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.

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.

RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes.

Eukaryotic cellular mRNAs possess a 5' cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4GII can associate with either eIF4A1 or eIF4A2 to recruit ribosomes to mRNA templates. In combination with eIF4E and eIF4E3, our results indicate the presence of up to eight eIF4F complexes that can operate in translation initiation.

Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-methylguanine nucleotides targeting cap-dependent translation.

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating translational control of the cell cycle. mRNA 5' cap analogs targeting eIF4E are small molecules with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical utility of typical cap analogs is limited because of their reduced cell membrane permeability. Transforming the active analogs into their pronucleotide derivatives is a promising approach to overcome this obstacle. 7-Benzylguanosine monophosphate (bn7GMP) is a cap analog that has been successfully transformed into a cell-penetrating pronucleotide by conjugation of the phosphate moiety with tryptamine. In this work, we explored whether a similar strategy is applicable to other cap analogs, particularly phosphate-modified 7-methylguanine nucleotides. We report the synthesis of six new tryptamine conjugates containing N7-methylguanosine mono- and diphosphate and their analogs modified with thiophosphate moiety. These new potential pronucleotides and the expected products of their activation were characterized by biophysical and biochemical methods to determine their affinity towards eIF4E, their ability to inhibit translation in vitro, their susceptibility to enzymatic degradation and their turnover in cell extract. The results suggest that compounds containing the thiophosphate moiety may act as pronucleotides that release low but sustainable concentrations of 7-methylguanosine 5'-phosphorothioate (m7GMPS), which is a translation inhibitor with in vitro potency higher than bn7GMP.

A cyclin-dependent kinase, CDK11/p58, represses cap-dependent translation during mitosis.

During mitosis, translation of most mRNAs is strongly repressed; none of the several explanatory hypotheses suggested can fully explain the molecular basis of this phenomenon. Here we report that cyclin-dependent CDK11/p58-a serine/threonine kinase abundantly expressed during M phase-represses overall translation by phosphorylating a subunit (eIF3F) of the translation factor eIF3 complex that is essential for translation initiation of most mRNAs. Ectopic expression of CDK11/p58 strongly repressed cap-dependent translation, and knockdown of CDK11/p58 nullified the translational repression during M phase. We identified the phosphorylation sites in eIF3F responsible for M phase-specific translational repression by CDK11/p58. Alanine substitutions of CDK11/p58 target sites in eIF3F nullified its effects on cell cycle-dependent translational regulation. The mechanism of translational regulation by the M phase-specific kinase, CDK11/p58, has deep evolutionary roots considering the conservation of CDK11 and its target sites on eIF3F from C. elegans to humans.

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.

Tracking the m7G-cap during translation initiation by crosslinking methods.

In eukaryotes, cap-dependent translation initiation is a sophisticated process that requires numerous trans-acting factors, the eukaryotic Initiation Factors (eIFs). Their main function is to assist the ribosome for accurate AUG start codon recognition. The whole process requires a 5'-3' scanning step and is therefore highly dynamic. Therefore translation requires a complex interplay between eIFs through assembly/release cycles. Here, we describe an original approach to assess the dynamic features of translation initiation. The principle is to use the m7Gcap located at the 5' extremity of mRNAs as a tracker to monitor RNA and protein components that are in its vicinity. Cap-binding molecules are trapped by chemical and UV crosslinking. The combination of cap crosslinking methods in cell-free translation systems with the use of specific translation inhibitors for different steps such as edeine, GMP-PNP or cycloheximide allowed assessing the cap fate during eukaryotic translation. Here, we followed the position of the cap in the histone H4 mRNA and the cap binding proteins during H4 mRNA translation.

Analysis of decapping scavenger cap complex using modified cap analogs reveals molecular determinants for efficient cap binding.

Decapping scavenger (DcpS) assists in precluding inhibition of cap-binding proteins by hydrolyzing cap species remaining after mRNA 3'→5' degradation. Its significance was reported in splicing, translation initiation and microRNA turnover. Here we examine the structure and binding mode of DcpS from Caenorhabditis elegans (CeDcpS) using a large collection of chemically modified methylenebis(phosphonate), imidodiphosphate and phosphorothioate cap analogs. We determine that CeDcpS is a homodimer and propose high accuracy structural models of apo- and m(7) GpppG-bound forms. The analysis of CeDcpS regioselectivity uncovers that the only site of hydrolysis is located between the ß and γ phosphates. Structure-affinity relationship studies of cap analogs for CeDcpS reveal molecular determinants for efficient cap binding: a strong dependence on the type of substituents in the phosphate chain, and reduced binding affinity for either methylated hydroxyl groups of m(7) Guo or an extended triphosphate chain. Docking analysis of cap analogs in the CeDcpS active site explains how both phosphate chain mobility and the orientation in the cap-binding pocket depend on the number of phosphate groups, the substituent type and the presence of the second nucleoside. Finally, the comparison of CeDcpS with its well known human homolog provides general insights into DcpS-cap interactions.

Structural and functional control of the eukaryotic mRNA decapping machinery.

The regulation of mRNA degradation is critical for proper gene expression. Many major pathways for mRNA decay involve the removal of the 5' 7-methyl guanosine (m(7)G) cap in the cytoplasm to allow for 5'-to-3' exonucleolytic decay. The most well studied and conserved eukaryotic decapping enzyme is Dcp2, and its function is aided by co-factors and decapping enhancers. A subset of these factors can act to enhance the catalytic activity of Dcp2, while others might stimulate the remodeling of proteins bound to the mRNA substrate that may otherwise inhibit decapping. Structural studies have provided major insights into the mechanisms by which Dcp2 and decapping co-factors activate decapping. Additional mRNA decay factors can function by recruiting components of the decapping machinery to target mRNAs. mRNA decay factors, decapping factors, and mRNA substrates can be found in cytoplasmic foci named P bodies that are conserved in eukaryotes, though their function remains unknown. In addition to Dcp2, other decapping enzymes have been identified, which may serve to supplement the function of Dcp2 or act in independent decay or quality control pathways. This article is part of a Special Issue entitled: RNA Decay mechanisms.

A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA.

A method is described for the detection of certain nucleotide modifications adjacent to the 5' 7-methyl guanosine cap of mRNAs from individual genes. The method quantitatively measures the relative abundance of 2'-O-methyl and N(6),2'-O-dimethyladenosine, two of the most common modifications. In order to identify and quantitatify the amounts of N(6),2'-O-dimethyladenosine, a novel method for the synthesis of modified adenosine phosphoramidites was developed. This method is a one step synthesis and the product can directly be used for the production of N(6),2'-O-dimethyladenosine containing RNA oligonucleotides. The nature of the cap-adjacent nucleotides were shown to be characteristic for mRNAs from individual genes transcribed in liver and testis.

The nematode eukaryotic translation initiation factor 4E/G complex works with a trans-spliced leader stem-loop to enable efficient translation of trimethylguanosine-capped RNAs.

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.

Crystal structure of the RRM domain of poly(A)-specific ribonuclease reveals a novel m(7)G-cap-binding mode.

Poly(A)-specific ribonuclease (PARN) is a processive 3'-exoribonuclease involved in the decay of eukaryotic mRNAs. Interestingly, PARN interacts not only with the 3' end of the mRNA but also with its 5' end as PARN contains an RRM domain that specifically binds both the poly(A) tail and the 7-methylguanosine (m(7)G) cap. The interaction of PARN with the 5' cap of mRNAs stimulates the deadenylation activity and enhances the processivity of this reaction. We have determined the crystal structure of the PARN-RRM domain with a bound m(7)G triphosphate nucleotide, revealing a novel binding mode for the m(7)G cap. The structure of the m(7)G binding pocket is located outside of the canonical RNA-binding surface of the RRM domain and differs significantly from that of other m(7)G-cap-binding proteins. The crystal structure also shows a remarkable conformational flexibility of the RRM domain, leading to a perfect exchange of two alpha-helices with an adjacent protein molecule in the crystal lattice.

Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania.

The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

mRNA transfection of dendritic cells: synergistic effect of ARCA mRNA capping with Poly(A) chains in cis and in trans for a high protein expression level.

The occurrence of translation mechanism in the cytosol offers advantages to mRNA transfer over DNA-based transfection in non-dividing cells. Here, we sought to optimize mRNA constructs allowing a high level of protein upon lipofection. We found that luciferase into mouse dendritic cells (JAWSII cells) was approximately 20-fold higher when the luciferase mRNA was capped with 3'-O-methyl-m7(5')Gppp5'G (anti-reverse cap analogue; ARCA) than with m7(5')Gppp5'G (CAP). Adding a Poly(A) tail of 100 instead of 64 adenosines in cis increased by approximately 35-fold more the protein level. Finally, ARCA-Luc-mRNA-A100 construct was 700-fold better efficient than the CAP-Luc-mRNA-A64 one. Moreover, co-transfection with free Poly(A) chains in trans enhanced by 100% the luciferase level. The efficiency of ARCA-mRNA-A100 construct was validated in immature and mature human CD34-derived dendritic cells. Such mRNA construct was also successful to obtain high level of MART-1 tumor antigen.

High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation.

The eukaryotic translation initiation factor 4F (eIF4F) consists of three polypeptides (eIF4A, eIF4G, and eIF4E) and is responsible for recruiting ribosomes to mRNA. eIF4E recognizes the mRNA 5'-cap structure (m7GpppN) and plays a pivotal role in control of translation initiation, which is the rate-limiting step in translation. Overexpression of eIF4E has a dramatic effect on cell growth and leads to oncogenic transformation. Therefore, an inhibitory agent to eIF4E, if any, might serve as a novel therapeutic against malignancies that are caused by aberrant translational control. Along these lines, we developed two RNA aptamers, aptamer 1 and aptamer 2, with high affinity for mammalian eIF4E by in vitro RNA selection-amplification. Aptamer 1 inhibits the cap binding to eIF4E more efficiently than the cap analog m7GpppN or aptamer 2. Consistently, aptamer 1 inhibits specifically cap-dependent in vitro translation while it does not inhibit cap-independent HCV IRES-directed translation initiation. The interaction between eIF4E and eIF4E-binding protein 1 (4E-BP1), however, was not inhibited by aptamer 1. Aptamer 1 is composed of 86 nucleotides, and the high affinity to eIF4E is affected by deletions at both termini. Moreover, relatively large areas in the aptamer 1 fold are protected by eIF4E as determined by ribonuclease footprinting. These findings indicate that aptamers can achieve high affinity to a specific target protein via global conformational recognition. The genetic mutation and affinity study of variant eIF4E proteins suggests that aptamer 1 binds to eIF4E adjacent to the entrance of the cap-binding slot and blocks the cap-binding pocket, thereby inhibiting translation initiation.

Effective non-viral leader for cap-independent translation in a eukaryotic cell-free system.

The 61 nt 5'-untranslated region (5'-UTR) of mRNA encoding for a light-emitting protein of hydroid polyp Obelia longissima, obelin, is shown to provide a high level of cap-independent translation of heterologous mRNAs in cell-free translation systems based on wheat germ extracts. The inhibition of translation typically observed when excess mRNA is present or produced in a eukaryotic system (the so-called self-inhibition phenomenon) is found abated with mRNA constructs carrying the obelin mRNA leader. The role of the sequestration of a limiting initiation factor, probably eIF4F, in the self-inhibition phenomenon and the possible independence of the obelin mRNA leader from eIF4F are discussed. We propose the obelin mRNA leader be used for effective cap-independent translation in eukaryotic cell-free systems, including combined transcription-translation systems with uncontrolled phage polymerase-catalyzed accumulation of mRNA.

Quantitative analysis of influenza virus RNP interaction with RNA cap structures and comparison to human cap binding protein eIF4E.

Influenza virus polymerase uses capped RNA primers for transcription initiation in infected cells. This unique mechanism involves the specific binding of the polymerase to capped mRNA precursors in the nucleus of infected cells. These host RNAs are then cleaved by a polymerase associated endonuclease at a position 10-15 nucleotides downstream of the cap structure. The resulting capped RNA oligonucleotides function as primers for transcription initiation. The viral cap binding site has previously been mapped to the PB2 subunit of the trimeric influenza polymerase complex. We have established a quantitative assay system for the analysis of cap interaction with PB2 as part of the native, viral ribonucleoprotein complex (RNP) using a specific UV cross-linking approach. Cap binding was not affected by the RNase pretreatment of the capped RNA substrate and cap binding was not inhibited by excess uncapped RNA, indicating that under the assay conditions, the majority of the binding energy was contributed by the interaction with the cap structure. Binding to 7-methyl-GTP was found to involve synergistic interaction with 7-methyl guanosine and triphosphate binding subsites. A similar mode of interaction with 7-methyl-GTP was found for human cap binding protein eIF4E. However, the potency of 7-methyl-GTP for cap binding inhibition was 200-fold stronger with eIF4E and had a higher contribution from the triphosphate moiety as compared to influenza RNP. Due to this difference in cap subsite interaction, it was possible to identify novel cap analogues, which selectively interact with influenza virus, but not human cap binding protein.

Kinetic proofreading scanning models for eukaryotic translational initiation: the cap and poly(A) tail dependency of translation.

Two simplified kinetic proofreading scanning (KPS) models were proposed to describe the 5' cap and 3' poly(A) tail dependency of eukaryotic translation initiation. In Model I, the initiation factor complex starts scanning and unwinding the secondary structure of the 5' untranslated region (UTR) from the 5' terminus of mRNA. In Model II, the initiation factor complex starts scanning from any binding site in the 5' UTR. In both models, following ATP hydrolysis, the initiation factor complex either dissociates from mRNA or continues to scan and unwind RNA secondary structure in the 5' UTR. This step repeats n times until the AUG codon is reached. These two models show very different cap and/or poly(A) tail dependency of translation initiation. The models predict that both cap and poly(A) tail dependencies of translation, and translatability of mRNAs are coupled with the structure of 5' UTR: the translation of mRNA with structured 5' UTR is strongly cap- and poly(A) tail-dependent; while translation of mRNA with unstructured 5' UTR is less cap- and poly(A) tail-dependent. We use these two models to explain: (1) the cap and poly(A) tail dependence of translation; (2) the effect of exogenous poly(A) on translation; (3) repression of host mRNA and translation of late adenovirus mRNA in the late phase of adenovirus infection; (4) repression of host mRNA and translation of Vaccinia virus mRNA in virus-infected cell; (5) heat shock repression of translation of normal mRNA and stimulation of translation of hsp mRNA; and (6) the synergistic effect of cap and poly(A) tail on stimulating translation. The kinetic proofreading scanning models provide a coherent interpretation of those phenomena.

Construction of an efficient expression vector for coupled transcription/translation in a wheat germ cell-free system.

Using the expression vector, pEU, which we have constructed, highly efficient in vitro protein synthesis can be achieved: The system works for 150 hours and without further template addition once the reaction has started, yielding 5 mg of enzymatically active protein in a 1 ml reaction.

Evidence that the genomic RNA of hepatitis E virus is capped.

Hepatitis E virus (HEV) is an unclassified virus with a positive-sense RNA genome and an undefined replication strategy. In order to determine whether the HEV genome is capped or not, we developed a reverse transcription-PCR assay that is based on the ability of a monoclonal antibody to recognize 7-methylguanosine (m(7)G). Antibody to m(7)G bound RNA extracted from virions of two different HEV genotypes. The cap analog competitively inhibited the binding of virion RNAs, demonstrating that HEV has a capped RNA genome.