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.

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.

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.

A universal fluorescence polarization high throughput screening assay to target the SAM-binding sites of SARS-CoV-2 and other viral methyltransferases.

SARS-CoV-2 has caused a global pandemic with significant humanity and economic loss since 2020. Currently, only limited options are available to treat SARS-CoV-2 infections for vulnerable populations. In this study, we report a universal fluorescence polarization (FP)-based high throughput screening (HTS) assay for SAM-dependent viral methyltransferases (MTases), using a fluorescent SAM-analogue, FL-NAH. We performed the assay against a reference MTase, NSP14, an essential enzyme for SARS-CoV-2 to methylate the N7 position of viral 5'-RNA guanine cap. The assay is universal and suitable for any SAM-dependent viral MTases such as the SARS-CoV-2 NSP16/NSP10 MTase complex and the NS5 MTase of Zika virus (ZIKV). Pilot screening demonstrated that the HTS assay was very robust and identified two candidate inhibitors, NSC 111552 and 288387. The two compounds inhibited the FL-NAH binding to the NSP14 MTase with low micromolar IC50. We used three functional MTase assays to unambiguously verified the inhibitory potency of these molecules for the NSP14 N7-MTase function. Binding studies indicated that these molecules are bound directly to the NSP14 MTase with similar low micromolar affinity. Moreover, we further demonstrated that these molecules significantly inhibited the SARS-CoV-2 replication in cell-based assays at concentrations not causing cytotoxicity. Furthermore, NSC111552 significantly synergized with known SARS-CoV-2 drugs including nirmatrelvir and remdesivir. Finally, docking suggested that these molecules bind specifically to the SAM-binding site on the NSP14 MTase. Overall, these molecules represent novel and promising candidates to further develop broad-spectrum inhibitors for the management of viral infections.

mRNA ageing shapes the Cap2 methylome in mammalian mRNA.

The mRNA cap structure is a major site of dynamic mRNA methylation. mRNA caps exist in either the Cap1 or Cap2 form, depending on the presence of 2'-O-methylation on the first transcribed nucleotide or both the first and second transcribed nucleotides, respectively1,2. However, the identity of Cap2-containing mRNAs and the function of Cap2 are unclear. Here we describe CLAM-Cap-seq, a method for transcriptome-wide mapping and quantification of Cap2. We find that unlike other epitranscriptomic modifications, Cap2 can occur on all mRNAs. Cap2 is formed through a slow continuous conversion of mRNAs from Cap1 to Cap2 as mRNAs age in the cytosol. As a result, Cap2 is enriched on long-lived mRNAs. Large increases in the abundance of Cap1 leads to activation of RIG-I, especially in conditions in which expression of RIG-I is increased. The methylation of Cap1 to Cap2 markedly reduces the ability of RNAs to bind to and activate RIG-I. The slow methylation rate of Cap2 allows Cap2 to accumulate on host mRNAs, yet ensures that low levels of Cap2 occur on newly expressed viral RNAs. Overall, these results reveal an immunostimulatory role for Cap1, and that Cap2 functions to reduce activation of the innate immune response.

Intrinsically Unstructured Sequences in the mRNA 3' UTR Reduce the Ability of Poly(A) Tail to Enhance Translation.

The 5' cap and 3' poly(A) tail of mRNA are known to synergistically stimulate translation initiation via the formation of the cap•eIF4E•eIF4G•PABP•poly(A) complex. Most mRNA sequences have an intrinsic propensity to fold into extensive intramolecular secondary structures that result in short end-to-end distances. The inherent compactness of mRNAs might stabilize the cap•eIF4E•eIF4G•PABP•poly(A) complex and enhance cap-poly(A) translational synergy. Here, we test this hypothesis by introducing intrinsically unstructured sequences into the 5' or 3' UTRs of model mRNAs. We found that the introduction of unstructured sequences into the 3' UTR, but not the 5' UTR, decreases mRNA translation in cell-free wheat germ and yeast extracts without affecting mRNA stability. The observed reduction in protein synthesis results from the diminished ability of the poly(A) tail to stimulate translation. These results suggest that base pair formation by the 3' UTR enhances the cap-poly(A) synergy in translation initiation.

A gel electrophoresis-based assay for measuring enzymatic RNA decapping activity.

RNA 5' ends are remarkably heterogeneous. In addition to the eukaryotic 5' methyl-7-Guanosine (m7G) cap, a number of primarily metabolite-based cap structures have been identified both in prokaryotic and eukaryotic systems. These metabolite caps include Nicotinamide Adenine Dinucleotide (NAD+/NADH), dephosphoCoenzyme A (dpCoA), Flavin Adenine Dinucleotide (FAD), dinucleotide polyphosphates and Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) (Chen et al., 2009; Kowtoniuk et al., 2009; Wang et al., 2019). The most highly studied of these new cap structures, 5' NAD, has significant effects on RNA stability (Bird et al., 2016; Jiao et al., 2017). Both prokaryotes and eukaryotes have decapping enzymes specific to these metabolite caps and decapping is an integral step in the control of RNA stability (Cahová et al., 2015; Jiao et al., 2017; Sharma et al., 2020; Zhang et al., 2020). To better study how these 5' metabolite RNAs are decapped, we present a method to (1) generate radiolabeled dinucleotide and "full length" 5' capped RNA substrates for use in decapping assays, (2) a simple decapping assay to test the activity of various enzymes on different 5' capped transcripts and (3) a gel electrophoresis-based method for the visualization and differentiation of 5' capped transcripts.

CMTr mediated 2'-O-ribose methylation status of cap-adjacent nucleotides across animals.

Cap methyltransferases (CMTrs) O methylate the 2' position of the ribose (cOMe) of cap-adjacent nucleotides of animal, protist, and viral mRNAs. Animals generally have two CMTrs, whereas trypanosomes have three, and many viruses encode one in their genome. In the splice leader of mRNAs in trypanosomes, the first four nucleotides contain cOMe, but little is known about the status of cOMe in animals. Here, we show that cOMe is prominently present on the first two cap-adjacent nucleotides with species- and tissue-specific variations in Caenorhabditis elegans, honeybees, zebrafish, mouse, and human cell lines. In contrast, Drosophila contains cOMe primarily on the first cap-adjacent nucleotide. De novo RoseTTA modeling of CMTrs reveals close similarities of the overall structure and near identity for the catalytic tetrad, and for cap and cofactor binding for human, Drosophila and C. elegans CMTrs. Although viral CMTrs maintain the overall structure and catalytic tetrad, they have diverged in cap and cofactor binding. Consistent with the structural similarity, both CMTrs from Drosophila and humans methylate the first cap-adjacent nucleotide of an AGU consensus start. Because the second nucleotide is also methylated upon heat stress in Drosophila, these findings argue for regulated cOMe important for gene expression regulation.

Mature Rotavirus Particles Contain Equivalent Amounts of 7meGpppG-Capped and Noncapped Viral Positive-Sense RNAs.

Viruses have evolved different strategies to overcome their recognition by the host innate immune system. The addition of caps at their 5' RNA ends is an efficient mechanism not only to ensure escape from detection by the innate immune system but also to ensure the efficient synthesis of viral proteins. Rotavirus mRNAs contain a type 1 cap structure at their 5' end that is added by the viral capping enzyme VP3, which is a multifunctional protein with all the enzymatic activities necessary to add the cap and also functions as an antagonist of the 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway. Here, the relative abundances of capped and noncapped viral RNAs during the replication cycle of rotavirus were determined. We found that both classes of rotaviral plus-sense RNAs (+RNAs) were encapsidated and that they were present in a 1:1 ratio in the mature infectious particles. The capping of viral +RNAs was dynamic, since different ratios of capped and noncapped RNAs were detected at different times postinfection. Similarly, when the relative amounts of capped and uncapped viral +RNAs produced in an in vitro transcription system were determined, we found that the proportions were very similar to those in the mature viral particles and in infected cells, suggesting that the capping efficiency of VP3, both in vivo and in vitro, might be close to 50%. Unexpectedly, when the effect of simultaneously knocking down the expression of VP3 and RNase L on the cap status of viral +RNAs was evaluated, we found that, even though at late times postinfection there was an increased proportion of capped viral RNAs in infected cells, the viral particles isolated from this condition contained equal ratios of capped and noncapped viral RNA, suggesting that there might be selective packaging of capped and noncapped RNAs. IMPORTANCE Rotaviruses have a genome composed of 11 segments of double-stranded RNA. Whether all 5' ends of the positive-sense genomic RNAs contained in the mature viral particles are modified by a cap structure is unknown. In this work, we characterized the relative proportions of capped and noncapped viral RNAs in rotavirus-infected cells and in viral particles by using a direct quantitative assay. We found that, independent of the relative proportions of capped/noncapped RNAs present in rotavirus-infected cells, there were similar proportions of these two kinds of 5'-modified positive-sense RNAs in the viral particles.

The mechanism of RNA capping by SARS-CoV-2.

The RNA genome of SARS-CoV-2 contains a 5' cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2'-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2'-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.

THRONE: A New Approach for Accurate Prediction of Human RNA N7-Methylguanosine Sites.

N7-methylguanosine (m7G) is an essential, ubiquitous, and positively charged modification at the 5' cap of eukaryotic mRNA, modulating its export, translation, and splicing processes. Although several machine learning (ML)-based computational predictors for m7G have been developed, all utilized specific computational framework. This study is the first instance we explored four different computational frameworks and identified the best approach. Based on that we developed a novel predictor, THRONE (A three-layer ensemble predictor for identifying human RNA N7-methylguanosine sites) to accurately identify m7G sites from the human genome. THRONE employs a wide range of sequence-based features inputted to several ML classifiers and combines these models through ensemble learning. The three-step ensemble learning is as follows: 54 baseline models were constructed in the first layer and the predicted probability of m7G was considered as a new feature vector for the sequential step. Subsequently, six meta-models were created using the new feature vector and their predicted probability was yet again considered as novel features. Finally, random forest was deemed as the best super classifier learner for the final prediction using a systematic approach incorporated with novel features. Interestingly, THRONE outperformed other existing methods in the prediction of m7G sites on both cross-validation analysis and independent evaluation. The proposed method is publicly accessible at: http://thegleelab.org/THRONE/ and expects to help the scientific community identify the putative m7G sites and formulate a novel testable biological hypothesis.

Identification and Characterization of the Interaction Between the Methyl-7-Guanosine Cap Maturation Enzyme RNMT and the Cap-Binding Protein eIF4E.

The control of RNA metabolism is an important aspect of molecular biology with wide-ranging impacts on cells. Central to processing of coding RNAs is the addition of the methyl-7 guanosine (m7G) "cap" on their 5' end. The eukaryotic translation initiation factor eIF4E directly binds the m7G cap and through this interaction plays key roles in many steps of RNA metabolism including nuclear RNA export and translation. eIF4E also stimulates capping of many transcripts through its ability to drive the production of the enzyme RNMT which methylates the G-cap to form the mature m7G cap. Here, we found that eIF4E also physically associated with RNMT in human cells. Moreover, eIF4E directly interacted with RNMT in vitro. eIF4E is only the second protein reported to directly bind the methyltransferase domain of RNMT, the first being its co-factor RAM. We combined high-resolution NMR methods with biochemical studies to define the binding interfaces for the RNMT-eIF4E complex. Further, we found that eIF4E competes for RAM binding to RNMT and conversely, RNMT competes for binding of well-established eIF4E-binding partners such as the 4E-BPs. RNMT uses novel structural means to engage eIF4E. Finally, we observed that m7G cap-eIF4E-RNMT trimeric complexes form, and thus RNMT-eIF4E complexes may be employed so that eIF4E captures newly capped RNA. In all, we show for the first time that the cap-binding protein eIF4E directly binds to the cap-maturation enzyme RNMT.

NAD-seq for profiling the NAD+ capped transcriptome of Arabidopsis thaliana.

Eukaryotic RNAs can be modified with a non-canonical 5' nicotinamide adenine dinucleotide (NAD+) cap. NAD-seq identifies transcriptome-wide NAD+ capped RNAs. NAD-seq takes advantage of click chemistry to allow the capture of NAD+ capped RNAs. Unlike other approaches, NAD-seq does not require DNA synthesis on beads, but this technique uses full NAD+ capped transcripts eluted from beads as the substrates for strand-specific RNA sequencing library preparation. For complete details on the use and execution of this protocol, please refer to Yu et al. (2021).

Studying the Structural Organization of Polyribosomes with Alexander S. Spirin.

"Would it be possible to analyze molecular mechanisms and structural organisation of polyribosome assemblies using cryo electron tomography?" - we asked through a longstanding collaboration between my research group and that of Alexander S. Spirin. Indeed, it was: we found that double-row polyribosomes can have both circular and linear arrangements of their mRNA [Afonina, Z. A., et al. (2013) Biochemistry (Moscow)], we figured out how eukaryotic ribosomes assemble on an mRNA to form supramolecular left-handed helices [Myasnikov, A. G., et al. (2014) Nat. Commun.], that the circularization of polyribosomes is poly-A and cap-independent [Afonina, Z. A., et al. (2014) Nucleic Acids Res.], and that intermediary polyribosomes with open structures exist after a transition from a juvenile phase to strongly translating polysomes of medium size [Afonina, Z. A., et al. (2015) Nucleic Acids Res.] until they form densely packed helical structures with reduced activity. Our joint fruitful exchanges, hence, led to major advances in the field, which are reviewed here from a personal and historical perspective in memory of Alexander S. Spirin.

5'-Cap sequestration is an essential determinant of HIV-1 genome packaging.

HIV-1 selectively packages two copies of its 5'-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5' leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (ΨCES) is unexpectedly poorly packaged when assayed in competition with the intact 5' leader. ΨCES lacks a 5'-tandem hairpin element that sequesters the 5' cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5' ribozyme to ΨCES to enable cotranscriptional shedding of the 5' cap promoted ΨCES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5' cap and exposure of NC binding sites that reside fully within the ΨCES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5'-capped RNA genomes.

Translation Initiation Regulated by RNA-Binding Protein in Mammals: The Modulation of Translation Initiation Complex by Trans-Acting Factors.

Protein synthesis is tightly regulated at each step of translation. In particular, the formation of the basic cap-binding complex, eukaryotic initiation factor 4F (eIF4F) complex, on the 5' cap structure of mRNA is positioned as the rate-limiting step, and various cis-elements on mRNA contribute to fine-tune spatiotemporal protein expression. The cis-element on mRNAs is recognized and bound to the trans-acting factors, which enable the regulation of the translation rate or mRNA stability. In this review, we focus on the molecular mechanism of how the assembly of the eIF4F complex is regulated on the cap structure of mRNAs. We also summarize the fine-tuned regulation of translation initiation by various trans-acting factors through cis-elements on mRNAs.