piRNA processing by a trimeric Schlafen-domain nuclease.

Transposable elements are genomic parasites that expand within and spread between genomes1. PIWI proteins control transposon activity, notably in the germline2,3. These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5'-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5'-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (m7G-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors4, and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5'-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity5, exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements.

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.

Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines.

The recent FDA approval of the mRNA vaccine for severe acute respiratory syndrome coronavirus (SARS-CoV-2) emphasizes the importance of mRNA as a powerful tool for therapeutic applications. The chemically modified mRNA cap analogs contain a unique cap structure, m7 G[5']ppp[5']N (where N=G, A, C or U), present at the 5'-end of many eukaryotic cellular and viral RNAs and several non-coding RNAs. The chemical modifications on cap analog influence orientation's nature, translational efficiency, nuclear stability, and binding affinity. The recent invention of a trinucleotide cap analog provides groundbreaking research in the area of mRNA analogs. Notably, trinucleotide cap analogs outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleotide containing a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog containing N1 modification, cap analog containing N2 modification, dinucleotide containing fluorescence probe and TAT, bacterial caps, and trinucleotide cap analogs. In addition, the biological applications of these novel cap analogs are delineated.

Characterization of an Atypical eIF4E Ortholog in Leishmania, LeishIF4E-6.

Leishmania parasites are digenetic protists that shuffle between sand fly vectors and mammalian hosts, transforming from flagellated extracellular promastigotes that reside within the intestinal tract of female sand flies to the obligatory intracellular and non-motile amastigotes within mammalian macrophages. Stage differentiation is regulated mainly by post-transcriptional mechanisms, including translation regulation. Leishmania parasites encode six different cap-binding proteins, LeishIF4E1-6, that show poor conservation with their counterparts from higher eukaryotes and among themselves. In view of the changing host milieu encountered throughout their life cycle, we propose that each LeishIF4E has a unique role, although these functions may be difficult to determine. Here we characterize LeishIF4E-6, a unique eIF4E ortholog that does not readily associate with m7GTP cap in either of the tested life forms of the parasite. We discuss the potential effect of substituting two essential tryptophan residues in the cap-binding pocket, expected to be involved in the cap-binding activity, as judged from structural studies in the mammalian eIF4E. LeishIF4E-6 binds to LeishIF4G-5, one of the five eIF4G candidates in Leishmania. However, despite this binding, LeishIF4E-6 does not appear to function as a translation factor. Its episomal overexpression causes a general reduction in the global activity of protein synthesis, which was not observed in the hemizygous deletion mutant generated by CRISPR-Cas9. This genetic profile suggests that LeishIF4E-6 has a repressive role. The interactome of LeishIF4E-6 highlights proteins involved in RNA metabolism such as the P-body marker DHH1, PUF1 and an mRNA-decapping enzyme that is homologous to the TbALPH1.

2'-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography.

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.

Development of bis-ANS-based modified fluorescence titration assay for IFIT/RNA studies.

The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding proteins that are very highly expressed during antiviral response of immune system. IFIT proteins recognize and tightly bind foreign RNA particles. These are primarily viral RNAs ended with triphosphate at the 5' or lacking methylation of the first cap-proximal nucleotide but also in vitro transcribed RNA synthesized in the laboratory. Recognition of RNA by IFIT proteins leads to the formation of stable RNA/IFIT complexes and translational shut off of non-self transcripts. Here, we present a fluorescent-based assay to study the interaction between RNA molecules and IFIT family proteins. We have particularly focused on two representatives of this family: IFIT1 and IFIT5. We found a probe that competitively with RNA binds the positively charged tunnel in these IFIT proteins. The use of this probe for IFIT titration allowed us to evaluate the differences in binding affinities of mRNAs with different variants of 5' ends.

High-resolution structures of the SARS-CoV-2 2'-O-methyltransferase reveal strategies for structure-based inhibitor design.

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.

1H, 13C, and 15N backbone chemical shift assignments of m7GTP cap-bound Leishmania initiation factor 4E-1.

Most of the translational control of gene expression in higher eukaryotes occurs during the initiation step of protein synthesis. While this process is well characterized in mammalian cells, it is less defined in parasites, including the ones that cause human Leishmaniasis. The Leishmania cap-binding isoform 1 (LeishIF4E-1) is the only isoform that binds the specific trypanosomatids-specific hypermethylated 5' cap, called cap-4, in the human stage of the parasite life cycle. We report here the extensive NMR resonance assignment of LeishIF4E-1 bound to a cap analog, m7GTP. The chemical shift data constitute a prerequisite to understanding specific translation initiation mechanisms used in Leishmania parasites and to developing antiparasitic drugs targeting their translation initiation factors.

The yeast scavenger decapping enzyme DcpS and its application for in vitro RNA recapping.

Eukaryotic mRNAs are modified at their 5' end early during transcription by the addition of N7-methylguanosine (m7G), which forms the "cap" on the first 5' nucleotide. Identification of the 5' nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effect of various reaction conditions on the activity of the yeast scavenger mRNA decapping enzyme DcpS and examined decapping of 30 chemically distinct cap structures varying the state of methylation, sugar, phosphate linkage, and base composition on 25mer RNA oligonucleotides. Contrary to the generally accepted belief that DcpS enzymes only decap short oligonucleotides, we found that the yeast scavenger decapping enzyme decaps RNA transcripts as long as 1400 nucleotides. Further, we validated the application of yDcpS for enriching capped RNA using a strategy of specifically tagging the 5' end of capped RNA by first decapping and then recapping it with an affinity-tagged guanosine nucleotide.

Structure of a functional cap-binding domain in Rift Valley fever virus L protein.

Rift Valley fever virus (RVFV) belongs to the family of Phenuiviridae within the order of Bunyavirales. The virus may cause fatal disease both in livestock and humans, and therefore, is of great economical and public health relevance. In analogy to the influenza virus polymerase complex, the bunyavirus L protein is assumed to bind to and cleave off cap structures of cellular mRNAs to prime viral transcription. However, even though the presence of an endonuclease in the N-terminal domain of the L protein has been demonstrated for several bunyaviruses, there is no evidence for a cap-binding site within the L protein. We solved the structure of a C-terminal 117 amino acid-long domain of the RVFV L protein by X-ray crystallography. The overall fold of the domain shows high similarity to influenza virus PB2 cap-binding domain and the putative non-functional cap-binding domain of reptarenaviruses. Upon co-crystallization with m7GTP, we detected the cap-analogue bound between two aromatic side chains as it has been described for other cap-binding proteins. We observed weak but specific interaction with m7GTP rather than GTP in vitro using isothermal titration calorimetry. The importance of m7GTP-binding residues for viral transcription was validated using a RVFV minigenome system. In summary, we provide structural and functional evidence for a cap-binding site located within the L protein of a virus from the Bunyavirales order.

N1-Propargylguanosine Modified mRNA Cap Analogs: Synthesis, Reactivity, and Applications to the Study of Cap-Binding Proteins.

The mRNA 5' cap consists of N7-methylguanosine bound by a 5',5'-triphosphate bridge to the first nucleotide of the transcript. The cap interacts with various specific proteins and participates in all key mRNA-related processes, which may be of therapeutic relevance. There is a growing demand for new biophysical and biochemical methods to study cap-protein interactions and identify the factors which inhibit them. The development of such methods can be aided by the use of properly designed fluorescent molecular probes. Herein, we synthesized a new class of m7Gp3G cap derivatives modified with an alkyne handle at the N1-position of guanosine and, using alkyne-azide cycloaddition, we functionalized them with fluorescent tags to obtain potential probes. The cap derivatives and probes were evaluated in the context of two cap-binding proteins, eukaryotic translation initiation factor (eIF4E) and decapping scavenger (DcpS). Biochemical and biophysical studies revealed that N1-propargyl moiety did not significantly disturb cap-protein interaction. The fluorescent properties of the probes turned out to be in line with microscale thermophoresis (MST)-based binding assays.

Crystal structure of the Trypanosoma cruzi EIF4E5 translation factor homologue in complex with mRNA cap-4.

Association of the initiation factor eIF4E with the mRNA cap structure is a key step for translation. Trypanosomatids present six eIF4E homologues, showing a low conservation and also differing significantly from the IF4Es of multicellular eukaryotes. On the mRNA side, while in most eukaryotes the mRNA contains cap-0 (7-methyl-GTP), the trypanosomatid mRNA features a cap-4, which is formed by a cap-0, followed by the AACU sequence containing 2'-O-ribose methylations and base methylations on nucleotides 1 and 4. The studies on eIF4E-cap-4 interaction have been hindered by the difficulty to synthesize this rather elaborated cap-4 sequence. To overcome this problem, we applied a liquid-phase oligonucleotide synthesis strategy and describe for the first time the crystal structure of a trypanosomatid eIF4E (T. cruzi EIF4E5) in complex with cap-4. The TcEIF4E5-cap-4 structure allowed a detailed description of the binding mechanism, revealing the interaction mode for the AACU sequence, with the bases packed in a parallel stacking conformation and involved, together with the methyl groups, in hydrophobic contacts with the protein. This binding mechanism evidences a distinct cap interaction mode in comparison with previously described eIF4E structures and may account for the difference of TcEIF4E5-cap-4 dissociation constant in comparison with other eIF4E homologues.

Exploring the potential of phosphotriazole 5' mRNA cap analogues as efficient translation initiators.

Augmenting the mRNA translation efficiency and stability by replacing the standard 7-methylguanosine 5'-cap with properly designed analogues is a viable strategy for increasing the in vivo expression of proteins from exogenously delivered mRNA. However, the development of novel cap analogues with superior biological properties is hampered by the challenges associated with the synthesis of such highly modified nucleotides. To provide a simpler alternative to traditional methods for cap analogue preparation, we have recently proposed a click-chemistry-based strategy for the synthesis of dinucleotide cap analogues and identified several triazole-containing compounds with promising biochemical properties. Here, we further explored the concept of CuAAC-mediated cap synthesis by designing and studying 'second generation' triazole-modified caps, which were derived from the most promising 'first generation' compounds by modifying the oligophosphate chain length, altering the position of the triazole moiety, or replacing chemically labile P-N bonds with P-O bonds. The biochemical properties of the new analogues were evaluated by determining their affinity for eIF4E, susceptibility to hDcp2-catalysed decapping, and translation efficiencies in vitro and in cultured cells. The results led to identification of cap analogues that have superior translational properties compared to standard caps and the parent triazole-modified compounds as well as provided directions for future improvements.

Structural motifs in eIF4G and 4E-BPs modulate their binding to eIF4E to regulate translation initiation in yeast.

The interaction of the eukaryotic initiation factor 4G (eIF4G) with the cap-binding protein eIF4E initiates cap-dependent translation and is regulated by the 4E-binding proteins (4E-BPs), which compete with eIF4G to repress translation. Metazoan eIF4G and 4E-BPs interact with eIF4E via canonical and non-canonical motifs that bind to the dorsal and lateral surface of eIF4E in a bipartite recognition mode. However, previous studies pointed to mechanistic differences in how fungi and metazoans regulate protein synthesis. We present crystal structures of the yeast eIF4E bound to two yeast 4E-BPs, p20 and Eap1p, as well as crystal structures of a fungal eIF4E-eIF4G complex. We demonstrate that the core principles of molecular recognition of eIF4E are in fact highly conserved among translational activators and repressors in eukaryotes. Finally, we reveal that highly specialized structural motifs do exist and serve to modulate the affinity of protein-protein interactions that regulate cap-dependent translation initiation in fungi.

A molecular dynamics simulation study decodes the Zika virus NS5 methyltransferase bound to SAH and RNA analogue.

Since 2015, widespread Zika virus outbreaks in Central and South America have caused increases in microcephaly cases, and this acute problem requires urgent attention. We employed molecular dynamics and Gaussian accelerated molecular dynamics techniques to investigate the structure of Zika NS5 protein with S-adenosyl-L-homocysteine (SAH) and an RNA analogue, namely 7-methylguanosine 5'-triphosphate (m7GTP). For the binding motif of Zika virus NS5 protein and SAH, we suggest that the four Zika NS5 substructures (residue orders: 101-112, 54-86, 127-136 and 146-161) and the residues (Ser56, Gly81, Arg84, Trp87, Thr104, Gly106, Gly107, His110, Asp146, Ile147, and Gly148) might be responsible for the selectivity of the new Zika virus drugs. For the binding motif of Zika NS5 protein and m7GTP, we suggest that the three Zika NS5 substructures (residue orders: 11-31, 146-161 and 207-218) and the residues (Asn17, Phe24, Lys28, Lys29, Ser150, Arg213, and Ser215) might be responsible for the selectivity of the new Zika virus drugs.

Hydrolytic activity of human Nudt16 enzyme on dinucleotide cap analogs and short capped oligonucleotides.

Human Nudt16 (hNudt16) is a member of the Nudix family of hydrolases, comprising enzymes catabolizing various substrates including canonical (d)NTPs, oxidized (d)NTPs, nonnucleoside polyphosphates, and capped mRNAs. Decapping activity of the Xenopus laevis (X29) Nudt16 homolog was observed in the nucleolus, with a high specificity toward U8 snoRNA. Subsequent studies have reported cytoplasmic localization of mammalian Nudt16 with cap hydrolysis activity initiating RNA turnover, similar to Dcp2. The present study focuses on hNudt16 and its hydrolytic activity toward dinucleotide cap analogs and short capped oligonucleotides. We performed a screening assay for potential dinucleotide and oligonucleotide substrates for hNudt16. Our data indicate that dinucleotide cap analogs and capped oligonucleotides containing guanine base in the first transcribed nucleotide are more susceptible to enzymatic digestion by hNudt16 than their counterparts containing adenine. Furthermore, unmethylated dinucleotides (GpppG and ApppG) and respective oligonucleotides (GpppG-16nt and GpppA-16nt) were hydrolyzed by hNudt16 with greater efficiency than were m7GpppG and m7GpppG-16nt. In conclusion, we found that hNudt16 hydrolysis of dinucleotide cap analogs and short capped oligonucleotides displayed a broader spectrum specificity than is currently known.

Capped RNA primer binding to influenza polymerase and implications for the mechanism of cap-binding inhibitors.

Influenza polymerase uses short capped primers snatched from nascent Pol II transcripts to initiate transcription of viral mRNAs. Here we describe crystal structures of influenza A and B polymerase bound to a capped primer in a configuration consistent with transcription initiation ('priming state') and show by functional assays that conserved residues from both the PB2 midlink and cap-binding domains are important for positioning the capped RNA. In particular, mutation of PB2 Arg264, which interacts with the triphosphate linkage in the cap, significantly and specifically decreases cap-dependent transcription. We also compare the configuration of the midlink and cap-binding domains in the priming state with their very different relative arrangement (called the 'apo' state) in structures where the potent cap-binding inhibitor VX-787, or a close analogue, is bound. In the 'apo' state the inhibitor makes additional interactions to the midlink domain that increases its affinity beyond that to the cap-binding domain alone. The comparison suggests that the mechanism of resistance of certain mutations that allow virus to escape from VX-787, notably PB2 N510T, can only be rationalized if VX-787 has a dual mode of action, direct inhibition of capped RNA binding as well as stabilization of the transcriptionally inactive 'apo' state.

Kinetic analyses of phosphorylated and non-phosphorylated eIFiso4E binding to mRNA cap analogues.

Phosphorylation of eukaryotic initiation factors was previously shown to interact with m7G cap and play an important role in the regulation of translation initiation of protein synthesis. To gain further insight into the phosphorylation process of plant protein synthesis, the kinetics of phosphorylated wheat eIFiso4E binding to m7G cap analogues were examined. Phosphorylation of wheat eIFiso4E showed similar kinetic effects to human eIF4E binding to m7-G cap. Phosphorylation of eIFiso4E decreased the kinetic rate (2-fold) and increased the dissociation rate (2-fold) as compared to non-phosphorylated eIFiso4E binding to both mono- and di-nucleotide analogues at 22°C. Phosphorylated and non-phosphorylated eIFiso4E-m7G cap binding rates were found to be independent of concentration, suggesting conformational changes were rate limiting. Rate constant for phosphorylated and non-phosphorylated eIFiso4E binding to m7-G cap increased with temperature. Phosphorylation of eIFiso4E decreased (2-fold) the activation energy for both m7-G cap analogues binding as compared to non-phosphorylated eIFiso4E. The reduced energy barrier for the formation of eIFiso4E-m7-G cap complex suggests a more stable platform for further initiation complex formation and possible means of adapting variety of environmental conditions. Furthermore, the formation of phosphorylated eIFiso4E-cap complex may contribute to modulation of the initiation of protein synthesis in plants.

Structural basis for mutually exclusive co-transcriptional nuclear cap-binding complexes with either NELF-E or ARS2.

Pol II transcribes diverse classes of RNAs that need to be directed into the appropriate nuclear maturation pathway. All nascent Pol II transcripts are 5'-capped and the cap is immediately sequestered by the nuclear cap-binding complex (CBC). Mutually exclusive interactions of CBC with different partner proteins have been implicated in transcript fate determination. Here, we characterise the direct interactions between CBC and NELF-E, a subunit of the negative elongation factor complex, ARS2 and PHAX. Our biochemical and crystal structure results show that the homologous C-terminal peptides of NELF-E and ARS2 bind identically to CBC and in each case the affinity is enhanced when CBC is bound to a cap analogue. Furthermore, whereas PHAX forms a complex with CBC and ARS2, NELF-E binding to CBC is incompatible with PHAX binding. We thus define two mutually exclusive complexes CBC-NELF-E and CBC-ARS2-PHAX, which likely act in respectively earlier and later phases of transcription.

PERK Signal-Modulated Protein Translation Promotes the Survivability of Dengue 2 Virus-Infected Mosquito Cells and Extends Viral Replication.

Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5'-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells. This suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. When the PERK signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. This suggested that ER stress response was alleviated through the PERK-mediated shutdown of global proteins in DENV2-infected C6/36 cells. In the meantime, the activities of caspases-9 and -3 and the apoptosis-related cell death rate increased in C6/36 cells with PERK inhibition. This reflected that the PERK-signaling pathway is involved in determining cell survival, presumably by reducing DENV2-induced ER stress. Looking at the PERK downstream target, α-subunit of eukaryotic initiation factor 2 (eIF2α), an increased phosphorylation status was only shown in infected C6/36 cells. This indicated that recruitment of ribosome binding to the mRNA 5'-cap structure could have been impaired in cap-dependent translation. It turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to DENV2 infection. As synthesis of viral proteins was not affected by the PERK signal pathway, an alternate mode other than cap-dependent translation may be utilized. This finding provides insights into elucidating how the PERK signal pathway modulates dynamic translation of proteins and helps mosquito cells survive continuous replication of the DENV2. It was ecologically important for virus amplification in mosquitoes and transmission to humans.