eIF4E-independent translation is largely eIF3d-dependent.

Translation initiation is a highly regulated step needed for protein synthesis. Most cell-based mechanistic work on translation initiation has been done using non-stressed cells growing in medium with sufficient nutrients and oxygen. This has yielded our current understanding of 'canonical' translation initiation, involving recognition of the mRNA cap by eIF4E1 followed by successive recruitment of initiation factors and the ribosome. Many cells, however, such as tumor cells, are exposed to stresses such as hypoxia, low nutrients or proteotoxic stress. This leads to inactivation of mTORC1 and thereby inactivation of eIF4E1. Hence the question arises how cells translate mRNAs under such stress conditions. We study here how mRNAs are translated in an eIF4E1-independent manner by blocking eIF4E1 using a constitutively active version of eIF4E-binding protein (4E-BP). Via ribosome profiling we identify a subset of mRNAs that are still efficiently translated when eIF4E1 is inactive. We find that these mRNAs preferentially release eIF4E1 when eIF4E1 is inactive and bind instead to eIF3d via its cap-binding pocket. eIF3d then enables these mRNAs to be efficiently translated due to its cap-binding activity. In sum, our work identifies eIF3d-dependent translation as a major mechanism enabling mRNA translation in an eIF4E-independent manner.

Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation.

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

If the 5' cap fits (wear it) - Non-canonical RNA capping.

RNA capping is a prominent RNA modification that influences RNA stability, metabolism, and function. While it was long limited to the study of the most abundant eukaryotic canonical m7G cap, the field recently went through a large paradigm shift with the discovery of non-canonical RNA capping in bacteria and ultimately all domains of life. The repertoire of non-canonical caps has expanded to encompass metabolite caps, including NAD, FAD, CoA, UDP-Glucose, and ADP-ribose, alongside alarmone dinucleoside polyphosphate caps, and methylated phosphate cap-like structures. This review offers an introduction into the field, presenting a summary of the current knowledge about non-canonical RNA caps. We highlight the often still enigmatic biological roles of the caps together with their processing enzymes, focusing on the most recent discoveries. Furthermore, we present the methods used for the detection and analysis of these non-canonical RNA caps and thus provide an introduction into this dynamic new field.

The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

CapTrap-seq: a platform-agnostic and quantitative approach for high-fidelity full-length RNA sequencing.

Long-read RNA sequencing is essential to produce accurate and exhaustive annotation of eukaryotic genomes. Despite advancements in throughput and accuracy, achieving reliable end-to-end identification of RNA transcripts remains a challenge for long-read sequencing methods. To address this limitation, we develop CapTrap-seq, a cDNA library preparation method, which combines the Cap-trapping strategy with oligo(dT) priming to detect 5' capped, full-length transcripts. In our study, we evaluate the performance of CapTrap-seq alongside other widely used RNA-seq library preparation protocols in human and mouse tissues, employing both ONT and PacBio sequencing technologies. To explore the quantitative capabilities of CapTrap-seq and its accuracy in reconstructing full-length RNA molecules, we implement a capping strategy for synthetic RNA spike-in sequences that mimics the natural 5'cap formation. Our benchmarks, incorporating the Long-read RNA-seq Genome Annotation Assessment Project (LRGASP) data, demonstrate that CapTrap-seq is a competitive, platform-agnostic RNA library preparation method for generating full-length transcript sequences.

Oomycete Nudix effectors display WY-Nudix conformation and mRNA decapping activity.

Oomycete Nudix effectors have characteristics of independent evolution, but adopt a conserved WY-Nudix conformation. Furthermore, multiple oomycete Nudix effectors exhibit mRNA decapping activity.

CK2 phosphorylation of CMTR1 promotes RNA cap formation and influenza virus infection.

The RNA cap methyltransferase CMTR1 methylates the first transcribed nucleotide of RNA polymerase II transcripts, impacting gene expression mechanisms, including during innate immune responses. Using mass spectrometry, we identify a multiply phosphorylated region of CMTR1 (phospho-patch [P-Patch]), which is a substrate for the kinase CK2 (casein kinase II). CMTR1 phosphorylation alters intramolecular interactions, increases recruitment to RNA polymerase II, and promotes RNA cap methylation. P-Patch phosphorylation occurs during the G1 phase of the cell cycle, recruiting CMTR1 to RNA polymerase II during a period of rapid transcription and RNA cap formation. CMTR1 phosphorylation is required for the expression of specific RNAs, including ribosomal protein gene transcripts, and promotes cell proliferation. CMTR1 phosphorylation is also required for interferon-stimulated gene expression. The cap-snatching virus, influenza A, utilizes host CMTR1 phosphorylation to produce the caps required for virus production and infection. We present an RNA cap methylation control mechanism whereby CK2 controls CMTR1, enhancing co-transcriptional capping.

Modeling the structure and DAP5-binding site of the FGF-9 5'-UTR RNA utilized in cap-independent translation.

Cap-independent or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5' untranslated regions (5' UTRs) that directly bind DAP5 or eIF4GI. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem-loops, and the RNA-helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. However, the DAP5-binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which require an unpaired, accessible 5' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186 nt FGF-9 5'-UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV cross-linking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5'-UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5-binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF-binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.

Structural basis of the monkeypox virus mRNA cap N7 methyltransferase complex.

The global outbreak of Mpox, caused by the monkeypox virus (MPXV), has attracted international attention and become another major infectious disease event after COVID-19. The mRNA cap N7 methyltransferase (RNMT) of MPXV methylates the N7 position of the added guanosine to the 5'-cap structure of mRNAs and plays a vital role in evading host antiviral immunity. MPXV RNMT is composed of the large subunit E1 and the small subunit E12. How E1 and E12 of MPXV assembly remains unclear. Here, we report the crystal structures of E12, the MTase domain of E1 with E12 (E1CTD-E12) complex, and the E1CTD-E12-SAM ternary complex, revealing the detailed conformations of critical residues and the structural changes upon E12 binding to E1. Functional studies suggest that E1CTD N-terminal extension (Asp545-Arg562) and the small subunit E12 play an essential role in the binding process of SAM. Structural comparison of the AlphaFold2-predicted E1, E1CTD-E12 complex, and the homologous D1-D12 complex of vaccinia virus (VACV) indicates an allosteric activating effect of E1 in MPXV. Our findings provide the structural basis for the MTase activity stimulation of the E1-E12 complex and suggest a potential interface for screening the anti-poxvirus inhibitors.

The impact of mRNA poly(A) tail length on eukaryotic translation stages.

The poly(A) tail plays an important role in maintaining mRNA stability and influences translation efficiency via binding with PABP. However, the impact of poly(A) tail length on mRNA translation remains incompletely understood. This study explores the effects of poly(A) tail length on human translation. We determined the translation rates in cell lysates using mRNAs with different poly(A) tails. Cap-dependent translation was stimulated by the poly(A) tail, however, it was largely independent of poly(A) tail length, with an exception observed in the case of the 75 nt poly(A) tail. Conversely, cap-independent translation displayed a positive correlation with poly(A) tail length. Examination of translation stages uncovered the dependence of initiation and termination on the presence of the poly(A) tail, but the efficiency of initiation remained unaffected by poly(A) tail extension. Further study unveiled that increased binding of eRFs to the ribosome with the poly(A) tail extension induced more efficient hydrolysis of peptidyl-tRNA. Building upon these findings, we propose a crucial role for the 75 nt poly(A) tail in orchestrating the formation of a double closed-loop mRNA structure within human cells which couples the initiation and termination phases of translation.

Generation and Characterization of In Vitro Transcribed mRNA.

Here we describe the in vitro preparation of mRNA from DNA templates, including setting up the transcription reaction, mRNA capping, and mRNA labeling. We then describe methods used for mRNA characterization, including UV and fluorescence spectrophotometry, as well as gel electrophoresis. Moreover, characterization of the in vitro transcribed RNA using the Bioanalyzer instrument is described, allowing a higher resolution analysis of the target molecules. For the in vitro testing of the mRNA molecules, we include protocols for the transfection of various primary cell cultures and the confirmation of translation by intracellular staining and western blotting.

Plasmodium RNA triphosphatase validation as antimalarial target.

Target-based approaches have traditionally been used in the search for new anti-infective molecules. Target selection process, a critical step in Drug Discovery, identifies targets that are essential to establish or maintain the infection, tractable to be susceptible for inhibition, selective towards their human ortholog and amenable for large scale purification and high throughput screening. The work presented herein validates the Plasmodium falciparum mRNA 5' triphosphatase (PfPRT1), the first enzymatic step to cap parasite nuclear mRNAs, as a candidate target for the development of new antimalarial compounds. mRNA capping is essential to maintain the integrity and stability of the messengers, allowing their translation. PfPRT1 has been identified as a member of the tunnel, metal dependent mRNA 5' triphosphatase family which differs structurally and mechanistically from human metal independent mRNA 5' triphosphatase. In the present study the essentiality of PfPRT1 was confirmed and molecular biology tools and methods for target purification, enzymatic assessment and target engagement were developed, with the goal of running a future high throughput screening to discover PfPRT1 inhibitors.

HIV-1 Infection Reduces NAD Capping of Host Cell snRNA and snoRNA.

Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5' cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g., U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity.

Structural snapshots of phenuivirus cap-snatching and transcription.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a human pathogen that is now endemic to several East Asian countries. The viral large (L) protein catalyzes viral transcription by stealing host mRNA caps via a process known as cap-snatching. Here, we establish an in vitro cap-snatching assay and present three high-quality electron cryo-microscopy (cryo-EM) structures of the SFTSV L protein in biologically relevant, transcription-specific states. In a priming-state structure, we show capped RNA bound to the L protein cap-binding domain (CBD). The L protein conformation in this priming structure is significantly different from published replication-state structures, in particular the N- and C-terminal domains. The capped-RNA is positioned in a way that it can feed directly into the RNA-dependent RNA polymerase (RdRp) ready for elongation. We also captured the L protein in an early-elongation state following primer-incorporation demonstrating that this priming conformation is retained at least in the very early stages of primer extension. This structural data is complemented by in vitro biochemical and cell-based assays. Together, these insights further our mechanistic understanding of how SFTSV and other bunyaviruses incorporate stolen host mRNA fragments into their viral transcripts thereby allowing the virus to hijack host cell translation machinery.

Identification and in vitro characterization of UDP-GlcNAc-RNA cap-modifying and decapping enzymes.

In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied, with multiple decapping enzymes identified and efficient capture and sequencing protocols developed for nicotinamide adenine dinucleotide (NAD)-RNA, which allowed for a stepwise elucidation of capping functions. Despite being identified as an abundant noncanonical RNA-cap, UDP-sugar-capped RNA remains poorly understood, which is partly due to its complex in vitro preparation. Here, we describe a scalable synthesis of sugar-capped uridine-guanosine dinucleotides from readily available protected building blocks and their enzymatic conversion into several cell wall precursor-capped dinucleotides. We employed these capped dinucleotides in T7 RNA polymerase-catalyzed in vitro transcription reactions to efficiently generate RNAs capped with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), its N-azidoacetyl derivative UDP-GlcNAz, and various cell wall precursors. We furthermore identified four enzymes capable of processing UDP-GlcNAc-capped RNA in vitro: MurA, MurB and MurC from Escherichia coli can sequentially modify the sugar-cap structure and were used to introduce a bioorthogonal, clickable moiety, and the human Nudix hydrolase Nudt5 was shown to efficiently decap UDP-GlcNAc-RNA. Our findings underscore the importance of efficient synthetic methods for capped model RNAs. Additionally, we provide useful enzymatic tools that could be utilized in the development and application of UDP-GlcNAc capture and sequencing protocols. Such protocols are essential for deepening our understanding of the widespread yet enigmatic GlcNAc modification of RNA and its physiological significance.

Structures of co-transcriptional RNA capping enzymes on paused transcription complex.

The 5'-end capping of nascent pre-mRNA represents the initial step in RNA processing, with evidence demonstrating that guanosine addition and 2'-O-ribose methylation occur in tandem with early steps of transcription by RNA polymerase II, especially at the pausing stage. Here, we determine the cryo-EM structures of the paused elongation complex in complex with RNGTT, as well as the paused elongation complex in complex with RNGTT and CMTR1. Our findings show the simultaneous presence of RNGTT and the NELF complex bound to RNA polymerase II. The NELF complex exhibits two conformations, one of which shows a notable rearrangement of NELF-A/D compared to that of the paused elongation complex. Moreover, CMTR1 aligns adjacent to RNGTT on the RNA polymerase II stalk. Our structures indicate that RNGTT and CMTR1 directly bind the paused elongation complex, illuminating the mechanism by which 5'-end capping of pre-mRNA during transcriptional pausing.

Viral RNA capping: Mechanisms and antiviral therapy.

RNA capping is an essential trigger for protein translation in eukaryotic cells. Many viruses have evolved various strategies for initiating the translation of viral genes and generating progeny virions in infected cells via synthesizing cap structure or stealing the RNA cap from nascent host messenger ribonucleotide acid (mRNA). In addition to protein translation, a new understanding of the role of the RNA cap in antiviral innate immunity has advanced the field of mRNA synthesis in vitro and therapeutic applications. Recent studies on these viral RNA capping systems have revealed startlingly diverse ways and molecular machinery. A comprehensive understanding of how viruses accomplish the RNA capping in infected cells is pivotal for designing effective broad-spectrum antiviral therapies. Here we systematically review the contemporary insights into the RNA-capping mechanisms employed by viruses causing human and animal infectious diseases, while also highlighting its impact on host antiviral innate immune response. The therapeutic applications of targeting RNA capping against viral infections and the development of RNA-capping inhibitors are also summarized.

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.

Toll/interleukin-1 receptor (TIR) domain-containing proteins have NAD-RNA decapping activity.

The occurrence of NAD+ as a non-canonical RNA cap has been demonstrated in diverse organisms. TIR domain-containing proteins present in all kingdoms of life act in defense responses and can have NADase activity that hydrolyzes NAD+. Here, we show that TIR domain-containing proteins from several bacterial and one archaeal species can remove the NAM moiety from NAD-capped RNAs (NAD-RNAs). We demonstrate that the deNAMing activity of AbTir (from Acinetobacter baumannii) on NAD-RNA specifically produces a cyclic ADPR-RNA, which can be further decapped in vitro by known decapping enzymes. Heterologous expression of the wild-type but not a catalytic mutant AbTir in E. coli suppressed cell propagation and reduced the levels of NAD-RNAs from a subset of genes before cellular NAD+ levels are impacted. Collectively, the in vitro and in vivo analyses demonstrate that TIR domain-containing proteins can function as a deNAMing enzyme of NAD-RNAs, raising the possibility of TIR domain proteins acting in gene expression regulation.

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.