A highly optimized human in vitro translation system.

In vitro translation is an important method for studying fundamental aspects of co- and post-translational gene regulation, as well as for protein expression in the laboratory and on an industrial scale. Here, by re-examining and improving a human in vitro translation system (HITS), we were able to develop a minimal system where only four components are needed to supplement human cell lysates. Functional characterization of our improved HITS revealed the synergistic effect of mRNA capping and polyadenylation. Furthermore, we found that mRNAs are translated with an efficiency equal to or higher than existing state-of-the-art mammalian in vitro translation systems. Lastly, we present an easy preparation procedure for cytoplasmic extracts from cultured HeLa cells, which can be performed in any cell culture laboratory. These methodological advances will allow HITSs to become a widespread tool in basic molecular biology research.

Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.

5'-cap RNA/SAM mimetic conjugates as bisubstrate inhibitors of viral RNA cap 2'-O-methyltransferases.

Viral RNA cap 2'-O-methyltransferases are considered promising therapeutic targets for antiviral treatments, as they play a key role in the formation of viral RNA cap-1 structures to escape the host immune system. A better understanding of how they interact with their natural substrates (RNA and the methyl donor SAM) would enable the rational development of potent inhibitors. However, as few structures of 2'-O-MTases in complex with RNA have been described, little is known about substrate recognition by these MTases. For this, chemical tools mimicking the state in which the cap RNA substrate and SAM cofactor are bound in the enzyme's catalytic pocket may prove useful. In this work, we designed and synthesized over 30 RNA conjugates that contain a short oligoribonucleotide (ORN with 4 or 6 nucleotides) with the first nucleotide 2'-O-attached to an adenosine by linkers of different lengths and containing S or N-heteroatoms, or a 1,2,3-triazole ring. These ORN conjugates bearing or not a cap structure at 5'-extremity mimic the methylation transition state with RNA substrate/SAM complex as bisubstrates of 2'-O-MTases. The ORN conjugates were synthesized either by the incorporation of a dinucleoside phosphoramidite during RNA elongation or by click chemistry performed on solid-phase post-RNA elongation. Their ability to inhibit the activity of the nsp16/nsp10 complex of SARS-CoV-2 and the NS5 protein of dengue and Zika viruses was assessed. Significant submicromolar IC50 values and Kd values in the µM range were found, suggesting a possible interaction of some ORN conjugates with these viral 2'-O-MTases.

On-column capping of poly dT media-tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase.

The messenger RNA (mRNA) 5'-cap structure is indispensable for mRNA translation initiation and stability. Despite its importance, large-scale production of capped mRNA through in vitro transcription (IVT) synthesis using vaccinia capping enzyme (VCE) is challenging, due to the requirement of tedious and multiple pre-and-post separation steps causing mRNA loss and degradation. Here in the present study, we found that the VCE together with 2'-O-methyltransferase can efficiently catalyze the capping of poly dT media-tethered mRNA to produce mRNA with cap-1 structure under an optimized condition. We have therefore designed an integrated purification and solid-based capping protocol, which involved capturing the mRNA from the IVT system by using poly dT media through its affinity binding for 3'-end poly-A in mRNA, in situ capping of mRNA 5'-end by supplying the enzymes, and subsequent eluting of the capped mRNA from the poly dT media. Using mRNA encoding the enhanced green fluorescent protein as a model system, we have demonstrated that the new strategy greatly simplified the mRNA manufacturing process and improved its overall recovery without sacrificing the capping efficiency, as compared with the conventional process, which involved at least mRNA preseparation from IVT, solution-based capping, and post-separation and recovering steps. Specifically, the new process accomplished a 1.76-fold (84.21% over 47.79%) increase in mRNA overall recovery, a twofold decrease in operation time (70 vs. 140 min), and similar high capping efficiency (both close to 100%). Furthermore, the solid-based capping process greatly improved mRNA stability, such that the integrity of the mRNA could be well kept during the capping process even in the presence of exogenously added RNase; in contrast, mRNA in the solution-based capping process degraded almost completely. Meanwhile, we showed that such a strategy can be operated both in a batch mode and in an on-column continuous mode. The results presented in this work demonstrated that the new on-column capping process developed here can accomplish high capping efficiency, enhanced mRNA recovery, and improved stability against RNase; therefore, can act as a simple, efficient, and cost-effective platform technology suitable for large-scale production of capped mRNA.

Targeting cap1 RNA methyltransferases as an antiviral strategy.

Methylation is one of the critical modifications that regulates numerous biological processes. Guanine capping and methylation at the 7th position (m7G) have been shown to mature mRNA for increased RNA stability and translational efficiency. The m7G capped cap0 RNA remains immature and requires additional methylation at the first nucleotide (N1-2'-O-Me), designated as cap1, to achieve full maturation. This cap1 RNA with N1-2'-O-Me prevents its recognition by innate immune sensors as non-self. Viruses have also evolved various strategies to produce self-like capped RNAs with the N1-2'-O-Me that potentially evades the antiviral response and establishes an efficient replication. In this review, we focus on the importance of the presence of N1-2'-O-Me in viral RNAs and discuss the potential for drug development by targeting host and viral N1-2'-O-methyltransferases.

Ceg1 depletion reveals mechanisms governing degradation of non-capped RNAs in Saccharomyces cerevisiae.

Most functional eukaryotic mRNAs contain a 5' 7-methylguanosine (m7G) cap. Although capping is essential for many biological processes including mRNA processing, export and translation, the fate of uncapped transcripts has not been studied extensively. Here, we employed fast nuclear depletion of the capping enzymes in Saccharomyces cerevisiae to uncover the turnover of the transcripts that failed to be capped. We show that although the degradation of cap-deficient mRNA is dominant, the levels of hundreds of non-capped mRNAs increase upon depletion of the capping enzymes. Overall, the abundance of non-capped mRNAs is inversely correlated to the expression levels, altogether resembling the effects observed in cells lacking the cytoplasmic 5'-3' exonuclease Xrn1 and indicating differential degradation fates of non-capped mRNAs. The inactivation of the nuclear 5'-3' exonuclease Rat1 does not rescue the non-capped mRNA levels indicating that Rat1 is not involved in their degradation and consequently, the lack of the capping does not affect the distribution of RNA Polymerase II on the chromatin. Our data indicate that the cap presence is essential to initiate the Xrn1-dependent degradation of mRNAs underpinning the role of 5' cap in the Xrn1-dependent buffering of the cellular mRNA levels.

Chikungunya virus nonstructural protein 1 is a versatile RNA capping and decapping enzyme.

Chikungunya virus (CHIKV) nonstructural protein 1 (nsP1) contains both the N7-guanine methyltransferase and guanylyltransferase activities and catalyzes the 5' end cap formation of viral RNAs. To further understand its catalytic activity and role in virus-host interaction, we demonstrate that purified recombinant CHIKV nsP1 can reverse the guanylyl transfer reaction and remove the m7GMP from a variety of capped RNA substrates including host mRNAs. We then provide the structural basis of this function with a high-resolution cryo-EM structure of nsP1 in complex with the unconventional cap-1 substrate RNA m7GpppAmU. We show that the 5'ppRNA species generated by decapping can trigger retinoic acid-inducible gene I-mediated interferon response. We further demonstrate that the decapping activity is conserved among the alphaviral nsP1s. To our knowledge, this is a new mechanism through which alphaviruses activate the antiviral immune response. This decapping activity could promote cellular mRNA degradation and facilitate viral gene expression, which is functionally analogous to the cap-snatching mechanism by influenza virus.

Future Perspectives for the Identification and Sequencing of Nicotinamide Adenine Dinucleotide-Capped RNAs.

Ribonucleic acid (RNA) is composed primarily of four canonical building blocks. In addition, more than 170 modifications contribute to its stability and function. Metabolites like nicotinamide adenine dinucleotide (NAD) were found to function as 5'-cap structures of RNA, just like 7-methylguanosine (m7G). The identification of NAD-capped RNA sequences was first made possible by NAD captureSeq, a multistep protocol for the specific targeting, purification, and sequencing of NAD-capped RNAs, developed in the authors' laboratory in the year 2015. In recent years, a number of NAD-RNA identification protocols have been developed by researchers around the world. They have enabled the discovery and identification of NAD-RNAs in bacteria, archaea, yeast, plants, mice, and human cells, and they play a key role in studying the biological functions of NAD capping. We introduce the four parameters of yield, specificity, evaluability, and throughput and describe to the reader how an ideal NAD-RNA identification protocol would perform in each of these disciplines. These parameters are further used to describe and analyze existing protocols that follow two general methodologies: the capture approach and the decapping approach. Capture protocols introduce an exogenous moiety into the NAD-cap structure in order to either specifically purify or sequence NAD-capped RNAs. In decapping protocols, the NAD cap is digested to 5'-monophosphate RNA, which is then specifically targeted and sequenced. Both approaches, as well as the different protocols within them, have advantages and challenges that we evaluate based on the aforementioned parameters. In addition, we suggest improvements in order to meet the future needs of research on NAD-modified RNAs, which is beginning to emerge in the area of cell-type specific samples. A limiting factor of the capture approach is the need for large amounts of input RNA. Here we see a high potential for innovation within the key targeting step: The enzymatic modification reaction of the NAD-cap structure catalyzed by ADP-ribosyl cyclase (ADPRC) is a major contributor to the parameters of yield and specificity but has mostly seen minor changes since the pioneering protocol of NAD captureSeq and needs to be more stringently analyzed. The major challenge of the decapping approach remains the specificity of the decapping enzymes, many of which act on a variety of 5'-cap structures. Exploration of new decapping enzymes or engineering of already known enzymes could lead to improvements in NAD-specific protocols. The use of a curated set of decapping enzymes in a combinatorial approach could allow for the simultaneous detection of multiple 5'-caps. The throughput of both approaches could be greatly improved by early sample pooling. We propose that this could be achieved by introducing a barcode RNA sequence before or immediately after the NAD-RNA targeting steps. With increased processing capacity and a potential decrease in the cost per sample, protocols will gain the potential to analyze large numbers of samples from different growth conditions and treatments. This will support the search for biological roles of NAD-capped RNAs in all types of organisms.

The Nuclear Cap-Binding Complex, a multitasking binding partner of RNA polymerase II transcripts.

In eukaryotic cells, RNAs transcribed by RNA polymerase-II receive the modification at the 5' end. This structure is called the cap structure. The cap structure has a fundamental role for translation initiation by recruiting eukaryotic translation initiation factor 4F (eIF4F). The other important mediator of the cap structure is a nuclear cap-binding protein complex (CBC). CBC consists of two proteins, which are renamed as NCBP1 and NCBP2 (previously called as CBP80/NCBP and CBP20/NIP1, respectively). This review article discusses the multiple roles CBC mediates and co-ordinates in several gene expression steps in eukaryotes.

Identification of tanshinone I as cap-dependent endonuclease inhibitor with broad-spectrum antiviral effect.

IMPORTANCE: The spread of avian-borne, tick-borne, and rodent-borne pathogens has the potential to pose a serious threat to human health, and candidate vaccines as well as therapeutics for these pathogens are urgently needed. Tanshinones, especially tanshinone I, were identified as a cap-dependent endonuclease inhibitor with broad-spectrum antiviral effects on negative-stranded, segmented RNA viruses including bandavirus, orthomyxovirus, and arenavirus from natural products, implying an important resource of candidate antivirals from the traditional Chinese medicines. This study supplies novel candidate antivirals for the negative-stranded, segmented RNA virus and highlights the endonuclease involved in the cap-snatching process as a reliable broad-spectrum antiviral target.

Biochemical characterization of mRNA capping enzyme from Faustovirus.

The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here, we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity toward RNA containing secondary structures and a free 5' end, and a broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.

Crystal structure of a cap-independent translation enhancer RNA.

In eukaryotic messenger RNAs, the 5' cap structure binds to the translation initiation factor 4E to facilitate early stages of translation. Although many plant viruses lack the 5' cap structure, some contain cap-independent translation elements (CITEs) in their 3' untranslated region. The PTE (Panicum mosaic virus translation element) class of CITEs contains a G-rich asymmetric bulge and a C-rich helical junction that were proposed to interact via formation of a pseudoknot. SHAPE analysis of PTE homologs reveals a highly reactive guanosine residue within the G-rich region proposed to mediate eukaryotic initiation factor 4E (eIF4E) recognition. Here we have obtained the crystal structure of the PTE from Pea enation mosaic virus 2 (PEMV2) RNA in complex with our structural chaperone, Fab BL3-6. The structure reveals that the G-rich and C-rich regions interact through a complex network of interactions distinct from those expected for a pseudoknot. The motif, which contains a short parallel duplex, provides a structural mechanism for how the guanosine is extruded from the core stack to enable eIF4E recognition. Homologous PTE elements harbor a G-rich bulge and a three-way junction and exhibit covariation at crucial positions, suggesting that the PEMV2 tertiary architecture is conserved among these homologs.

A systems-level mass spectrometry-based technique for accurate and sensitive quantification of the RNA cap epitranscriptome.

Chemical modifications of transcripts with a 5' cap occur in all organisms and function in many aspects of RNA metabolism. To facilitate analysis of RNA caps, we developed a systems-level mass spectrometry-based technique, CapQuant, for accurate and sensitive quantification of the cap epitranscriptome. The protocol includes the addition of stable isotope-labeled cap nucleotides (CNs) to RNA, enzymatic hydrolysis of endogenous RNA to release CNs, and off-line enrichment of CNs by ion-pairing high-pressure liquid chromatography, followed by a 17 min chromatography-coupled tandem quadrupole mass spectrometry run for the identification and quantification of individual CNs. The total time required for the protocol can be up to 7 d. In this approach, 26 CNs can be quantified in eukaryotic poly(A)-tailed RNA, bacterial total RNA and viral RNA. This protocol can be modified to analyze other types of RNA and RNA from in vitro sources. CapQuant stands out from other methods in terms of superior specificity, sensitivity and accuracy, and it is not limited to individual caps nor does it require radiolabeling. Thanks to its unique capability of accurately and sensitively quantifying RNA caps on a systems level, CapQuant can reveal both the RNA cap landscape and the transcription start site distribution of capped RNA in a broad range of settings.

DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction.

Recent discoveries of noncanonical RNA caps, such as nicotinamide adenine dinucleotide (NAD+) and 3'-dephospho-coenzyme A (dpCoA), have expanded our knowledge of RNA caps. Although dpCoA has been known to cap RNAs in various species, the identities of its capped RNAs (dpCoA-RNAs) remained unknown. To fill this gap, we developed a method called dpCoA tagSeq, which utilized a thiol-reactive maleimide group to label dpCoA cap with a tag RNA serving as the 5' barcode. The barcoded RNAs were isolated using a complementary DNA strand of the tag RNA prior to direct sequencing by nanopore technology. Our validation experiments with model RNAs showed that dpCoA-RNA was efficiently tagged and captured using this protocol. To confirm that the tagged RNAs are capped by dpCoA and no other thiol-containing molecules, we used a pyrophosphatase NudC to degrade the dpCoA cap to adenosine monophosphate (AMP) moiety before performing the tagSeq protocol. We identified 44 genes that transcribe dpCoA-RNAs in mouse liver, demonstrating the method's effectiveness in identifying and characterizing the capped RNAs. This strategy provides a viable approach to identifying dpCoA-RNAs that allows for further functional investigations of the cap.

A unique mRNA decapping complex in trypanosomes.

Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts.

Blocking tri-methylguanosine synthase 1 (TGS1) stops anchorage-independent growth of canine sarcomas.

Tri methylguanosine synthase 1 (TGS1) is the enzyme that hyper methylates the hallmark 7-methyl-guanosine cap (m7G-cap) appended to the transcription start site of RNAs. The m7G-cap and the eIF4E-cap binding protein guide canonical cap-dependent translation of mRNAs, whereas hyper methylated cap, m2,2,7G-cap (TMG) lacks adequate eIF4E affinity and licenses entry into a different translation initiation pathway. The potential role for TGS1 and TMG-capped mRNA in neoplastic growth is unknown. Canine sarcoma has high translational value to the human disease. Cumulative downregulation of protein synthesis in osteosarcoma OSCA-40 was achieved cooperatively by siTGS1 and Torin-1. Torin-1 inhibited the proliferation of three canine sarcoma explants in a reversible manner that was eliminated by siRNA-downregulation of TGS1. TGS1 failure prevented the anchorage-independent growth of osteo- and hemangio-sarcomas and curtailed sarcoma recovery from mTOR inhibition. RNA immunoprecipitation studies identified TMG-capped mRNAs encoding TGS1, DHX9 and JUND. TMG-tgs1 transcripts were downregulated by leptomycin B and TGS1 failure was compensated by eIF4E mRNP-dependent tgs1 mRNA translation affected by mTOR. The evidence documents TMG-capped mRNAs are hallmarks of the investigated neoplasms and synergy between TGS1 specialized translation and canonical translation is involved in sarcoma recovery from mTOR inhibition. Therapeutic targeting of TGS1 activity in cancer is ripe for future exploration.

DXO gears mRNA with alternative NAD and m7G caps.

NAD is a noncanonical mRNA cap that challenges our traditional dogma of N7-methylguanosine (m7G)-capped eukaryotic mRNAs. The relationship between NAD and m7G caps has been elusive. Xiao et al. find that the deNADding enzyme DXO promotes maturation of m7G caps, suggesting that DXO fine-tunes the dynamic balance between alternative RNA cap structures.

Contribution of Nudt12 enzyme to differentially methylated dinucleotides of 5'RNA cap structure.

Recent findings have substantially broadened our knowledge about the diversity of modifications of the 5'end of RNAs, an issue generally attributed to mRNA cap structure (m7GpppN). Nudt12 is one of the recently described new enzymatic activities involved in cap metabolism. However, in contrast to its roles in metabolite-cap turnover (e.g., NAD-cap) and NADH/NAD metabolite hydrolysis, little is known regarding its hydrolytic activity towards dinucleotide cap structures. In order to gain further insight into this Nudt12 activity, comprehensive analysis with a spectrum of cap-like dinucleotides was performed with respect to different nucleotide types adjacent to the (m7)G moiety and its methylation status. Among the tested compounds, GpppA, GpppAm, and Gpppm6Am were identified as novel potent Nudt12 substrates, with KM values in the same range as that of NADH. Interestingly, substrate inhibition of Nudt12 catalytic activity was detected in the case of the GpppG dinucleotide, a phenomenon not reported to date. Finally, comparison of Nudt12 with DcpS and Nud16, two other enzymes with known activity on dinucleotide cap structures, revealed their overlapping and more specific substrates. Altogether, these findings provide a basis for clarifying the role of Nudt12 in cap-like dinucleotide turnover.

The RNA cap methyltransferases RNMT and CMTR1 co-ordinate gene expression during neural differentiation.

Regulation of RNA cap formation has potent impacts on gene regulation, controlling which transcripts are expressed, processed and translated into protein. Recently, the RNA cap methyltransferases RNA guanine-7 methyltransferase (RNMT) and cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1 (CMTR1) have been found to be independently regulated during embryonic stem (ES) cell differentiation controlling the expression of overlapping and distinct protein families. During neural differentiation, RNMT is repressed and CMTR1 is up-regulated. RNMT promotes expression of the pluripotency-associated gene products; repression of the RNMT complex (RNMT-RAM) is required for repression of these RNAs and proteins during differentiation. The predominant RNA targets of CMTR1 encode the histones and ribosomal proteins (RPs). CMTR1 up-regulation is required to maintain the expression of histones and RPs during differentiation and to maintain DNA replication, RNA translation and cell proliferation. Thus the co-ordinate regulation of RNMT and CMTR1 is required for different aspects of ES cell differentiation. In this review, we discuss the mechanisms by which RNMT and CMTR1 are independently regulated during ES cell differentiation and explore how this influences the co-ordinated gene regulation required of emerging cell lineages.

NADcapPro and circNC: methods for accurate profiling of NAD and non-canonical RNA caps in eukaryotes.

Accurate identification of NAD-capped RNAs is essential for delineating their generation and biological function. Previous transcriptome-wide methods used to classify NAD-capped RNAs in eukaryotes contain inherent limitations that have hindered the accurate identification of NAD caps from eukaryotic RNAs. In this study, we introduce two orthogonal methods to identify NAD-capped RNAs more precisely. The first, NADcapPro, uses copper-free click chemistry and the second is an intramolecular ligation-based RNA circularization, circNC. Together, these methods resolve the limitations of previous methods and allowed us to discover unforeseen features of NAD-capped RNAs in budding yeast. Contrary to previous reports, we find that 1) cellular NAD-RNAs can be full-length and polyadenylated transcripts, 2) transcription start sites for NAD-capped and canonical m7G-capped RNAs can be different, and 3) NAD caps can be added subsequent to transcription initiation. Moreover, we uncovered a dichotomy of NAD-RNAs in translation where they are detected with mitochondrial ribosomes but minimally on cytoplasmic ribosomes indicating their propensity to be translated in mitochondria.