 RNAP was prepared from ribed in .E. coli RNAP core enzyme was prepared from E. coli strain NiCo21(DE3) transformed with plasmid pIA900 (d pIA900  using cud pIA900 .S. cerevisiae RNA polymerase II core enzyme (gift of Craig Kaplan) was prepared as described in (ribed in .E. coli NudC was prepared from E. coli strain NiCo21(DE3) transformed with plasmid pET NudC-His (NudC-His  using meNudC-His .RNA 5' pyrophosphohydrolase (RppH) and T4 polynucleotide kinase (PNK) were purchased from New England Biolabs (NEB). FastAP Thermosensitive Alkaline Phosphatase was purchased from Thermo Fisher Scientific. Molar concentrations of purified proteins were determined by light absorbance at 280 nm and the calculated respective molar extinction coefficients.Sequences of the oligodeoxyribonucleotides used in this work are provided in Key Resources Table. All oligodeoxyribonucleotides were purchased from Integrated DNA Technologies (IDT) with standard desalting purification unless otherwise specified.Linear in vitro transcription templates used for transcription assays shown in Transcription templates used to generate in vitro RNA standards for Northern analysis  and 5 we32P]-ATP .The radiolabeled 10-nt marker labeled ‘M’ in gel images shown in S. cerevisiae mtRNAP were based on procedures described in . A mixture containing the initiating nucleotide  and extending nucleotide  was added, and assays were incubated at 25°C for 30 min. For assays in For initial RNA product assays in +, or 1 mM NADH) and extending nucleotide  was added, and assays were incubated at 25°C for 60 min. Radiolabeled initial RNA products were isolated using a Nanosep 3 kDa cutoff spin concentrator .For the initial RNA product assays in A portion of the recovered initial RNA products were mixed with either 10 U of RppH or 400 nM NudC and incubated at 37°C for 30 min. Reactions were stopped by addition of 10 µl RNA loading dye. Samples were analyzed by electrophoresis on 7.5 M urea, 1x TBE, 20% polyacrylamide gels , followed by storage-phosphor imaging .S. cerevisiae mtRNAP, and 1 µM Mtf1 were incubated at 25°C for 10 min in Sce-mtRNAP reaction buffer. A mixture containing the initiating nucleotide .For full-length product assays in +, or 1 mM NADH for 32P]-ATP  or 1 mM NAD+ plus 20 µCi [α32P]-NAD+  for 32P]-UTP  for For full-length product assays in Full-length RNA products were incubated at 37°C for 30 min with 400 nM NudC alone , 0.25 U S. cerevisiae mtRNAP; TFAM and TFB2M for human mtRNAP) were incubated at 25°C for 10 min in Sce-mtRNAP or human reaction buffer. A mixture containing 200 µM ATP, 200 µM UTP, 200 µM non-radiolabeled GTP, and 6 mCi [α32P]-GTP at 3000 Ci/mmol and NCIN  was added, and assays were incubated at 25°C for 30 min. Reactions were stopped by addition of an equal volume of RNA loading dye. Samples were analyzed by electrophoresis on 7.5 M urea, 1x TBE, 20% polyacrylamide gels  supplemented with 0.2% 3-acrylamidophenylboronic acid (Boron Molecular), followed by storage-phosphor imaging .For experiments in cat/KM)NCIN / (kcat/KM)ATP is equal to 1/x. Data for determination of relative efficiencies are means of three technical replicates.Bands corresponding to uncapped (pppRNA) and NCIN-capped (NCIN-RNA) full-length products were quantified using ImageQuant software. The ratio of NCIN-RNA to total RNA [NCIN-RNA / (pppRNA + NCIN RNA)] was plotted vs. the relative concentrations of NCIN vs. ATP ([NCIN] / [ATP]) on a semi-log plot (SigmaPlot) and non-linear regression was used to fit the data to the equation: y = (ax) / (b + x); where y is [NCIN-RNA / (pppRNA + NCIN RNA)], x is ([NCIN] / [ATP]), and a and b are regression parameters. The resulting fit yields the value of x for which y = 0.5. The relative efficiency  were incubated at 25°C for 10 min in Sce-mtRNAP, human mtRNAP reaction buffer, or T7 RNAP reaction buffer . A mixture containing 1 mM NCIN, ATP , 20 µM non-radiolabeled GTP, and 6 mCi [α32P]-GTP at 3000 Ci/mmol was added, and assays were incubated at 25°C for 30 min. Reactions were stopped by addition of an equal volume of RNA loading dye. Samples were analyzed by electrophoresis on 7.5 M urea, 1x TBE, 20% polyacrylamide gels  supplemented with 0.2% 3-acrylamidophenylboronic acid (Boron Molecular), followed by storage-phosphor imaging .For experiments in S. cerevisiae mtRNAP, human mtRNAP, S. cerevisiae RNAP II, T7 RNAP, or E. coli RNAP) were incubated at 25°C for 15 min in reaction buffer containing 10 mM Tris pH 8.0, 50 mM potassium glutamate, 10 mM MgCl2, 2 mM DTT, and 50 ug/ml BSA. A mixture containing NCIN , ATP , 10 µM non-radiolabeled CTP, and 6 mCi [α32P]-CTP  was added, and assays were incubated at 25°C for 1 hr. Reactions were stopped by addition of an equal volume of RNA loading dye. Samples were analyzed by electrophoresis on 7.5 M urea, 1x TBE, 20% polyacrylamide gels , followed by storage-phosphor imaging .For experiments in cat/KM)NCIN / (kcat/KM)ATP is equal to 1/x. Data for determination of relative efficiencies are means of three technical replicates.Bands corresponding to uncapped (pppApC) and NCIN-capped (NCINpC) initial RNA products were quantified using ImageQuant software. The ratio of NCINpC to total RNA (NCINpC / [pppApC + NCINpC]) was plotted vs. the relative concentrations of NCIN vs. ATP ([NCIN] / [ATP]) on a semi-log plot (SigmaPlot) and non-linear regression was used to fit the data to the equation: y = (ax) / (b + x); where y is [NCINpC / (pppApC + NCINpC)], x is ([NCIN] / [ATP]), and a and b are regression parameters. The resulting fit yields the value of x for which y = 0.5. The relative efficiency  in 125 ml flasks (Bellco) shaken at 220 rpm. When cell density reached an OD600 ~1.8 (approximately 24 hr) the cell suspension was centrifuged to collect cells , supernatants were removed, and cell pellets were resuspended in 0.8 mL RNA extraction buffer .For analysis of NAD246.1.1  in 125 ml flasks (Bellco) with airlocks to prevent oxygenation without shaking for 42 hr. The cell suspension was centrifuged to collect cells , supernatants were removed, and cell pellets were resuspended in 0.8 mL RNA extraction buffer .For analysis of NAD2O.To extract RNA, an equal volume of acid phenol:chloroform  was added to cells in resuspension buffer and mixed by vortexing. Samples were incubated at 65°C for 5 min, −80°C for 5 min, then centrifuged  to separate the aqueous and organic phases. The aqueous phase was collected and acid phenol:chloroform extraction was performed two more times on this solution. RNA transcripts were recovered by ethanol precipitation and resuspended in RNase free H2 at 37°C in DMEM medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum , 100 units/ml penicillin, and 100 µg/ml streptomycin. HEK293T cells were seeded in 100 mm tissue-culture treated plates and grown for 72 hr at 37°C or seeded in 100 mm tissue-culture treated plates, grown for 24 hr at 37°C, treated with 5 nM FK866 (Sigma Aldrich), and grown for an additional 48 hr at 37°C. Total cellular RNA was isolated with TRIzol Reagent according to the manufacture’s protocol (Thermo Fisher Scientific).Human embryonic kidney HEK293T cells  were maintained under 5% COS. cerevisiae mitochondrial RNA, 40 µg of total cellular RNA was mixed with 1 µM DNAzyme  in buffer containing 10 mM Tris pH 8.0, 50 mM NaCl, 2 mM DTT, and 10 mM MgCl2 . When present, NudC was added to 400 nM. Reactions were incubated for 60 min at 37°C and 100 µl of stop solution and 500 µl ethanol was added. Samples were centrifuged , the supernatant removed, and the pellet resuspended in RNA loading dye.For analysis of NCIN capping of 2 was added to a final concentration of 10 mM and, when present, NudC was added to 400 nM. Reactions were incubated for 60 min at 37°C and 100 µl of stop solution and 500 µl ethanol was added. Samples were centrifuged , the supernatant removed, and the pellet resuspended in RNA loading dye.For analysis of NCIN capping of human mitochondrial RNA, 40 µg of total cellular RNA was mixed with 1 µM DNAzyme (JB559 for human LSP-generated RNA) in buffer containing 10 mM Tris pH 8.0, 50 mM NaCl, 2 mM DTT . Samples were heated to 85°C for 5 min, cooled to 37°C. MgCl+ or NADH), 200 µM GTP, 200 µM UTP, 200 µM CTP, and 20 µM ATP. Reactions were incubated for 60 min at 25°C, stopped by addition of stop solution, extracted with acid phenol:chloroform  and ethanol precipitated. Full-length RNAs were resuspended in buffer containing 10 mM Tris pH 8.0, 50 mM NaCl, 2 mM DTT, and 10 mM MgCl2 and treated with DNAzyme as described above.To prepare synthetic RNA standards non-radiolabeled full-length RNA products were generated by in vitro transcription reactions containing 1 µM of template DNA, 1 µM of mtRNAP, 1 µM transcription factor(s), 1 mM initiating nucleotide ; (v) high-stringency washing, procedures as in  electrophoresis on 7.5 M urea, 1x TBE, 10% polyacrylamide gels supplemented with 0.2% 3-acrylamidophenylboronic acid (Boron Molecular); (ii) transfer of nucleic acids to a Nytran supercharge nylon membrane  using a semidry transfer apparatus (Bio-Rad); (iii) immobilization of nucleic acids by UV crosslinking; (iv) incubation with a es as in ; and (vi+-capped RNA (5'-NAD+), or NADH-capped RNA (5'-NADH) to total RNA were determined from three biological replicates.Bands corresponding to uncapped and NCIN-capped DNAzyme-generated subfragments were quantified using ImageQuant software. The percentages of uncapped RNA (5'-ppp), NADCells were lysed in 400 μl of NAD/H Extraction Buffer  and total proteins were extracted with two freeze-thaw cycles . Lysates were centrifuged , the supernatant was isolated, and the protein concentration was determined. NAD(H) was measured using a NAD/H Quantitation Kit (Sigma) from a volume of supernatant containing 10 μg protein.3 for HEK293T cells and assuming a homogenous distribution of NAD(H) in the cell. The volume of HEK293T cells was calculated using a value of 13 µm for the diameter of HEK293T cells, assuming trypsinized cells adopt a spherical shape. NAD/H concentrations were determined from three biological replicates.Molecules of NAD(H) per cell were calculated using a value of 300 pg total protein per cell. The cellular concentration of NAD(H) was then estimated by using a cellular volume of ~1200 µm In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by James Manley as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ann Hochschild (Reviewer #2).Thank you for submitting your article "Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:E. coli and T7 RNA Pol enzymes, they show by careful enzyme kinetics that the mt enzymes are considerably more efficient than the yeast and E. coli enzymes at NAD+ capping in vitro. They further show that the mt enzymes and T7 Pol, all single polypeptide polymerases, exhibit the same promoter sequence specificity for the reaction demonstrated previously for the E. coli multi-subunit enzyme, requiring a T on the template strand for the TSS (+1 position) and a purine:pyrimidine base pair on the non-template:template strands at the -1 position. They introduce a significant advance in the techniques for quantifying the occurrence of NAD+ and NADH, vs uncapped mt transcripts produced in vivo, and show that the proportions of NAD+/NADH vs. uncapped transcripts for two yeast and one human mt transcripts are much higher than observed previously for bacterial transcripts and yeast cytoplasmic mRNAs. Finally, they show that the ratio of NAD+ vs NADH capped mRNAs for the two yeast mt mRNAs vary in response to altered growth conditions expected to change the NAD+/NADH ratio in cells, providing evidence that mt RNAP could be utilized as a sensor of energy metabolism that would generate altered ratios of NAD+ vs NADH-capped mt mRNAs. In view of their findings that a large fraction of the mt transcripts harbor one or the other cap, this has the potential of altering mt gene expression through possible influences of these caps on mRNA translation or turnover.This paper uses a combination of in-depth biochemical analysis and a novel in vivo approach to provide important new insights into NAD/NADH capping of eukaryotic mitochondrial transcripts and of this capping process in general. Using purified RNA polymerases from yeast or human mitochondria (mt), as well yeast nuclear, Although NAD+ capped yeast mt mRNAs had been detected previously, this work is significant in providing direct evidence that mt RNAPs carry out this reaction and utilize the same promoter sequences found for multi-subunit RNAPs to direct capping; and they also function much more efficiently than do the latter. The results also provide evidence that the capped mt transcripts detected in cells represent capping during transcription initiation vs. capping of degradation products. By measuring the precise proportions of NAD+-capped, NADH-capped, and uncapped mt mRNAs in cells-which is much higher than seen previously for bacterial or yeast cytoplasmic mRNAs-they show that the ratio of NAD+ to NADH-capped mRNAs varies with the energy status of yeast cells.Essential revisions:E. coli cells in addition to the different capping efficiencies of the respective RNA polymerases, and cite any information available about these concentrations.It is requested that you discuss how the relative levels of NAD/NADH capping of mitochondrial versus cytoplasmic mRNAs could be affected by different concentrations of NAD/NADH in the mitochondria versus eukaryotic nuclei or It is requested that you provide a much better explanation of how the "fork-junction" template works and how it bypasses the requirement for sequence-specific RNAP-DNA interactions and transcription-initiation factor-DNA interactions for transcription initiation, as neither the text nor cartoon were found to be adequate.It is requested that you state more explicitly how the conclusion that "mitochondrial RNAs undergo NAD+ capping at 5' ends generated by transcription initiation . Second, levels of NAD+ and NADH relative to ATP in mitochondria are substantially higher than levels of NAD+ and NADH relative to ATP in bacteria and eukaryotic nuclei .”“First, mtRNAPs are substantially more efficient at NADIt is requested that you provide a much better explanation of how the "fork-junction" template works and how it bypasses the requirement for sequence-specific RNAP-DNA interactions and transcription-initiation factor-DNA interactions for transcription initiation, as neither the text nor cartoon were found to be adequate.We have revised the text to indicate that the template used for these assays is a “tailed” template and provided references establishing use of a tailed template to bypass the requirement for sequence-specific RNAP-DNA interactions and transcription-initiation factor-DNA interactions. The revised text is:“…we performed transcription assays using a “tailed” template  that bypasses the requirement for sequence-specific RNAP-DNA interactions and transcription-initiation factor-DNA interactions for transcription initiation .”It is requested that you state more explicitly how the conclusion that "mitochondrial RNAs undergo NAD+ capping at 5' ends generated by transcription initiation , the detected NADIt is requested that you discuss what is known about whether the capping ratio affects the amount of transcript, the stability of the transcript, or the efficiency with which it is translated.We have modified the text at the beginning of the second paragraph of the Discussion. The revised text is:“We and others previously have shown that NCIN capping by cellular RNAPs has functional consequences, including modulating RNA stability and modulating RNA translatability.”