Full-Length P2X7 Structures Reveal How Palmitoylation Prevents Channel Desensitization.

P2X receptors are trimeric, non-selective cation channels activated by extracellular ATP. The P2X7 receptor subtype is a pharmacological target because of involvement in apoptotic, inflammatory, and tumor progression pathways. It is the most structurally and functionally distinct P2X subtype, containing a unique cytoplasmic domain critical for the receptor to initiate apoptosis and not undergo desensitization. However, lack of structural information about the cytoplasmic domain has hindered understanding of the molecular mechanisms underlying these processes. We report cryoelectron microscopy structures of full-length rat P2X7 receptor in apo and ATP-bound states. These structures reveal how one cytoplasmic element, the C-cys anchor, prevents desensitization by anchoring the pore-lining helix to the membrane with palmitoyl groups. They show a second cytoplasmic element with a unique fold, the cytoplasmic ballast, which unexpectedly contains a zinc ion complex and a guanosine nucleotide binding site. Our structures provide first insights into the architecture and function of a P2X receptor cytoplasmic domain.

Molecular basis for the reversible ADP-ribosylation of guanosine bases.

Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as antitoxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP-ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indicating a wide distribution of reversible guanine modifications beyond DarTG systems.

METTL1-mediated m7G modification of Arg-TCT tRNA drives oncogenic transformation.

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.

N7-Methylguanosine tRNA modification enhances oncogenic mRNA translation and promotes intrahepatic cholangiocarcinoma progression.

Cancer cells selectively promote translation of specific oncogenic transcripts to facilitate cancer survival and progression, but the underlying mechanisms are poorly understood. Here, we find that N7-methylguanosine (m7G) tRNA modification and its methyltransferase complex components, METTL1 and WDR4, are significantly upregulated in intrahepatic cholangiocarcinoma (ICC) and associated with poor prognosis. We further reveal the critical role of METTL1/WDR4 in promoting ICC cell survival and progression using loss- and gain-of-function assays in vitro and in vivo. Mechanistically, m7G tRNA modification selectively regulates the translation of oncogenic transcripts, including cell-cycle and epidermal growth factor receptor (EGFR) pathway genes, in m7G-tRNA-decoded codon-frequency-dependent mechanisms. Moreover, using overexpression and knockout mouse models, we demonstrate the crucial oncogenic function of Mettl1-mediated m7G tRNA modification in promoting ICC tumorigenesis and progression in vivo. Our study uncovers the important physiological function and mechanism of METTL1-mediated m7G tRNA modification in the regulation of oncogenic mRNA translation and cancer progression.

Mammalian 5'-capped microRNA precursors that generate a single microRNA.

MicroRNAs (miRNAs) are short RNA gene regulators typically produced from primary transcripts that are cleaved by the nuclear microprocessor complex, with the resulting precursor miRNA hairpins exported by exportin 5 and processed by cytoplasmic Dicer to yield two (5p and 3p) miRNAs. Here, we document microprocessor-independent 7-methylguanosine (m(7)G)-capped pre-miRNAs, whose 5' ends coincide with transcription start sites and 3' ends are most likely generated by transcription termination. By establishing a small RNA Cap-seq method that employs the cap-binding protein eIF4E, we identified a group of murine m(7)G-capped pre-miRNAs genome wide. The m(7)G-capped pre-miRNAs are exported via the PHAX-exportin 1 pathway. After Dicer cleavage, only the 3p-miRNA is efficiently loaded onto Argonaute to form a functional microRNP. This unusual miRNA biogenesis pathway, which differs in pre-miRNA synthesis, nuclear-cytoplasmic transport, and guide strand selection, enables the development of shRNA expression constructs that produce a single 3p-siRNA.

A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

TLR7 gain-of-function genetic variation causes human lupus.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease1-7, evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA8,9 and binds to guanosine10-12. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP10-12, and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.

METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation.

7-methylguanosine (m7G) is present at mRNA caps and at defined internal positions within tRNAs and rRNAs. However, its detection within low-abundance mRNAs and microRNAs (miRNAs) has been hampered by a lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in miRNAs. Using this technique (Borohydride Reduction sequencing [BoRed-seq]) alongside RNA immunoprecipitation, we identify m7G within a subset of miRNAs that inhibit cell migration. We show that the METTL1 methyltransferase mediates m7G methylation within miRNAs and that this enzyme regulates cell migration via its catalytic activity. Using refined mass spectrometry methods, we map m7G to a single guanosine within the let-7e-5p miRNA. We show that METTL1-mediated methylation augments let-7 miRNA processing by disrupting an inhibitory secondary structure within the primary miRNA transcript (pri-miRNA). These results identify METTL1-dependent N7-methylation of guanosine as a new RNA modification pathway that regulates miRNA structure, biogenesis, and cell migration.

Transcriptome-wide Mapping of Internal N7-Methylguanosine Methylome in Mammalian mRNA.

N7-methylguanosine (m7G) is a positively charged, essential modification at the 5' cap of eukaryotic mRNA, regulating mRNA export, translation, and splicing. m7G also occurs internally within tRNA and rRNA, but its existence and distribution within eukaryotic mRNA remain to be investigated. Here, we show the presence of internal m7G sites within mammalian mRNA. We then performed transcriptome-wide profiling of internal m7G methylome using m7G-MeRIP sequencing (MeRIP-seq). To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively converts internal m7G sites into abasic sites, inducing misincorporation at these sites during reverse transcription. This base-resolution m7G-seq enabled transcriptome-wide mapping of m7G in human tRNA and mRNA, revealing distribution features of the internal m7G methylome in human cells. We also identified METTL1 as a methyltransferase that installs a subset of m7G within mRNA and showed that internal m7G methylation could affect mRNA translation.

Forodesine Enhances Immune Responses through Guanosine-Mediated TLR7 Activation while Preventing Graft-versus-Host Disease.

Recent evidence indicates that specific types of nuclear acids, including guanosine and its derivatives, act as natural ligands for TLR7. This led us to hypothesize that purine nucleoside phosphorylase inhibitors not only can induce apoptosis of T cells but also can lead to TLR7 activation by accumulation of guanine nucleosides, in particular under systemic inflammation, where damaged tissues release a large amount of nucleotides. We demonstrate in the present study that a purine nucleoside phosphorylase inhibitor, forodesine, can reduce the disease severity and prolong the survival in a xenogeneic mouse model of graft-versus-host disease (GVHD). Guanine nucleosides were undetectable in mice during GVHD but increased significantly following forodesine treatment. Our in vitro experiments showed that forodesine enhanced guanosine-mediated cytokine production from APCs, including alveolar macrophages and plasmacytoid dendritic cells, through TLR7 signaling. Forodesine also enhanced Ag-presenting capacity, as demonstrated by increased CD8+ T cell proliferation and higher secretion of IFN-γ and IL-12p40 in an MLR with plasmacytoid dendritic cells. Furthermore, forodesine stimulated IFN-γ production from activated T cells in the presence of a low concentration of guanosine while inhibiting their proliferation and inducing apoptotic cell death. Although forodesine ameliorated GVHD severity, mice treated with forodesine showed significantly higher levels of multiple proinflammatory cytokines and chemokines in plasma, suggesting in vivo upregulation of TLR7 signaling. Our study suggests that forodesine may activate a wide range of immune cells, including T cells, through TLR7 stimulation while inhibiting GVHD by inducing apoptosis of T cells, after allogeneic hematopoietic stem cell transplant.

Mettl1/Wdr4-Mediated m7G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation.

tRNAs are subject to numerous modifications, including methylation. Mutations in the human N7-methylguanosine (m7G) methyltransferase complex METTL1/WDR4 cause primordial dwarfism and brain malformation, yet the molecular and cellular function in mammals is not well understood. We developed m7G methylated tRNA immunoprecipitation sequencing (MeRIP-seq) and tRNA reduction and cleavage sequencing (TRAC-seq) to reveal the m7G tRNA methylome in mouse embryonic stem cells (mESCs). A subset of 22 tRNAs is modified at a "RAGGU" motif within the variable loop. We observe increased ribosome occupancy at the corresponding codons in Mettl1 knockout mESCs, implying widespread effects on tRNA function, ribosome pausing, and mRNA translation. Translation of cell cycle genes and those associated with brain abnormalities is particularly affected. Mettl1 or Wdr4 knockout mESCs display defective self-renewal and neural differentiation. Our study uncovers the complexity of the mammalian m7G tRNA methylome and highlights its essential role in ESCs with links to human disease.

m7GHub V2.0: an updated database for decoding the N7-methylguanosine (m7G) epitranscriptome.

With recent progress in mapping N7-methylguanosine (m7G) RNA methylation sites, tens of thousands of experimentally validated m7G sites have been discovered in various species, shedding light on the significant role of m7G modification in regulating numerous biological processes including disease pathogenesis. An integrated resource that enables the sharing, annotation and customized analysis of m7G data will greatly facilitate m7G studies under various physiological contexts. We previously developed the m7GHub database to host mRNA m7G sites identified in the human transcriptome. Here, we present m7GHub v.2.0, an updated resource for a comprehensive collection of m7G modifications in various types of RNA across multiple species: an m7GDB database containing 430 898 putative m7G sites identified in 23 species, collected from both widely applied next-generation sequencing (NGS) and the emerging Oxford Nanopore direct RNA sequencing (ONT) techniques; an m7GDiseaseDB hosting 156 206 m7G-associated variants (involving addition or removal of an m7G site), including 3238 disease-relevant m7G-SNPs that may function through epitranscriptome disturbance; and two enhanced analysis modules to perform interactive analyses on the collections of m7G sites (m7GFinder) and functional variants (m7GSNPer). We expect that m7Ghub v.2.0 should serve as a valuable centralized resource for studying m7G modification. It is freely accessible at: www.rnamd.org/m7GHub2.

HIV-1 RNAs whose transcription initiates from the third deoxyguanosine of GGG tract in the 5' long terminal repeat serve as a dominant genome for efficient provirus DNA formation.

Unspliced HIV-1 RNAs function as messenger RNAs for Gag or Gag-Pol polyproteins and progeny genomes packaged into virus particles. Recently, it has been reported that fate of the RNAs might be primarily determined, depending on transcriptional initiation sites among three consecutive deoxyguanosine residues (GGG tract) downstream of TATA-box in the 5' long terminal repeat (LTR). Although HIV-1 RNA transcription starts mostly from the first deoxyguanosine of the GGG tract and often from the second or third deoxyguanosine, RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine, were predominant in HIV-1 particles. Despite selective packaging of G1-form RNAs into virus particles, its biological impact during viral replication remains to be determined. In this study, we revealed that G1-form RNAs are primarily selected as a template for provirus DNA rather than other RNAs. In competitions between HIV-1 and lentiviral vector transcripts in virus-producing cells, approximately 80% of infectious particles were found to generate provirus using HIV-1 transcripts, while lentiviral vector transcripts were conversely selected when we used HIV-1 mutants in which the third deoxyguanosine in the GGG tract was replaced with deoxythymidine or deoxycytidine (GGT or GGC mutants, respectively). In the other analyses of proviral sequences after infection with an HIV-1 mutant in which the GGG tract in 3' LTR was replaced with TTT, most proviral sequences of the GGG-tract region in 5' LTR were found to be TTG, which is reasonably generated using the G1-form transcripts. Our results indicate that the G1-form RNAs serve as a dominant genome to establish provirus DNA.IMPORTANCESince the promoter for transcribing HIV-1 RNA is unique, all viral elements including genomic RNA and viral proteins have to be generated by the unique transcripts through ingenious mechanisms including RNA splicing and frameshifting during protein translation. Previous studies suggested a new mechanism for diversification of HIV-1 RNA functions by heterogeneous transcriptional initiation site usage; HIV-1 RNAs whose transcription initiates from a certain nucleotide were predominant in virus particles. In this study, we established two methods to analyze heterogenous transcriptional initiation site usage by HIV-1 during viral infection and showed that RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine of the GGG tract in 5' LTR, were primarily selected as viral genome in infectious particles and thus are used as a template to generate provirus for continuous replication. This study provides insights into the mechanism for diversification of unspliced RNA functions and requisites of lentivirus infectivity.

Structural Analysis Reveals that Toll-like Receptor 7 Is a Dual Receptor for Guanosine and Single-Stranded RNA.

Toll-like receptor 7 (TLR7) is a single-stranded RNA (ssRNA) sensor in innate immunity and also responds to guanosine and chemical ligands, such as imidazoquinoline compounds. However, TLR7 activation mechanism by these ligands remain largely unknown. Here, we generated crystal structures of three TLR7 complexes, and found that all formed an activated m-shaped dimer with two ligand-binding sites. The first site conserved in TLR7 and TLR8 was used for small ligand-binding essential for its activation. The second site spatially distinct from that of TLR8 was used for a ssRNA-binding that enhanced the affinity of the first-site ligands. The first site preferentially recognized guanosine and the second site specifically bound to uridine moieties in ssRNA. Our structural, biochemical, and mutagenesis studies indicated that TLR7 is a dual receptor for guanosine and uridine-containing ssRNA. Our findings have important implications for understanding of TLR7 function, as well as for therapeutic manipulation of TLR7 activation.

CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids.

An enzymatic method has been successfully established enabling the generation of partially base-modified RNA (previously named RZA) constructs, in which all G residues were replaced by isomorphic fluorescent thienoguanosine (thG) analogs, as well as fully modified RZA featuring thG, 5-bromocytosine, 7-deazaadenine and 5-chlorouracil. The transcriptional efficiency of emissive fully modified RZA was found to benefit from the use of various T7 RNA polymerase variants. Moreover, dthG could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing thG as well as thG together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N1-Methylpseudouridine was also demonstrated to be a faithful non-canonical substitute of uridine to direct Cas9 nuclease cleavage when incorporated in sgRNA. The Cas9 inactivation by 7-deazapurines indicated the importance of the 7-nitrogen atom of purines in both sgRNA and PAM site for achieving efficient Cas9 cleavage. Additional aspects of this study are discussed in relation to the significance of sgRNA-protein and PAM--protein interactions that were not highlighted by the Cas9-sgRNA-DNA complex crystal structure. These findings could expand the impact and therapeutic value of CRISPR-Cas9 and other RNA-based technologies.

With the advent of CRISPR-Cas9 gene editing, we now have to hand a simple two-component system amendable to silencing and knock-in editing effectively any gene. Yet we must not forget that the implications of immunotoxicity along with the poor stability and specificity of canonical nucleic acids hold enormous challenges for in vivo applications, especially in gene therapy. Our study endorses the feasibility of the enzymatic approach to incorporate nucleobase modifications into the CRISPR-Cas9 system unveiling the tolerance of Cas9 to N1-methylpseudouridine (m1Ψ)- and emissive thienoguanosine (thG)-modified sgRNA as well as thus far uncharted structural requirements for ensuring proper PAM recognition.

Crystal structure of a cap-independent translation enhancer RNA.

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

Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation.

Phenotypic heterogeneity of microbial populations can facilitate survival in dynamic environments by generating sub-populations of cells that may have differential fitness in a future environment. Bacillus subtilis cultures experiencing nutrient limitation contain distinct sub-populations of cells exhibiting either comparatively high or low protein synthesis activity. This heterogeneity requires the production of phosphorylated guanosine nucleotides (pp)pGpp by three synthases: SasA, SasB, and RelA. Here we show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population of cells exhibiting low protein synthesis whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. Previously, it was reported that a RelA product allosterically activates SasB and we find that a SasA product competitively inhibits this activation. Finally, we provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This work therefore identifies the mechanism underlying phenotypic heterogeneity in protein synthesis.

HIV-1 hypermethylated guanosine cap licenses specialized translation unaffected by mTOR.

Appended to the 5' end of nascent RNA polymerase II transcripts is 7-methyl guanosine (m7G-cap) that engages nuclear cap-binding complex (CBC) to facilitate messenger RNA (mRNA) maturation. Mature mRNAs exchange CBC for eIF4E, the rate-limiting translation factor that is controlled through mTOR. Experiments in immune cells have now documented HIV-1 incompletely processed transcripts exhibited hypermethylated m7G-cap and that the down-regulation of the trimethylguanosine synthetase-1-reduced HIV-1 infectivity and virion protein synthesis by several orders of magnitude. HIV-1 cap hypermethylation required nuclear RNA helicase A (RHA)/DHX9 interaction with the shape of the 5' untranslated region (UTR) primer binding site (PBS) segment. Down-regulation of RHA or the anomalous shape of the PBS segment abrogated hypermethylated caps and derepressed eIF4E binding for virion protein translation during global down-regulation of host translation. mTOR inhibition was detrimental to HIV-1 proliferation and attenuated Tat, Rev, and Nef synthesis. This study identified mutually exclusive translation pathways and the calibration of virion structural/accessory protein synthesis with de novo synthesis of the viral regulatory proteins. The hypermethylation of select, viral mRNA resulted in CBC exchange to heterodimeric CBP80/NCBP3 that expanded the functional capacity of HIV-1 in immune cells.

The role of GTP hydrolysis by EF-G in ribosomal translocation.

Translocation of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome is catalyzed by the GTPase elongation factor G (EF-G) in bacteria. Although guanosine-5'-triphosphate (GTP) hydrolysis accelerates translocation and is required for dissociation of EF-G, its fundamental role remains unclear. Here, we used ensemble Förster resonance energy transfer (FRET) to monitor how inhibition of GTP hydrolysis impacts the structural dynamics of the ribosome. We used FRET pairs S12-S19 and S11-S13, which unambiguously report on rotation of the 30S head domain, and the S6-L9 pair, which measures intersubunit rotation. Our results show that, in addition to slowing reverse intersubunit rotation, as shown previously, blocking GTP hydrolysis slows forward head rotation. Surprisingly, blocking GTP hydrolysis completely abolishes reverse head rotation. We find that the S13-L33 FRET pair, which has been used in previous studies to monitor head rotation, appears to report almost exclusively on intersubunit rotation. Furthermore, we find that the signal from quenching of 3'-terminal pyrene-labeled mRNA, which is used extensively to follow mRNA translocation, correlates most closely with reverse intersubunit rotation. To account for our finding that blocking GTP hydrolysis abolishes a rotational event that occurs after the movements of mRNA and tRNAs are essentially complete, we propose that the primary role of GTP hydrolysis is to create an irreversible step in a mechanism that prevents release of EF-G until both the tRNAs and mRNA have moved by one full codon, ensuring productive translocation and maintenance of the translational reading frame.

Biogenesis of telomerase RNA from a protein-coding mRNA precursor.

Telomerase is a eukaryotic ribonucleoprotein (RNP) enzyme that adds DNA repeats onto chromosome ends to maintain genomic stability and confer cellular immortality in cancer and stem cells. The telomerase RNA (TER) component is essential for telomerase catalytic activity and provides the template for telomeric DNA synthesis. The biogenesis of TERs is extremely divergent across eukaryotic kingdoms, employing distinct types of transcription machinery and processing pathways. In ciliates and plants, TERs are transcribed by RNA polymerase III (Pol III), while animal and ascomycete fungal TERs are transcribed by RNA Pol II and share biogenesis pathways with small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA), respectively. Here, we report an unprecedented messenger RNA (mRNA)-derived biogenesis pathway for the 1,291 nucleotide TER from the basidiomycete fungus Ustilago maydis. The U. maydis TER (UmTER) contains a 5'-monophosphate, distinct from the 5' 2,2,7-trimethylguanosine (TMG) cap common to animal and ascomycete fungal TERs. The mature UmTER is processed from the 3'-untranslated region (3'-UTR) of a larger RNA precursor that possesses characteristics of mRNA including a 5' 7-methyl-guanosine (m7G) cap, alternative splicing of introns, and a poly(A) tail. Moreover, this mRNA transcript encodes a protein called Early meiotic induction protein 1 (Emi1) that is conserved across dikaryotic fungi. A recombinant UmTER precursor expressed from an mRNA promoter is processed correctly to yield mature UmTER, confirming an mRNA-processing pathway for producing TER. Our findings expand the plethora of TER biogenesis mechanisms and demonstrate a pathway for producing a functional long noncoding RNA from a protein-coding mRNA precursor.