Characterization of tRNA splicing enzymes RNA ligase and tRNA 2'-phosphotransferase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trl1 enzymes are present in many human fungal pathogens and are promising targets for antifungal drug discovery because their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. Here we report that Mucorales species (deemed high-priority human pathogens by WHO) elaborate a noncanonical tRNA splicing apparatus in which a monofunctional RNA ligase enzyme is encoded separately from any end-healing enzymes. We show that Mucor circinelloides RNA ligase (MciRNL) is active in tRNA splicing in vivo in budding yeast in lieu of the Trl1 ligase domain. Biochemical and kinetic characterization of recombinant MciRNL underscores its requirement for a 2'-PO4 terminus in the end-joining reaction, whereby the 2'-PO4 enhances the rates of RNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3) by ∼125-fold and ∼6200-fold, respectively. In the canonical fungal tRNA splicing pathway, the splice junction 2'-PO4 installed by RNA ligase is removed by a dedicated NAD+-dependent RNA 2'-phosphotransferase Tpt1. Here we identify and affirm by genetic complementation in yeast the biological activity of Tpt1 orthologs from three Mucorales species. Recombinant M. circinelloides Tpt1 has vigorous NAD+-dependent RNA 2'-phosphotransferase activity in vitro.

Activity and substrate specificity of Candida, Aspergillus, and Coccidioides Tpt1: essential tRNA splicing enzymes and potential anti-fungal targets.

The enzyme Tpt1 is an essential agent of fungal tRNA splicing that removes an internal RNA 2'-PO4 generated by fungal tRNA ligase. Tpt1 performs a two-step reaction in which: (i) the 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-(ADP-ribose) intermediate; and (ii) transesterification of the ADP-ribose O2'' to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1'',2''-cyclic phosphate. Because Tpt1 does not participate in metazoan tRNA splicing, and Tpt1 knockout has no apparent impact on mammalian physiology, Tpt1 is considered a potential anti-fungal drug target. Here we characterize Tpt1 enzymes from four human fungal pathogens: Coccidioides immitis, the agent of Valley Fever; Aspergillus fumigatus and Candida albicans, which cause invasive, often fatal, infections in immunocompromised hosts; and Candida auris, an emerging pathogen that is resistant to current therapies. All four pathogen Tpt1s were active in vivo in complementing a lethal Saccharomyces cerevisiae tpt1∆ mutation and in vitro in NAD+-dependent conversion of a 2'-PO4 to a 2'-OH. The fungal Tpt1s utilized nicotinamide hypoxanthine dinucleotide as a substrate in lieu of NAD+, albeit with much lower affinity, whereas nicotinic acid adenine dinucleotide was ineffective. Fungal Tpt1s efficiently removed an internal ribonucleotide 2'-phosphate from an otherwise all-DNA substrate. Replacement of an RNA ribose-2'-PO4 nucleotide with arabinose-2'-PO4 diminished enzyme specific activity by ≥2000-fold and selectively slowed step 2 of the reaction pathway, resulting in transient accumulation of an ara-2'-phospho-ADP-ribosylated intermediate. Our results implicate the 2'-PO4 ribonucleotide as the principal determinant of fungal Tpt1 nucleic acid substrate specificity.

Substrate analogs that trap the 2'-phospho-ADP-ribosylated RNA intermediate of the Tpt1 (tRNA 2'-phosphotransferase) reaction pathway.

The enzyme Tpt1 removes an internal RNA 2'-PO4 via a two-step reaction in which: (i) the 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Because step 2 is much faster than step 1, the ADP-ribosylated RNA intermediate is virtually undetectable under normal circumstances. Here, by testing chemically modified nucleic acid substrates for activity with bacterial Tpt1 enzymes, we find that replacement of the ribose-2'-PO4 nucleotide with arabinose-2'-PO4 selectively slows step 2 of the reaction pathway and results in the transient accumulation of high levels of the reaction intermediate. We report that replacing the NMN ribose of NAD+ with 2'-fluoroarabinose (thereby eliminating the ribose O2″ nucleophile) results in durable trapping of RNA-2'-phospho-(ADP-fluoroarabinose) as a "dead-end" product of step 1. Tpt1 enzymes from diverse taxa differ in their capacity to use ara-2″F-NAD+ as a substrate.

NAD+-dependent RNA terminal 2' and 3' phosphomonoesterase activity of a subset of Tpt1 enzymes.

The enzyme Tpt1 removes the 2'-PO4 at the splice junction generated by fungal tRNA ligase; it does so via a two-step reaction in which (i) the internal RNA 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-ADP-ribosyl intermediate; and (ii) transesterification of the ribose O2″ to the 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. The role that Tpt1 enzymes play in taxa that have no fungal-type RNA ligase remains obscure. An attractive prospect is that Tpt1 enzymes might catalyze reactions other than internal RNA 2'-PO4 removal, via their unique NAD+-dependent transferase mechanism. This study extends the repertoire of the Tpt1 enzyme family to include the NAD+-dependent conversion of RNA terminal 2' and 3' monophosphate ends to 2'-OH and 3'-OH ends, respectively. The salient finding is that different Tpt1 enzymes vary in their capacity and positional specificity for terminal phosphate removal. Clostridium thermocellum and Aeropyrum pernix Tpt1 proteins are active on 2'-PO4 and 3'-PO4 ends, with a 2.4- to 2.6-fold kinetic preference for the 2'-PO4 The accumulation of a terminal 3'-phospho-ADP-ribosylated RNA intermediate during the 3'-phosphotransferase reaction suggests that the geometry of the 3'-p-ADPR adduct is not optimal for the ensuing transesterification step. Chaetomium thermophilum Tpt1 acts specifically on a terminal 2'-PO4 end and not with a 3'-PO4 In contrast, Runella slithyformis Tpt1 and human Tpt1 are ineffective in removing either a 2'-PO4 or 3'-PO4 end.

Two-step mechanism and step-arrest mutants of Runella slithyformis NAD+-dependent tRNA 2'-phosphotransferase Tpt1.

Tpt1 catalyzes the transfer of an internal 2'-monophosphate moiety (2'-PO4) from a "branched" 2'-PO4 RNA splice junction to NAD+ to form a "clean" 2'-OH, 3'-5' phosphodiester junction, ADP-ribose 1″-2″ cyclic phosphate, and nicotinamide. First discovered as an essential component of the Saccharomyces cerevisiae tRNA splicing machinery, Tpt1 is widely distributed in nature, including in taxa that have no yeast-like RNA splicing system. Here we characterize the RslTpt1 protein from the bacterium Runella slithyformis, in which Tpt1 is encoded within a putative RNA repair gene cluster. We find that (i) expression of RslTpt1 in yeast complements a lethal tpt1Δ knockout, and (ii) purified recombinant RslTpt1 is a bona fide NAD+-dependent 2'-phosphotransferase capable of completely removing an internal 2'-phosphate from synthetic RNAs. The in vivo activity of RslTpt1 is abolished by alanine substitutions for conserved amino acids Arg16, His17, Arg64, and Arg119. The R64A, R119A, and H17A mutants accumulate high levels of a 2'-phospho-ADP-ribosylated RNA reaction intermediate (2'-P-ADPR, evanescent in the wild-type RslTpt1 reaction), which is converted slowly to a 2'-OH RNA product. The R16A mutant is 300-fold slower than wild-type RslTpt1 in forming the 2'-P-ADPR intermediate. Whereas wild-type RsTpt1 rapidly converts the isolated 2'-P-ADPR intermediate to 2'-OH product in the absence of NAD+, the H17A, R119A, R64A, and R16A mutant are slower by factors of 3, 33, 210, and 710, respectively. Our results identify active site constituents involved in the catalysis of step 1 and step 2 of the Tpt1 reaction pathway.

Reproducible Dendronized PEG Hydrogels via SPAAC Cross-Linking.

A common issue with hydrogel formulations is batch-to-batch irreproducibility originating from poorly defined polymer precursors. Here, we report the use of dendritic polymer end-groups to address this issue and maintain reproducibility between batches of poly(ethylene glycol) (PEG) hydrogels. Specifically, we synthesized two end-functionalized PEG chains: one with azide-terminated first- and second-generation dendrons and the other with strained cyclooctynes. The two complementary azide and alkyne polymers react via strain-promoted alkyne-azide cycloaddition (SPAAC) to produce hydrogels quickly in the absence of additional reagents or catalyst at low polymer concentrations. Hydrogels made with first-generation dendrons gelled in minutes and exhibited a small degree of swelling when incubated in PBS buffer at 37 °C, whereas hydrogels made from second-generation dendrons gelled in seconds with almost no swelling upon incubation at 37 °C. In both cases, the hydrogels proved reproducible, resulting in identical Young's modulus values from different batches. The hydrogels prepared with second-generation dendrons were seeded with human mesenchymal stem cells and showed high cell viability as well as cell spreading over a two-week time frame. These studies show that the SPAAC hydrogels are noncytotoxic and are capable of supporting cell growth.

Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles.

CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5' handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2'-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2'-hydroxyl sensitivity. Modified 5' pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5' pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5' pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.

Structure of tRNA splicing enzyme Tpt1 illuminates the mechanism of RNA 2'-PO4 recognition and ADP-ribosylation.

Tpt1 is an essential agent of fungal tRNA splicing that removes the 2'-PO4 at the splice junction generated by fungal tRNA ligase. Tpt1 catalyzes a unique two-step reaction whereby the 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-ADP-ribosyl intermediate that undergoes transesterification to yield 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. Because Tpt1 is inessential in exemplary bacterial and mammalian taxa, Tpt1 is seen as an attractive antifungal target. Here we report a 1.4 Šcrystal structure of Tpt1 in a product-mimetic complex with ADP-ribose-1″-phosphate in the NAD+ site and pAp in the RNA site. The structure reveals how Tpt1 recognizes a 2'-PO4 RNA splice junction and the mechanism of RNA phospho-ADP-ribosylation. This study also provides evidence that a bacterium has an endogenous phosphorylated substrate with which Tpt1 reacts.