The interaction between ALKBH2 DNA repair enzyme and PCNA is direct, mediated by the hydrophobic pocket of PCNA and perturbed in naturally-occurring ALKBH2 variants

Human AlkB homolog 2 (ALKBH2) is a DNA repair enzyme that catalyzes the direct reversal of DNA methylation damage through oxidative demethylation. While ALKBH2 colocalizes with proliferating cell nuclear antigen (PCNA) in DNA replication foci, it remains unknown whether these two proteins alone form a complex or require additional components for interaction. Here, we demonstrate that ALKBH2 can directly interact with PCNA independent from other cellular factors, and we identify the hydrophobic pocket of PCNA as the key domain mediating this interaction. Moreover, we find that PCNA association with ALKBH2 increases significantly during DNA replication, suggesting that ALKBH2 forms a cell-cycle dependent complex with PCNA. Intriguingly, we show that an ALKBH2 germline variant, as well as a variant found in cancer, display altered interaction with PCNA. Our studies reveal the ALKBH2 binding interface of PCNA and indicate that both germline and somatic ALKBH2 variants could have cellular effects on ALKBH2 function in DNA repair.Swiss National Science Foundation (31003A_133100/1)National Institutes of Health (U.S.) (grant CA055042)National Institutes of Health (U.S.) (grant ES002109)Universität Züric

Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro.

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA

Elp3 and RlmN: A tale of two mitochondrial tail-anchored radical SAM enzymes in Toxoplasma gondii

Radical S-adenosylmethionine (rSAM) enzymes use a 5'-deoxyadensyl 5'-radical to methylate a wide array of diverse substrates including proteins, lipids and nucleic acids. One such enzyme, Elongator protein-3 (TgElp3), is an essential protein in Toxoplasma gondii, a protozoan parasite that can cause life-threatening opportunistic disease. Unlike Elp3 homologues which are present in all domains of life, TgElp3 localizes to the outer mitochondrial membrane (OMM) via a tail-anchored trafficking mechanism in Toxoplasma. Intriguingly, we identified a second tail-anchored rSAM domain containing protein (TgRlmN) that also localizes to the OMM. The transmembrane domain (TMD) on Toxoplasma Elp3 and RlmN homologues is required for OMM localization and has not been seen beyond the chromalveolates. Both TgElp3 and TgRlmN contain the canonical rSAM amino acid sequence motif (CxxxCxxC) necessary to form the 4Fe-4S cluster required for tRNA modifications. In E. coli, RlmN is responsible for the 2-methlyadenosine (m2A) synthesis at purine 37 in tRNA while in S. cerevisiae, Elp3 is necessary for the formation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at the wobble tRNA position. To investigate why these two rSAM enzymes localize to the mitochondrion in Toxoplasma, and whether or not TgRlmN and TgElp3 possess tRNA methyltransferase activity, a series of mutational and biochemical studies were performed. Overexpression of either TgElp3 or TgRlmN resulted in a significant parasite replication defect, but overexpression was tolerated if either the TMD or rSAM domain was mutated. Furthermore, we show the first evidence that Toxoplasma tRNAGlu contains the mcm5s2U modification, which is the putative downstream product generated by TgElp3 activity

Human AlkB Homolog ABH8 Is a tRNA Methyltransferase Required for Wobble Uridine Modification and DNA Damage Survival

tRNA nucleosides are extensively modified to ensure their proper function in translation. However, many of the enzymes responsible for tRNA modifications in mammals await identification. Here, we show that human AlkB homolog 8 (ABH8) catalyzes tRNA methylation to generate 5-methylcarboxymethyl uridine (mcm[superscript 5]U) at the wobble position of certain tRNAs, a critical anticodon loop modification linked to DNA damage survival. We find that ABH8 interacts specifically with tRNAs containing mcm5U and that purified ABH8 complexes methylate RNA in vitro. Significantly, ABH8 depletion in human cells reduces endogenous levels of mcm[superscript 5]U in RNA and increases cellular sensitivity to DNA-damaging agents. Moreover, DNA-damaging agents induce ABH8 expression in an ATM-dependent manner. These results expand the role of mammalian AlkB proteins beyond that of direct DNA repair and support a regulatory mechanism in the DNA damage response pathway involving modulation of tRNA modification.United States. National Institutes of Health (grant CA055042)United States. National Institutes of Health (grant ES002109)United States. National Institutes of Health (grant ES01701)National Institutes of Health (U.S.). Intramural Research ProgramWestaway Research FundNational Center for Research Resources (U.S.) (grant S10-RR023783

Balancing repair and tolerance of DNA damage caused by alkylating agents

Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for a favourable response of an organism to alkylating agents. Furthermore, the response of an individual to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity

Human telomerase and Cajal body ribonucleoproteins share a unique specificity of Sm protein association

Cajal bodies are nuclear structures that host RNA modification and assembly reactions. Some RNAs transit Cajal bodies, while others must concentrate in Cajal bodies to function. Here we report that at least a subfraction of human telomerase RNA and individual resident Cajal body RNAs is associated with Sm proteins. Surprisingly, of seven Sm proteins assembled into a heteroheptameric ring, only a subset copurifies telomerase and Cajal body ribonucleoproteins. We show that a Cajal body RNA localization motif determines this specificity. These discoveries expand the cellular repertoire of Sm protein assemblies and their involvement in ribonucleoprotein localization and function

Direct repair of 3,N[superscript 4]-ethenocytosine by the human ALKBH2 dioxygenase is blocked by the AAG/MPG glycosylase

Exocyclic ethenobases are highly mutagenic DNA lesions strongly implicated in inflammation and vinyl chloride-induced carcinogenesis. While the alkyladenine DNA glycosylase, AAG (or MPG), binds the etheno lesions 1,N[superscript 6]-ethenoadenine (ɛA) and 3,N[superscript 4]-ethenocytosine (ɛC) with high affinity, only ɛA can be excised to initiate base excision repair. Here, we discover that the human AlkB homolog 2 (ALKBH2) dioxygenase enzyme catalyzes direct reversal of ɛC lesions in both double- and single-stranded DNA with comparable efficiency to canonical ALKBH2 substrates. Notably, we find that in vitro, the non-enzymatic binding of AAG to ɛC specifically blocks ALKBH2-catalyzed repair of ɛC but not that of methylated ALKBH2 substrates. These results identify human ALKBH2 as a repair enzyme for mutagenic ɛC lesions and highlight potential consequences for substrate-binding overlap between the base excision and direct reversal DNA repair pathways.American Cancer Society (Postdoctoral Fellowship 116155-PF-08-255-01-GMC)National Institutes of Health (U.S.) (Grant CA055042)National Institutes of Health (U.S.) (Grant ES002109

Phosphorylation of human TRM9L integrates multiple stress-signaling pathways for tumor growth suppression

The human transfer RNA methyltransferase 9–like gene (TRM9L, also known as KIAA1456) encodes a negative regulator of tumor growth that is frequently silenced in many forms of cancer. While TRM9L can inhibit tumor cell growth in vivo, the molecular mechanisms underlying the tumor inhibition activity of TRM9L are unknown. We show that oxidative stress induces the rapid and dose-dependent phosphorylation of TRM9L within an intrinsically disordered domain that is necessary for tumor growth suppression. Multiple serine residues are hyperphosphorylated in response to oxidative stress. Using a chemical genetic approach, we identified a key serine residue in TRM9L that undergoes hyperphosphorylation downstream of the oxidative stress–activated MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal–regulated kinase)–RSK (ribosomal protein S6 kinase) signaling cascade. Moreover, we found that phosphorylated TRM9L interacts with the 14-3-3 family of proteins, providing a link between oxidative stress and downstream cellular events involved in cell cycle control and proliferation. Mutation of the serine residues required for TRM9L hyperphosphorylation and 14-3-3 binding abolished the tumor inhibition activity of TRM9L. Our results uncover TRM9L as a key downstream effector of the ERK signaling pathway and elucidate a phospho-signaling regulatory mechanism underlying the tumor inhibition activity of TRM9L

ALKBH7 drives a tissue and sex-specific necrotic cell death response following alkylation-induced damage

Regulated necrosis has emerged as a major cell death mechanism in response to different forms of physiological and pharmacological stress. The AlkB homolog 7 (ALKBH7) protein is required for regulated cellular necrosis in response to chemotherapeutic alkylating agents but its role within a whole organism is unknown. Here, we show that ALKBH7 modulates alkylation-induced cellular death through a tissue and sex-specific mechanism. At the whole-animal level, we find that ALKBH7 deficiency confers increased resistance to MMS-induced toxicity in male but not female mice. Moreover, ALKBH7-deficient mice exhibit protection against alkylation-mediated cytotoxicity in retinal photoreceptor and cerebellar granule cells, two cell types that undergo necrotic death through the initiation of the base excision repair pathway and hyperactivation of the PARP1/ARTD1 enzyme. Notably, the protection against alkylation-induced cerebellar degeneration is specific to ALKBH7-deficient male but not female mice. Our results uncover an in vivo role for ALKBH7 in mediating a sexually dimorphic tissue response to alkylation damage that could influence individual responses to chemotherapies based upon alkylating agents

Identification and rescue of a tRNA wobble inosine deficiency causing intellectual disability disorder

The deamination of adenosine to inosine at the wobble position of tRNA is an essential post-transcriptional RNA modification required for wobble decoding in bacteria and eukaryotes. In humans, the wobble inosine modification is catalyzed by the heterodimeric ADAT2/3 complex. Here, we describe novel pathogenic ADAT3 variants impairing adenosine deaminase activity through a distinct mechanism that can be corrected through expression of the heterodimeric ADAT2 subunit. The variants were identified in a family in which all three siblings exhibit intellectual disability linked to biallelic variants in the ADAT3 locus. The biallelic ADAT3 variants result in a missense variant converting alanine to valine at a conserved residue or the introduction of a premature stop codon in the deaminase domain. Fibroblast cells derived fromtwo ID-affected individuals exhibit a reduction in tRNA wobble inosine levels and severely diminished adenosine tRNA deaminase activity. Notably, the ADAT3 variants exhibit impaired interaction with the ADAT2 subunit and alterations in ADAT2-dependent nuclear localization. Based upon these findings, we find that tRNA adenosine deaminase activity and wobble inosine modification can be rescued in patient cells by overexpression of the ADAT2 catalytic subunit. These results uncover a key role for the inactive ADAT3 deaminase domain in proper assembly with ADAT2 and demonstrate that ADAT2/3 nuclear import is required for maintaining proper levels of the wobble inosine modification in tRNA