Search

Toxins of toxin-antitoxin systems use diverse mechanisms to inhibit bacterial growth. In this study, we characterize the translation inhibitor toxin MenT3 of Mycobacterium tuberculosis, the bacterium responsible for tuberculosis in humans. We show that MenT3 is a robust cytidine specific tRNA nucleotidyltransferase in vitro, capable of modifying the aminoacyl acceptor ends of most tRNA but with a marked preference for tRNA Ser , to which long stretches of cytidines are added. Furthermore, transcriptomic-wide analysis of MenT3 targets in M. tuberculosis identifies tRNA Ser as the sole target of MenT3 and reveals significant detoxification attempts by the essential CCA-adding enzyme PcnA in response to MenT3. Finally, under physiological conditions, only in the presence the native menAT3 operon, an active pool of endogenous MenT3 targeting tRNA Ser in M. tuberculosis is detected, likely reflecting the importance of MenT3 during infection., (© 2024. The Author(s).)

Continuous directed evolution of base editors (BEs) has been successful in bacteria cells, but not yet in mammalian cells. Here, we report the development of a Continuous Directed Evolution system in Mammalian cells (CDEM). CDEM enables the BE evolution in a full-length manner with Cas9 nickase. We harness CDEM to evolve the deaminases of cytosine base editor BE3 and adenine base editors, ABEmax and ABE8e. The evolved cytidine deaminase variants on BE4 architecture show not only narrowed editing windows, but also higher editing purity and low off-target activity without a trade-off in on-targeting activity. The evolved ABEmax and ABE8e variants exhibit narrowed or shifted editing windows to different extents, and lower off-target effects. The results illustrate that CDEM is a simple but powerful approach to continuously evolve BEs without size restriction in the mammalian environment, which is advantageous over continuous directed evolution system in bacteria cells., (© 2024. The Author(s).)

RNAs are often modified to invoke new activities. While many modifications are limited in frequency, restricted to non-coding RNAs, or present only in select organisms, 5-methylcytidine (m 5 C) is abundant across diverse RNAs and fitness-relevant across Domains of life, but the synthesis and impacts of m 5 C have yet to be fully investigated. Here, we map m 5 C in the model hyperthermophile, Thermococcus kodakarensis. We demonstrate that m 5 C is ~25x more abundant in T. kodakarensis than human cells, and the m 5 C epitranscriptome includes ~10% of unique transcripts. T. kodakarensis rRNAs harbor tenfold more m 5 C compared to Eukarya or Bacteria. We identify at least five RNA m 5 C methyltransferases (R5CMTs), and strains deleted for individual R5CMTs lack site-specific m 5 C modifications that limit hyperthermophilic growth. We show that m 5 C is likely generated through partial redundancy in target sites among R5CMTs. The complexity of the m 5 C epitranscriptome in T. kodakarensis argues that m 5 C supports life in the extremes., (© 2024. The Author(s).)

The epitranscriptome includes a diversity of RNA modifications that influence gene expression. N3-methylcytidine (m 3 C) mainly occurs in the anticodon loop (position C32) of certain tRNAs yet its role is poorly understood. Here, using HAC-Seq, we report comprehensive METTL2A/2B-, METTL6-, and METTL2A/2B/6-dependent m 3 C profiles in human cells. METTL2A/2B modifies tRNA-arginine and tRNA-threonine members, whereas METTL6 modifies the tRNA-serine family. However, decreased m 3 C32 on tRNA-Ser-GCT isodecoders is only observed with combined METTL2A/2B/6 deletion. Ribo-Seq reveals altered translation of genes related to cell cycle and DNA repair pathways in METTL2A/2B/6-deficient cells, and these mRNAs are enriched in AGU codons that require tRNA-Ser-GCT for translation. These results, supported by reporter assays, help explain the observed altered cell cycle, slowed proliferation, and increased cisplatin sensitivity phenotypes of METTL2A/2B/6-deficient cells. Thus, we define METTL2A/2B/6-dependent methylomes and uncover a particular requirement of m 3 C32 tRNA modification for serine codon-biased mRNA translation of cell cycle, and DNA repair genes., (© 2024. The Author(s).)

N-acetyltransferase 10 (NAT10) is an N 4 -acetylcytidine (ac 4 C) writer that catalyzes RNA acetylation at cytidine N 4 position on tRNAs, rRNAs and mRNAs. Recently, NAT10 and the associated ac 4 C have been reported to increase the stability of HIV-1 transcripts. Here, we show that NAT10 catalyzes ac 4 C addition to the polyadenylated nuclear RNA (PAN), a long non-coding RNA encoded by the oncogenic DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV), triggering viral lytic reactivation from latency. Mutagenesis of ac 4 C sites in PAN RNA in the context of KSHV infection abolishes PAN ac 4 C modifications, downregulates the expression of viral lytic genes and reduces virion production. NAT10 knockdown or mutagenesis erases ac 4 C modifications of PAN RNA and increases its instability, and prevents KSHV reactivation. Furthermore, PAN ac 4 C modification promotes NAT10 recruitment of IFN-γ-inducible protein-16 (IFI16) mRNA, resulting in its ac 4 C acetylation, mRNA stability and translation, and eventual inflammasome activation. These results reveal a novel mechanism of viral and host ac 4 C modifications and the associated complexes as a critical switch of KSHV replication and antiviral immunity., (© 2023. Springer Nature Limited.)

APOBEC3 (A3) proteins are host-encoded deoxycytidine deaminases that provide an innate immune barrier to retroviral infection, notably against HIV-1. Low levels of deamination are believed to contribute to the genetic evolution of HIV-1, while intense catalytic activity of these proteins can induce catastrophic hypermutation in proviral DNA leading to near-total HIV-1 restriction. So far, little is known about how A3 cytosine deaminases might impact HIV-1 proviral DNA integration sites in human chromosomal DNA. Using a deep sequencing approach, we analyze the influence of catalytic active and inactive APOBEC3F and APOBEC3G on HIV-1 integration site selections. Here we show that DNA editing is detected at the extremities of the long terminal repeat regions of the virus. Both catalytic active and non-catalytic A3 mutants decrease insertions into gene coding sequences and increase integration sites into SINE elements, oncogenes and transcription-silencing non-B DNA features. Our data implicates A3 as a host factor influencing HIV-1 integration site selection and also promotes what appears to be a more latent expression profile., (© 2023. The Author(s).)

Post-transcriptional RNA editing modulates gene expression in a condition-dependent fashion. We recently discovered C-to-Ψ editing in Vibrio cholerae tRNA. Here, we characterize the biogenesis, regulation, and functions of this previously undescribed RNA editing process. We show that an enzyme, TrcP, mediates the editing of C-to-U followed by the conversion of U to Ψ, consecutively. AlphaFold-2 predicts that TrcP consists of two globular domains (cytidine deaminase and pseudouridylase) and a long helical domain. The latter domain tethers tRNA substrates during both the C-to-U editing and pseudouridylation, likely enabling a substrate channeling mechanism for efficient catalysis all the way to the terminal product. C-to-Ψ editing both requires and suppresses other modifications, creating an interdependent network of modifications in the tRNA anticodon loop that facilitates coupling of tRNA modification states to iron availability. Our findings provide mechanistic insights into an RNA editing process that likely promotes environmental adaptation., (© 2022. The Author(s).)

CRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop (pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR ( http://z.umn.edu/spliceR ) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.

Cpf1-linked base editors broaden the targeting scope of programmable cytidine deaminases by recognizing thymidine-rich protospacer-adjacent motifs (PAM) without inducing DNA double-strand breaks (DSBs). Here we present an unbiased in vitro method for identifying genome-wide off-target sites of Cpf1 base editors via whole genome sequencing. First, we treat human genomic DNA with dLbCpf1-BE ribonucleoprotein (RNP) complexes, which convert C-to-U at on-target and off-target sites and, then, with a mixture of E. coli uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII, which removes uracil and produces single-strand breaks (SSBs) in vitro. Whole-genome sequencing of the resulting digested genome (Digenome-seq) reveals that, on average, dLbCpf1-BE induces 12 SSBs in vitro per crRNA in the human genome. Off-target sites with an editing frequency as low as 0.1% are successfully identified by this modified Digenome-seq method, demonstrating its high sensitivity. dLbCpf1-BEs and LbCpf1 nucleases often recognize different off-target sites, calling for independent analysis of each tool.

RNAs contain post-transcriptional modifications, which fulfill a variety of functions in translation, secondary structure stabilization and cellular stress survival. Here, 2-methylthiocytidine (ms 2 C) is identified in tRNA of E. coli and P. aeruginosa using NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) in combination with genetic screening experiments. ms 2 C is only found in 2-thiocytidine (s 2 C) containing tRNAs, namely tRNA Arg CCG , tRNA Arg ICG , tRNA Arg UCU and tRNA Ser GCU at low abundances. ms 2 C is not formed by commonly known tRNA methyltransferases. Instead, we observe its formation in vitro and in vivo during exposure to methylating agents. More than half of the s 2 C containing tRNA can be methylated to carry ms 2 C. With a pulse-chase NAIL-MS experiment, the repair mechanism by AlkB dependent sulfur demethylation is demonstrated in vivo. Overall, we describe ms 2 C as a bacterial tRNA modification and damage product. Its repair by AlkB and other pathways is demonstrated in vivo by our powerful NAIL-MS approach.

RNA-guided nucleases of the CRISPR/Cas type can be repurposed as programmable nucleotide deaminases to mediate targeted nucleotide substitutions. Such base editors have enormous potential in genome editing, gene therapy and precision breeding. However, current editors suffer from limited specificity in that they edit different and/or multiple bases within a larger sequence window. Using cytidine deaminase base editors that elicit C-to-T mutations, we show here that high editing precision can be achieved by engineering the connection between the deaminase domain and the Cas domain of the editor. By systematically testing different linker sequences and removing non-essential sequences from the deaminase, we obtain high-precision base editors with narrow activity windows that can selectively edit a single cytidine at a specific position with high accuracy and efficiency. These base editors will enable the use of genome editing in applications where single-nucleotide changes are required and off-target editing of adjacent nucleotides is not tolerable.

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N 1 of purines and the N 3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT 2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited.

Cytidine base editors (CBEs) and adenine base editors (ABEs), composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable the conversion of C·G to T·A or A·T to G·C base pair in organisms, respectively. Here, we show that BE3 and ABE7.10 systems can achieve a targeted mutation efficiency of 53-88% and 44-100%, respectively, in both blastocysts and Founder (F0) rabbits. Meanwhile, this strategy can be used to precisely mimic human pathologies by efficiently inducing nonsense or missense mutations as well as RNA mis-splicing in rabbit. In addition, the reduced frequencies of indels with higher product purity are also determined in rabbit blastocysts by BE4-Gam, which is an updated version of the BE3 system. Collectively, this work provides a simple and efficient method for targeted point mutations and generation of disease models in rabbit.

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A-ssDNA complex defines the 5'-3' directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics.