The 3'-5' RNA polymerase family consists of eukaryotic tRNAHis guanylyltransferase (Thg1) and Thg1 homologs known as Thg1-like proteins (TLPs) that exist in all three domains of life. Thg1 catalyzes an essential reaction adding a G-1 nucleotide to the 5' end of the tRNAHis, forming an identity element for tRNA aminoacylation. All TLPs studied, except Dictyostelium discoideum (Ddi) TLP2, perform in vitro Watson-Crick (WC) dependent addition of multiple nucleotides to repair truncated tRNA. DdiTLP2 has similar activity to Thg1, adding G-1 to mt-tRNAHis, but shares other biochemical properties with other TLPs, including a restriction to making WC base pairs during this reaction. We identified two regions in DdiTLP2 that lacked residues that are conserved in other Thg1/TLP enzymes. DdiTLP2 variants in both regions abolish enzymatic activity of DdiTLP2, indicating these regions are important for DdiTLP2 catalysis. Changing DdiThg1 D150 to the corresponding arginine found in DdiTLP2 causes an unexpected reversal of this enzyme's specificity, with a loss of its ability to incorporate a non-WC base-paired G-1 to its physiological substrate, while gaining the ability to add WC base-paired G-1 to mt-tRNAHis Biochemical study of other changes to D150, combined with structural models, suggests a previously unknown role for D150 in controlling substrate specificity at the adenylation step by providing a checkpoint for correct setup of a WC base pair in the active site. Thg1 also appears to have adapted the role of the ancestral D150 residue for a second function, promoting non-WC nucleotide addition to its eukaryotic tRNAHis substrate.

Polymerase α–primase (polα–primase) initiates DNA synthesis in eukaryotes through de novo synthesis of chimeric RNA-DNA primers necessary for replicative polymerases polδ and polε. The mechanism by which polα–primase synthesizes primers of defined length and RNA/DNA composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of polα–primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5′-end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer/template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα–primase.

Mitochondrial tRNA processing is a chronicle of molecular adaptability. The processing of structurally compromised tRNAs is unexpectedly rescued by a multienzyme complex shaped by constructive neutral evolution. This striking example of biological complexity arising from nonadaptive mechanisms showcases how a potential vulnerability is transformed into a robust, if precarious, innovation.

The evolutionarily conserved methyltransferase Trm10 modifies the N1 position of guanosine 9 (G9) in some tRNAs, but how the enzyme recognizes and modifies its substrate tRNAs remains unclear. Here, we used an S-adenosyl-L-methionine (SAM) analog to trap the Trm10-tRNAGly complex and enable determination of its structure in a post-catalytic state by cryogenic electron microscopy (cryo-EM). We observed three distinct complexes: two with a single Trm10 bound to tRNA that differ in their tRNA acceptor stem orientation ("closed" and "open") and a minor population with two Trm10s engaging the same tRNA. The monomeric complexes reveal a positively charged surface that guides the G9 into the catalytic site with key conserved residues forming "pincer"-like interactions that stabilize the flipped methylated nucleotide. In the open tRNA conformation, the acceptor stem is rotated away from the enzyme, weakening the tRNA-protein contacts, consistent with a product-release conformation. The dimeric complex, which is supported by tRNA-dependent protein crosslinking, reveals one Trm10 positioned similarly to the monomeric complexes and engaged with G9, while the other Trm10 contacts distal tRNA regions, suggesting a potential role in facilitating a key conformational transition during the process of catalysis or modified tRNA release. Finally, molecular dynamics simulations comparing G9- and A9-containing complexes reveal that G9 is efficiently stabilized in the binding pocket unlike A9, identifying the structural basis for guanosine selectivity. Overall, these findings reveal the structural determinants of G9-specific tRNA methylation by Trm10 and suggest a unique mechanism of action among RNA-modifying SPOUT methyltransferases.

The two-metal ion mechanism for catalysis of RNA and DNA synthesis by 5'-3' polymerases has been extensively characterized. The 3'-5' polymerase family of enzymes, consisting of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), perform a similar nucleotide addition reaction, but in the reverse direction, adding Watson-Crick base-paired NTPs to the 5'-ends of RNA substrates. However, the effect of divalent cations beyond magnesium has not been described. Here, we examined the effects of five divalent cations (Mg2+, Mn2+, Co2+, Ni2+ and Ca2+) on templated nucleotide addition activity and the kinetics of 5'-activation by ATP catalyzed by recombinantly purified, metal-free TLPs from organisms across diverse domains of life. This work revealed that different TLPs exhibit distinct dependencies on the concentration and identity of divalent metal ions that support effective catalysis. The patterns of metal ion usage demonstrated here for TLPs evince features that are characteristic of both canonical 5'-3' polymerases and DNA/RNA ligases. Similar to 5'-3' polymerases, some metals were also observed to be mutagenic in the context of TLP catalysis. Furthermore, we provide the first direct evidence that both ATP and the NTP poised for nucleotidyl transfer are present in the active site during the 5'-adenylylation. These results provide the first in-depth study of the role of the two-metal ion mechanism in TLP catalysis, which was initially suggested by structures of these enzymes.

Unlike most polymerases that act in the 5′ to 3′ direction, tRNAHis guanylyltransferase (Thg1) synthesizes RNA 3′ to 5′. Thg1 catalyzes an essential reaction during tRNAHis processing by adding a G nucleotide on the 5′ end of the tRNA in most eukaryotes, forming an identity element for aminoacylation of the tRNA. Recent characterization of Thg1 homologs with alternative specificities, known as Thg1-like proteins (TLPs), raises questions about how distinct substrates are recognized by different members of this highly conserved enzyme family. In the slime mold Dictyostelium discoideum (Ddi), DdiThg1 adds G to the 5′ end of cytosolic (cy-) tRNAHis, while DdiTLP2 catalyzes the same reaction, but only with mitochondrial (mt-) tRNAHis substrates. In vivo and in vitro, these two enzymes exhibit strict specificity for their respective tRNA substrates. Moreover, unlike Thg1, DdiTLP2 does not depend on the GUG anticodon for tRNAHis recognition, suggesting different mechanisms for tRNA recognition by these two enzymes. We aimed to determine the molecular basis for the distinct RNA substrate specificities of DdiThg1 and DdiTLP2, and thus to provide insight into the general mechanisms of RNA recognition utilized by distinct 3'-5' RNA polymerases. To answer this question, electrophoretic mobility shift assays (EMSA) were used to assess tRNA binding by DdiThg1 vs. DdiTLP2. These assays revealed no difference in either enzyme's ability to bind to different tRNAs, requiring additional catalytic factors to explain the selective in vitro activities of these enzymes. Sequence comparison was used to identify a unique residue in DdiTLP2 (R187) that is different from an absolutely conserved D/E residue at the analogous position in the rest of Thg1/TLP family members, including in DdiThg1. We hypothesized that this unusual R187 residue may be responsible for the unique biochemical properties exhibited by DdiTLP2. Indeed, replacement of the conserved D residue in DdiThg1 with the R found in DdiTLP2 caused a dramatic reversal of DdiThg1 substrate specificity. The DdiThg1 D150R variant lost the ability to catalyze G-1 addition with cy-tRNAHis and instead gained the ability to act on DdiTLP2's mt-tRNAHis substrate. Interestingly, kinetic and conservative amino acid replacement studies revealed that the change in DdiThg1 predominantly impacts the second step of the 3'-5' addition reaction, suggesting a direct role for the conserved D residue in this nucleotidyl transfer step. This result may also help to explain a previously observed in vivo growth defect associated with alanine replacement of the analogous D residue in Saccharomyces cerevisiae (Sc) Thg1, despite the fact that the ScThg1 D153A variant enzyme retains full in vitro catalytic activity. These results suggest that the DdiThg1 D153/ScThg1 D153 residue helps to control RNA substrate specificity and carry out the nucleotidyl transfer reaction step in this unusual family of 3'-5' RNA polymerases. Grace Johnecheck was supported by NIH T32 GM141955; this research was funded by R01 GM087543.

The Thg1/TLP family of enzymes are 3'-5' RNA polymerases that catalyze diverse reactions during processes such as tRNAHis maturation and 5' end repair of tRNAs during tRNA editing. The eukaryotic slime mold Dictyostelium discoideum encodes four enzymes from the Thg1/TLP family: DdiThg1, DdiTLP2, DdiTLP3, and DdiTLP4. Previous work revealed distinct biological functions for three out of the four enzymes, while the biological role for DdiTLP4 remains unknown. Moreover, although it is known that DdiTLP3 catalyzes tRNA repair reactions during tRNA editing, the identity of the nuclease that generates tRNA substrates for tRNA repair in D. discoideum has not been identified. Therefore, the specific characteristics of tRNAs that are substrates for DdiTLP3 in vivo have also not been defined. We hypothesize that each of these enzymes interacts selectively with one or more cellular RNAs in order to carry out their respective biological functions. In this investigation, we are seeking to identify these RNAs that associate selectively with DdiTLP3 and DdiTLP4 to help define the biological substrates for each enzyme and to uncover unknown mechanistic details of their 3′-5′ nucleotide addition reactions. To do this, we carried out an immunoprecipitation-based approach to identify D. discoideum RNAs that co-purify with DdiTLP3 and DdiTLP4 that can be identified by RNA sequencing. I created constructs with a FLAG epitope tag fused to each terminus of both DdiTLPs for expression and purification in E. coli, and used kinetic assays to demonstrate that the presence of a FLAG-tag does not negatively impact the activity of each TLP compared to each wild-type enzyme. Subsequently, two different cross-linking methods, formaldehyde and UV light, were tested for the efficiency and specificity of protein-RNA crosslinks between the FLAG-tagged proteins and RNA isolated from D. discoideum. After optimization of each protocol, we determined that UV cross-linking, while less efficient, appears to result in a higher specificity of RNA-protein crosslinking. Utilizing radioactive 5'-end labeling, we have observed immunoprecipitated RNA of various apparent sizes under our optimized in vitro UV-CLIP conditions and will use RNA isolated from the CLIP reactions to identify these potential RNA substrates with RNA-Seq. Complementary experiments to perform CLIP to isolate co-purifying substrates with FLAG-tagged enzymes expressed in vivo in D. discoideum are also underway. By understanding the exact roles of TLPs in D. discoideum, we can possibly apply our findings to the roles of TLPs in other eukaryotes where specific biological functions for these enzymes have not yet been demonstrated.

Members of the 3 '-5 ' RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5 '-3 ' RNA and DNA polymerases. The discovery of enzymes capable of this reaction raised the possibility of exploiting 3 '-5 ' polymerases for posttranscriptional incorporation of nucleotides to the 5 '-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other noncoding RNAs. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3 '-5 ' polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5 '-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in trans to guide 5 '-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to eight bases to 5 '-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5 '-end labeling tool.

The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of Xenopus laevis polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5' end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer-template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.

InSaccharomyces cerevisiae, a single homolog of the tRNA methyltransferase Trm10 performs m1G9 modification on 13 different tRNAs. Here we provide evidence that the m1G9 modification catalyzed byS. cerevisiaeTrm10 plays a biologically important role for one of these tRNA substrates, tRNATrp. Overexpression of tRNATrp(and not any of 38 other elongator tRNAs) rescues growth hypersensitivity of thetrm10Δstrain in the presence of the antitumor drug 5-fluorouracil (5FU). Mature tRNATrpis depleted intrm10Δcells, and its levels are further decreased upon growth in 5FU, while another Trm10 substrate (tRNAGly) is not affected under these conditions. Thus, m1G9 inS. cerevisiaeis another example of a tRNA modification that is present on multiple tRNAs but is only essential for the biological function of one of those species. In addition to the effects of m1G9 on mature tRNATrp, precursor tRNATrpspecies accumulate in the same strains, an effect that is due to at least two distinct mechanisms. The levels of mature tRNATrpare rescued in thetrm10Δmet22Δstrain, consistent with the known role of Met22 in tRNA quality control, where deletion ofmet22causes inhibition of 5′–3′ exonucleases that catalyze tRNA decay. However, none of the known Met22-associated exonucleases appear to be responsible for the decay of hypomodified tRNATrp, based on the inability of mutants of each enzyme to rescue the growth of thetrm10Δstrain in the presence of 5FU. Thus, the surveillance of tRNATrpappears to constitute a distinct tRNA quality control pathway inS. cerevisiae.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNAmodifying enzymes which must distinguish between structurally similar tRNA species.

Transfer ribonucleic acid (tRNA) is the most highly modified RNA species in the cell, and loss of tRNA modifications can lead to growth defects in yeast as well as metabolic, neurological, and mitochondrial disorders in humans. Significant progress has been made toward identifying the enzymes that are responsible for installing diverse modifications in tRNA, revealing a landscape of fascinating biological and mechanistic diversity that remains to be fully explored. Most early discoveries of tRNA modification enzymes were in model systems, where many enzymes were not strictly required for viability, an observation somewhat at odds with the extreme conservation of many of the same enzymes throughout multiple domains of life. Moreover, many tRNA modification enzymes act on more than one type of tRNA substrate, which is not necessarily surprising given the similar overall secondary and tertiary structures of tRNA, yet biochemical characterization has revealed interesting patterns of substrate specificity that can be challenging to rationalize on a molecular level. Questions about how many enzymes efficiently select a precise set of target tRNAs from among a structurally similar pool of molecules persist.The tRNA methyltransferase Trm10 provides an exciting paradigm to study the biological and mechanistic questions surrounding tRNA modifications. Even though the enzyme was originally characterized in Saccharomyces cerevisiae where its deletion causes no detectable phenotype under standard lab conditions, several more recently identified phenotypes provide insight into the requirement for this modification in the overall quality control of the tRNA pool. Studies of Trm10 in yeast also revealed another characteristic feature that has turned out to be a conserved feature of enzymes throughout the Trm10 family tree. We were initially surprised to see that purified S. cerevisiae Trm10 was capable of modifying tRNA substrates that were not detectably modified by the enzyme in vivo in yeast. This pattern has continued to emerge as we and others have studied Trm10 orthologs from Archaea and Eukarya, with enzymes exhibiting in vitro substrate specificities that can differ significantly from in vivo patterns of modification. While this feature complicates efforts to predict substrate specificities of Trm10 enzymes in the absence of appropriate genetic systems, it also provides an exciting opportunity for studying how enzyme activities can be regulated to achieve dynamic patterns of biological tRNA modification, which have been shown to be increasingly important for stress responses and human disease. Finally, the intriguing diversity in target nucleotide modification that has been revealed among Trm10 orthologs is distinctive among known tRNA modifying enzymes and necessitates unusual and likely novel catalytic strategies for methylation that are being revealed by biochemical and structural studies directed toward various family members. These efforts will no doubt yield more surprising discoveries in terms of tRNA modification enzymology.

The SpoU-TrmD (SPOUT) methyltransferase superfamily was designated when structural similarity was identified between the transfer RNA-modifying enzymes TrmH (SpoU) and TrmD. SPOUT methyltransferases are found in all domains of life and predominantly modify transfer RNA or ribosomal RNA substrates, though one instance of an enzyme with a protein substrate has been reported. Modifications placed by SPOUT methyltransferases play diverse roles in regulating cellular processes such as ensuring translational fidelity, altering RNA stability, and conferring bacterial resistance to antibiotics. This large collection of S-adenosyl-L-methionine-dependent methyltransferases is defined by a unique alpha/beta fold with a deep trefoil knot in their catalytic (SPOUT) domain. Herein, we describe current knowledge of SPOUT enzyme structure, domain architecture, and key elements of catalytic function, including S-adenosyl-L-methionine co-substrate binding, beginning with a new sequence alignment that divides the SPOUT methyltransferase superfamily into four major clades. Finally, a major focus of this review will be on our growing understanding of how these diverse enzymes accomplish the molecular feat of specific substrate recognition and modification, as highlighted by recent advances in our knowledge of protein-RNA complex structures and the discovery of the dependence of one SPOUT methyltransferase on metal ion binding for catalysis. Considering the broad biological roles of RNA modifications, developing a deeper understanding of the process of substrate recognition by the SPOUT enzymes will be critical for defining many facets of fundamental RNA biology with implications for human disease.

The application of in vitro kinetic tools has the potential to provide important insight into the molecular mechanisms of RNA modification enzymes. Utilizing quantitative biochemical approaches can reveal information about enzyme preferences for specific substrates that are relevant for understanding modification reactions in their biological contexts. Moreover, kinetic tools have been powerfully applied to identify and characterize roles for specific amino acid residues in catalysis, which can be essential information for understanding the molecular basis for human disease, as well as for targeting these enzymes for potential therapeutic interventions. RNA methyltransferases are a particularly interesting group of RNA modification enzymes because of the diversity in structure and mechanism that has been revealed among members of this group, even including some examples of enzymes that use entirely distinct reaction mechanisms to form identical methylated nucleotides in RNA. Yet, many questions remain unanswered about how these distinct catalytic strategies are facilitated by the relevant enzyme families. We have applied in vitro kinetic analysis to specifically focus on catalytically relevant ionizations in the context of tRNA methyltransferase reactions, by measuring rates under conditions of varied pH. This analysis can be applied broadly to RNA methyltransferases to expand our understanding of these important enzymes.

The tRNA His guanylyltransferase (Thg1) was originally discovered in Saccharomyces cerevisiae where it catalyzes 3′–5′ addition of a single nontemplated guanosine (G −1 ) to the 5′ end of tRNA His . In addition to this activity, S. cerevisiae Thg1 (SceThg1) also catalyzes 3′–5′ polymerization of Watson–Crick (WC) base pairs, utilizing nucleotides in the 3′-end of a tRNA as the template for addition. Subsequent investigation revealed an entire class of enzymes related to Thg1, called Thg1-like proteins (TLPs). TLPs are found in all three domains of life and preferentially catalyze 3′–5′ polymerase activity, utilizing this unusual activity to repair tRNA, among other functions. Although both Thg1 and TLPs utilize the same chemical mechanism, the molecular basis for differences between WC-dependent (catalyzed by Thg1 and TLPs) and non-WC-dependent (catalyzed exclusively by Thg1) reactions has not been fully elucidated. Here we investigate the mechanism of base-pair recognition by 3′–5′ polymerases using transient kinetic assays, and identify Thg1-specific residues that play a role in base-pair discrimination. We reveal that, regardless of the identity of the opposing nucleotide in the RNA “template,” addition of a non-WC G −1 residue is driven by a unique kinetic preference for GTP. However, a secondary preference for forming WC base pairs is evident for all possible templating residues. Similar to canonical 5′–3′ polymerases, nucleotide addition by SceThg1 is driven by the maximal rate rather than by NTP substrate affinity. Together, these data provide new insights into the mechanism of base-pair recognition by 3′–5′ polymerases.

Acute graft-versus-host disease (aGVHD) is a T cell-mediated immunological disorder and the leading cause of nonrelapse mortality in patients who receive allogeneic hematopoietic cell transplants. Based on recent observations that protein arginine methyltransferase 5 (PRMT5) and arginine methylation are upregulated in activated memory T cells, we hypothesized that PRMT5 is involved in the pathogenesis of aGVHD. Here, we show that PRMT5 expression and enzymatic activity were upregulated in activated T cells in vitro and in T cells from mice developing aGVHD after allogeneic transplant. PRMT5 expression was also upregulated in T cells of patients who developed aGVHD after allogeneic hematopoietic cell transplant compared with those who did not develop aGVHD. PRMT5 inhibition using a selective small-molecule inhibitor (C220) substantially reduced mouse and human allogeneic T cell proliferation and inflammatory IFN-γ and IL-17 cytokine production. Administration of PRMT5 small-molecule inhibitors substantially improves survival, reducing disease incidence and clinical severity in mouse models of aGVHD without adversely affecting engraftment. Importantly, we show that PRMT5 inhibition retained the beneficial graft-versus-leukemia effect by maintaining cytotoxic CD8+ T cell responses. Mechanistically, we show that PRMT5 inhibition potently reduced STAT1 phosphorylation as well as transcription of proinflammatory genes, including interferon-stimulated genes and IL-17. Additionally, PRMT5 inhibition deregulates the cell cycle in activated T cells and disrupts signaling by affecting ERK1/2 phosphorylation. Thus, we have identified PRMT5 as a regulator of T cell responses and as a therapeutic target in aGVHD.