Biology subjects

Lambowitz, A. M.

Publications and source records attributed to Lambowitz, A. M..

15 recordsLinked to original sources

bioRxiv2026

Real-Time Visualization of G2L4 Reverse Transcriptase in DNA Repair via Microhomology-Mediated End Joining

Double-strand break repair (DSBR) is essential for genome integrity, yet mechanistic details of error-prone microhomology-mediated end joining (MMEJ) remain unclear. A bacterial group II intron-like reverse transcriptase, G2L4 RT, has been implicated in MMEJ, but how it executes DSBR is unknown. Using high-speed atomic force microscopy (HS-AFM), we directly visualize G2L4 RT-mediated DSBR via MMEJ. We observe that G2L4 RT dimers exhibit RT3a plug protrusion upon DNA engagement and catalyze MMEJ by binding and stabilizing a 4-bp annealed microhomology and filling adjacent single-strand gaps with dNTPs. We also observe Mn2+-stimulated terminal transferase activity that generates elongated and branched DNA intermediates prior to ligation. With T4 DNA ligase, we visualize binding near nick sites and real-time nick sealing, which stabilizes the repaired products and suppresses off-pathway branching. These results reveal how G2L4 RT and ligase activities shape MMEJ intermediates and outcomes.

biophysics↗

bioRxiv2025

A bacterial PrimPol-reverse transcriptase hybrid protein has a proofreading exonuclease activity that can be transferred to other reverse transcriptases

Gene disruption analysis revealed that an E. coli PPRT protein, which has an N-terminal Primase-Polymerase (PrimPol) domain fused to a group II intron-like reverse transcriptase (RT) domain followed by a long C-terminal domain (CTD), contributes to a cellular oxidative DNA damage response in addition to its previously described function in phage defense. Biochemical analysis showed that the PrimPol domain has an error-prone DNA polymerase activity that enables read through of oxidation-induced DNA damage. Surprisingly, we found that the RT-like domain, in addition to synthesizing protein-primed DNAs for phage defense, has a 3 to 5 DNA exonuclease activity that functions in proofreading DNAs synthesized by the PrimPol domain. Extending these findings, we identified structural features that contribute to this proofreading activity, enabling us to associate it with both a group II intron-encoded and retroviral RT and suggesting general methods for incorporating proofreading activity into RTs.

molecular biology↗

bioRxiv2025

Structural basis for the evolution of a domesticated group II intron-like reverse transcriptase to function in host cell DNA repair

A previous study found that a domesticated bacterial group II intron-like reverse transcriptase (G2L4 RT) functions in double-strand break repair (DSBR) via microhomology-mediated end joining (MMEJ) and that a mobile group II intron-encoded RT has a basal DSBR activity that uses conserved structural features of non-LTR-retroelement RTs. Here, we determined G2L4 RT apoenzyme and snap-back DNA synthesis structures revealing novel structural adaptations that optimized its cellular function in DSBR. These included a unique RT3a structure that stabilizes the apoenzyme in an inactive conformation until encountering an appropriate substrate; a longer N-terminal extension/RT0-loop with conserved residues that together with a modified active site favors strand annealing; and a conserved dimer interface that localizes G2L4 RT homodimers to DSBR sites with both monomers positioned for MMEJ. Our findings reveal how a non-LTR-retroelement RT evolved a dedicated cellular function and suggest new ways of optimizing these RTs for genome engineering applications.

molecular biology↗

bioRxiv2024

Biochemical and structural insights into a 5' to 3' RNA ligase reveal a potential role in tRNA ligation

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here we identify C12orf29 as an atypical ATP-grasp enzyme that ligates RNA. Human C12orf29 and its homologs auto-adenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5-phosphate and a 3-hydroxyl. C12orf29 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Genetic depletion of c12orf29 in female mice alters global tRNA levels in brain. Furthermore, crystal structures of a C12orf29 homolog from Yasminevirus bound to nucleotides reveal a minimal and atypical RNA ligase fold with a unique active site architecture that participates in catalysis. Collectively, our results identify C12orf29 as an RNA ligase and suggest its involvement in tRNA biology. Significance StatementATP-grasp enzymes share an atypical ATP-binding fold and catalyze a diverse set of reactions involved in many essential cellular processes. We identified C12orf29 as an atypical ATP-grasp enzyme. Our biochemical and structural characterizations reveal this enzyme to be a 5 to 3 RNA ligase, structurally and functionally similar to the phage T4 RNA ligase. C12orf29 can ligate tRNAs in vitro and C12orf29 knockout female mice have altered tRNA levels in brain. We also report structures of C12orf29, which have revealed critical insights into the mode of ATP binding and catalysis. Our work suggests that C12orf29 may be a new player in the regulation of tRNAs.

biochemistry↗

bioRxiv2023

Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein

Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found Marinomonas mediterranea RT-Cas1/Cas2 adds short 3-DNA (dN) tails to RNA protospacers enabling their direct integration into CRISPR arrays as 3-dN-RNA/cDNA duplexes or 3-dN-RNAs at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers occurs by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of cDNAs from diverse RNAs without fixed sequence requirements. The integration of 3-dN-RNAs or single-stranded (ss) DNAs is favored over duplexes at higher protospacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage-defense nucleases. Our findings reveal novel mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications.

molecular biology↗

bioRxiv2022

Group II Intron-Like Reverse TranscriptasesFunction in Double-Strand Break Repair by Microhomology-Mediated End Joining

Bacteria encode free-standing reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. Here, we found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when transferred into Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase{theta} and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ) in vitro and in vivo. We also found that a group II intron RT can function similarly to G2L4 RT in DNA repair, with reciprocal substitutions at the active site showing an I residue favors MMEJ and an A residue favors primer extension in both enzymes. The DNA repair functions of these enzymes utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have an inherent ability to function in DSBR in a wide range of organisms.

molecular biology↗

bioRxiv2021

Arg-tRNA synthetase links inflammatory metabolism to RNA splicing via nuclear condensates

Cells respond to perturbations like inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Here, we found that depletion of arginine during inflammation decreased levels of a nuclear form of arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal protein and nuclear speckle component and that arginine depletion impacted both condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a novel mechanism whereby a tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.

molecular biology↗

bioRxiv2021

High-grade Ovarian Cancer Associated H/ACA snoRNAs Promote Cancer Cell Proliferation and Survival

Small nucleolar RNAs (snoRNAs) are an omnipresent class of non-coding RNAs involved in the modification and processing of ribosomal RNA (rRNA). As snoRNAs are required for ribosome production, the increase of which is a hallmark of cancer development, their expression would be expected to increase in proliferating cancer cells. However, the nature and extent of snoRNAs contribution to the biology of cancer cells remain largely unexplored. In this study, we examined the abundance patterns of snoRNA in high-grade serous ovarian carcinomas (HGSC) and serous borderline tumours (SBT) and identified a subset of snoRNA associated with increased invasiveness. This subgroup of snoRNA accurately discriminates between SBT and HGSC underlining their potential as biomarkers of tumour aggressiveness. Remarkably, knockdown of HGSC-associated H/ACA snoRNAs, but not their host genes, inhibits cell proliferation and induces apoptosis of model ovarian cancer cell lines. Wound healing and cell migration assays confirmed the requirement of these HGSC-associated snoRNA for cell invasion and increased tumour aggressiveness. Together our data indicate that H/ACA snoRNAs promote tumour aggressiveness through the induction of cell proliferation and resistance to apoptosis.

cancer biology↗

bioRxiv2021

Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase

Reverse transcriptases (RTs) can template switch during cDNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-seq, and may contribute to retroelement fitness by enabling continuous cDNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a template-switching complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template/DNA heteroduplex with a 1-nt 3-DNA overhang. The latter mimics a completed cDNA after non-templated addition (NTA) of a nucleotide complementary to the 3 nucleotide of the acceptor as required for efficient template switching. The structure showed that the 3 end of the acceptor RNA binds in a pocket formed by an N-terminal extension (NTE) present in non-long-terminal-repeat (LTR)-retroelement RTs and the RT fingertips loop, with the 3 nucleotide of the acceptor base paired to the 1-nt 3-DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structure-guided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine near the RT active site that mediates NTA. Mutation of the latter residue decreased multiple sequential template switches in RNA-seq. Our results provide new insights into the mechanisms of template switching and NTA by RTs, suggest how these reactions could be improved for RNA-seq, and reveal common structural features for template switching by non-LTR-retroelement RTs and viral RNA-dependent RNA polymerases.

biochemistry↗

bioRxiv2020

Human cells contain myriad excised linear intron RNAs with potential functions in gene regulation and utility as disease biomarkers

Previous Thermostable Group II Intron Reverse Transcriptase sequencing (TGIRT-seq) found that human plasma contains short ([≤]300 nt) structured full-length excised linear intron RNAs (FLEXIs) with potential utility as blood-based biomarkers. Here, TGIRT-seq identified >9,000 different FLEXIs in human cells, including relatively abundant FLEXIs with cell-type-specific expression patterns. Analysis of published CLIP-seq datasets identified 126 RNA-binding proteins (RBPs) that bind different FLEXIs, including 53 RBPs that bind [≥]30 FLEXIs. These RBPs included splicing factors, transcription factors, a chromatin remodeling protein, cellular growth regulators, and proteins with cytoplasmic functions. Analysis of published datasets identified subsets of these RBPs that bind at FLEXI splice sites and impact alternative splicing or FLEXI host gene mRNA levels. Hierarchical clustering identified 6 subsets of RBPs whose binding sites were co-enriched in different subsets of FLEXIs: AGO1-4 and DICER, including but not limited to annotated agotrons and mirtron pre-miRNAs; DKC1, NOLC1, SMNDC1, and AATF (Apoptosis Antagonizing Transcription Factor), including but not limited to FLEXIs encoding snoRNAs; two sets of alternative splicing factors; and two sets that included RBPs with cytoplasmic functions (e.g., LARP4, PABPC4, METAP2, and ZNF622) together with nuclear regulatory proteins. The subsets of host genes encoding FLEXIs that bind these RBPs were enriched with non-FLEXI other short and long introns that bind the same RBPs, and TGIRT-seq of nuclear and cytoplasmic fractions from 4 cell lines showed cytoplasmic enrichment of FLEXIs with binding sites for RBPs that function in the cytoplasm. Collectively, our findings suggest that different subsets of RBPs bind FLEXIs and other introns to coordinately regulate the expression of functionally related host genes and then co-localize with stably bound FLEXIs to different intracellular locations. The cell-type specific expression of relatively abundant FLEXIs suggests utility as cellular as well as blood-based RNA biomarkers.

genomics↗

bioRxiv2020

Identification of protein-protected mRNA fragments and structured excised intron RNAs in human plasma by TGIRT-seq peak calling

Human plasma contains >40,000 different coding and non-coding RNAs that are potential biomarkers for human diseases. Here, we used thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) combined with peak calling to simultaneously profile all RNA biotypes in apheresis-prepared human plasma pooled from healthy individuals. Extending previous TGIRT-seq analysis, we found that human plasma contains largely fragmented mRNAs from >19,000 protein-coding genes, abundant full-length, mature tRNAs and other structured small non-coding RNAs, and less abundant tRNA fragments and mature and pre-miRNAs. Many of the mRNA fragments identified by peak calling correspond to annotated protein-binding sites and/or have stable predicted secondary structures that could afford protection from plasma nucleases. Peak calling also identified novel repeat RNAs, miRNA-sized RNAs, and putatively structured intron RNAs of potential biological, evolutionary, and biomarker significance, including a family of full-length excised introns RNAs, subsets of which correspond to mirtron pre-miRNAs or agotrons.

genomics↗

bioRxiv2019

Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

The reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retro-elements differ from retroviral RTs in being able to template switch from the 5 end of one template to the 3 end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT; GsI-IIC RT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. The rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3-DNA overhang complementary to the 3 nucleotide of the acceptor RNA, suggesting a role for non-templated nucleotide addition of a complementary nucleotide to the 3 end of cDNAs synthesized from natural templates. Longer 3-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3 end of the acceptor template. The reliance on a single base pair with the 3 nucleotide of the acceptor together with discrimination against mismatches and the high processivity of the enzyme enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3 end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTR-retroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.

molecular biology↗

bioRxiv2019

Distinct mechanisms of microRNA sorting into cancer cell-derived extracellular vesicle subtypes

Extracellular vesicles (EVs) encompass a variety of vesicles secreted into the extracellular space. EVs have been implicated in promoting tumor metastasis, but the molecular composition of tumor-derived EV sub-types and the mechanisms by which molecules are sorted into EVs remain mostly unknown. We report the separation of two EV sub-populations from a metastatic breast cancer cell line, with biochemical features consistent with different sub-cellular origins. These EV sub-types use different mechanisms of miRNA sorting (selective and non-selective), suggesting that sorting occurs via fundamentally distinct processes, possibly dependent on EV origin. Using biochemical and genetic tools, we identified the Lupus La protein as mediating sorting of some selectively packaged miRNAs. We found that two motifs embedded in miR-122 are responsible for high-affinity binding to Lupus La and sorting into vesicles formed in a cell-free reaction. Thus, tumor cells can simultaneously deploy multiple EV species using distinct sorting mechanisms that may enable diverse functions in normal and cancer biology.

biochemistry↗

bioRxiv2018

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

Thermostable group II intron reverse transcriptases (TGIRTs) with high fidelity and processivity have been used for a variety of RNA sequencing (RNA-seq) applications, including comprehensive profiling of whole-cell, exosomal, and human plasma RNAs; quantitative tRNA-seq based on the ability of TGIRT enzymes to give full-length reads of tRNAs and other structured small ncRNAs; high-throughput mapping of post-transcriptional modifications; and RNA structure mapping. Here, we improved TGIRT-seq methods for comprehensive transcriptome profiling by (i) rationally designing RNA-seq adapters that minimize adapter dimer formation, and (ii) developing biochemical and computational methods that remediate 5- and 3-end biases. These improvements, some of which may be applicable to other RNA-seq methods, increase the efficiency of TGIRT-seq library construction and improve coverage of very small RNAs, such as miRNAs. Our findings provide insight into the biochemical basis of 5- and 3-end biases in RNA-seq and suggest general approaches for remediating biases and decreasing adapter dimer formation.

genomics↗

bioRxiv2018

Limitation of alignment-free tools in total RNA-seq quantification

BackgroundAlignment-free RNA quantification tools have significantly increased the speed of RNA-seq analysis. However, it is unclear whether these state-of-the-art RNA-seq analysis pipelines can quantify small RNAs as accurately as they do with long RNAs in the context of total RNA quantification.\n\nResultWe comprehensively tested and compared four RNA-seq pipelines on the accuracies of gene quantification and fold-change estimation on a novel total RNA benchmarking dataset, in which small non-coding RNAs are highly represented along with other long RNAs. The four RNA-seq pipelines were of two commonly-used alignment-free pipelines and two variants of alignment-based pipelines. We found that all pipelines showed high accuracies for quantifying the expressions of long and highly-abundant genes. However, alignment-free pipelines showed systematically poorer performances in quantifying lowly-abundant and small RNAs.\n\nConclusionWe have shown that alignment-free and traditional alignment-based quantification methods performed similarly for common gene targets, such as protein-coding genes. However, we identified a potential pitfall in analyzing and quantifying lowly-expressed genes and small RNAs with alignment-free pipelines, especially when these small RNAs contain mutations.

bioinformatics↗