Biology subjects

Garcia, B. A.

Publications and source records attributed to Garcia, B. A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation

Arginylation installed by arginyltransferase 1 (ATE1) features an addition of arginine (Arg) to the reactive amino acids (e.g., Glu and Asp) at the protein N-terminus or side chain. Systemic removal of arginylation after ATE1 knockout (KO) in mouse models resulted in heart defects leading to embryonic lethality. The biological importance of arginylation has motivated the discovery of arginylation sites on proteins using bottom-up approaches. While bottom-up proteomics is powerful in localizing peptide arginylation, it lacks the ability to quantify proteoforms at the protein level. Here we developed a top-down proteomics workflow for characterizing and quantifying calreticulin (CALR) arginylation. To generate fully arginylated CALR (R-CALR), we have inserted an R residue after the signaling peptide (AA1-17). Upon overexpression in ATE1 KO cells, CALR and R-CALR were purified by affinity purification and analyzed by LCMS in positive mode. Both proteoforms showed charge states ranging from 27-68 with charge 58 as the most intense charge state. Their MS2 spectra from electron-activated dissociation (EAD) showed preferential fragmentation at the protein N-terminals which yielded sufficient c ions facilitating precise localization of the arginylation sites. The calcium-binding domain (CBD) gave minimum characteristic ions possibly due to the abundant presence of >100 D and E residues. Ultraviolet photodissociation (UVPD) compared with EAD and ETD significantly improved the sequence coverage of CBD. This method can identify and quantify CALR arginylation at absence, endogenous (low), and high levels. To our knowledge, our work is the first application of top-down proteomics in characterizing post-translational arginylation in vitro and in vivo.

biochemistry↗

bioRxiv2024

An Unbiased Proteomic Platform for Activity-based Arginylation Profiling

Protein arginylation is an essential posttranslational modification (PTM) catalyzed by arginyl-tRNA-protein transferase 1 (ATE1) in mammalian systems. Arginylation features a post-translational conjugation of an arginyl to a protein, making it extremely challenging to differentiate from translational arginine residues with the same mass in a protein sequence. Here we present a general ATE1-based arginylation profiling platform for the unbiased discovery of arginylation substrates and their precise modification sites. This method integrates isotopic arginine labeling into an ATE1 assay utilizing biological lysates (ex vivo) rather than live cells, thus eliminating translational bias derived from the ribosomal activity and enabling bona fide arginylation identification using isotopic features. The method has been successfully applied to an array of peptide, protein, cell, patient, and animal tissue samples using 20 {micro}g sample input, with 235 unique arginylation sites revealed from human proteomes. Representative sites were validated and followed up for their biological functions. The developed platform is globally applicable to the aforementioned sample types and therefore paves the way for functional studies of this difficult-to-characterize protein modification.

biochemistry↗

bioRxiv2024

Chromatin profiling identifies putative dual roles for H3K27me3 in regulating transposons and cell type specific genes in choanoflagellates

Gene expression is tightly controlled during animal development to allow the formation of specialized cell types. Our understanding of how animals evolved this exquisite regulatory control remains elusive, but evidence suggests that changes in chromatin-based mechanisms may have contributed. To investigate this possibility, here we examine chromatin-based gene regulatory features in the closest relatives of animals, choanoflagellates. Using Salpingoeca rosetta as a model system, we examined chromatin accessibility and histone modifications at the genome scale and compared these features to gene expression. We first observed that accessible regions of chromatin are primarily associated with gene promoters and found no evidence of distal gene regulatory elements resembling the enhancers that animals deploy to regulate developmental gene expression. Remarkably, a histone modification deposited by polycomb repressive complex 2, histone H3 lysine 27 trimethylation (H3K27me3), appeared to function similarly in S. rosetta to its role in animals, because this modification decorated genes with cell type-specific expression. Additionally, H3K27me3 marked transposons, retaining what appears to be an ancestral role in regulating these elements. We further uncovered a putative new bivalent chromatin state at cell type-specific genes that consists of H3K27me3 and histone H3 lysine 4 mono-methylation (H3K4me1). Together, our discoveries support the scenario that gene-associated histone modification states that underpin development emerged before the evolution of animal multicellularity.

evolutionary biology↗

bioRxiv2024

Lineage-specific proteome remodeling of diverse lung cancer cells by targeted epigenetic inhibitors

Epigenetic inhibitors exhibit powerful antiproliferative and anticancer activities. However, cellular responses to small-molecule epigenetic inhibition are heterogeneous and dependent on factors such as the genetic background and metabolic state of cells, as well as on-/off-target engagement of individual small-molecule compounds. The molecular study of the extent of this heterogeneity often measures changes in a single cell line. To more comprehensively profile the effects of small-molecule perturbations and their influence on heterogeneous cellular responses, we present a molecular resource based on the quantification of chromatin, proteome, and transcriptome remodeling due to histone deacetylase inhibitors (HDACi) in non-isogenic cell lines. Through quantitative molecular profiling of 10,621 proteins, these data reveal coordinated molecular remodeling of HDACi treated cancer cells. HDACi-regulated proteins differ greatly across cell lines with consistent (JUN, MAP2K3, CDKN1A) and divergent (CCND3, ASF1B, BRD7) cell-state effectors. Together these data provide valuable insight into cell-type driven and heterogeneous responses that must be taken into consideration when monitoring molecular perturbations in culture models. We have also built a web interface for the extensive amount of data to allow users to explore the data as a resource for understanding chemical perturbation of diverse cell types.

cancer biology↗

bioRxiv2024

ACSS2 contributes to transcriptional regulation in Cajal-Retzius cells in a mouse model of Alzheimer's disease

Dysregulation of histone acetylation in the brain has emerged as a major contributor to human Alzheimers disease (AD). The mechanisms by which these protective or risk-conferring epigenetic marks are established and maintained are under intense investigation. ACSS2 (Acetyl-CoA Synthetase 2) is a key metabolic enzyme that is chromatin-associated in neurons. ACSS2 is recruited to specific promoters and generates a local pool of acetyl-CoA from acetate, thereby fueling histone acetylation and driving the expression of neuronal genes that regulate learning and memory. Here, we examine the contribution of ACSS2-mediated histone acetylation to AD-related molecular and behavioral outcomes. Using a mouse model of human pathological AD-Tau injection, we show that loss of ACSS2 exacerbates Tau-related memory impairments, while dietary supplementation of acetate rescues learning in an ACSS2-dependent manner. Combining state-of-the-art proteomic and genomic approaches, we demonstrate that this effect is accompanied by ACSS2-dependent incorporation of acetate into hippocampal histone acetylation, which facilitates gene expression programs related to learning. Further, we identify Cajal-Retzius neurons as a critical hippocampal neuronal population affected, exhibiting the largest epigenetic and transcriptional dysregulation. Overall, these results reveal ACSS2 as a key neuroprotective metabolic enzyme, dysregulation of which might play an important role in the etiology of human AD, and guide the development of future therapies for AD and related dementia.

neuroscience↗

bioRxiv2024

Early-life stress alters chromatin modifications in VTA to prime stress sensitivity

Early-life stress increases sensitivity to subsequent stress, which has been observed at behavioral, neural activity, and gene expression levels. However, the molecular mechanisms underlying such long-lasting sensitivity are poorly understood. We tested the hypothesis that persistent changes in transcription and transcriptional potential were maintained at the level of the epigenome, through changes in chromatin. We used a combination of bottom-up mass spectrometry, viral-mediated epigenome-editing, RNA-sequencing, patch clamp electrophysiology of dopamine neurons, and behavioral quantification in a mouse model of early-life stress, focusing on the ventral tegmental area (VTA), a dopaminergic brain region critically implicated in motivation, reward learning, stress response, and mood and drug disorders. We found that early-life stress alters histone dynamics in VTA, including enrichment of histone-3 lysine-4 monomethylation -- associated with open chromatin and primed or active enhancers -- and the H3K4 monomethylase Setd7. Mimicking early-life stress through postnatal overexpression of Setd7 and enrichment of H3K4me1 in VTA sensitizes transcriptional, physiological, and behavioral response to adult stress. These findings link early-life stress experience to long-term stress hypersensitivity within the brains dopaminergic circuitry, providing a mechanism by which early-life stress increases risk for mood and anxiety disorders later in life.

neuroscience↗

bioRxiv2024

Development and application of GlycanDIA workflow for glycomic analysis

Glycans modify protein, lipid, and even RNA molecules to form the regulatory outer coat on cells called the glycocalyx. The changes in glycosylation have been linked to the initiation and progression of many diseases. Thus, while the significance of glycosylation is well established, a lack of accessible methods to characterize glycans has hindered the ability to understand their biological functions. Mass spectrometry (MS)-based methods have generally been at the core of most glycan profiling efforts; however, modern data-independent acquisition (DIA), which could increase sensitivity and simplify workflows, has not been benchmarked for analyzing glycans. Herein, we developed a DIA-based glycomic workflow, termed GlycanDIA, to identify and quantify glycans with high sensitivity and accuracy. The GlycanDIA workflow combined higher energy collisional dissociation (HCD)-MS/MS and staggered windows for glycomic analysis, which facilitates the sensitivity in identification and the accuracy in quantification compared to conventional data-dependent acquisition (DDA)-based glycomics. To facilitate its use, we also developed a generic search engine, GlycanDIA Finder, incorporating an iterative decoy searching for confident glycan identification and quantification from DIA data. The results showed that GlycanDIA can distinguish glycan composition and isomers from N-glycans, O-glycans, and human milk oligosaccharides (HMOs), while it also reveals information on low-abundant modified glycans. With the improved sensitivity, we performed experiments to profile N-glycans from RNA samples, which have been underrepresented due to their low abundance. Using this integrative workflow to unravel the N-glycan profile in cellular and tissue glycoRNA samples, we found that RNA-glycans have specific forms as compared to protein-glycans and are also tissue-specific differences, suggesting distinct functions in biological processes. Overall, GlycanDIA can provide comprehensive information for glycan identification and quantification, enabling researchers to obtain in-depth and refined details on the biological roles of glycosylation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/584702v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@18c0400org.highwire.dtl.DTLVardef@15ada57org.highwire.dtl.DTLVardef@1b4b28forg.highwire.dtl.DTLVardef@36d150_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma

Cancer immunotherapies have produced remarkable results in B-cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor specimens along with normal tissues to identify biologically relevant cell surface proteins that can serve as immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer of the developing nervous system. We apply this approach to human-derived cell lines (N=9) and cell/patient-derived xenograft (N=12) models of neuroblastoma. Plasma membrane-enriched mass spectrometry identified 1,461 cell surface proteins in cell lines and 1,401 in xenograft models, respectively. Additional proteogenomic analyses revealed 60 high-confidence candidate immunotherapeutic targets and we prioritized Delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlated with the presence of a super-enhancer spanning the DLK1 locus. Robust cell surface expression of DLK1 was validated by immunofluorescence, flow cytometry, and immunohistochemistry. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells resulted in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), showed potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Moreover, DLK1 is highly expressed in several adult cancer types, including adrenocortical carcinoma (ACC), pheochromocytoma/paraganglioma (PCPG), hepatoblastoma, and small cell lung cancer (SCLC), suggesting potential clinical benefit beyond neuroblastoma. Taken together, our study demonstrates the utility of comprehensive cancer surfaceome characterization and credentials DLK1 as an immunotherapeutic target. HighlightsO_LIPlasma membrane enriched proteomics defines surfaceome of neuroblastoma C_LIO_LIMulti-omic data integration prioritizes DLK1 as a candidate immunotherapeutic target in neuroblastoma and other cancers C_LIO_LIDLK1 expression is driven by a super-enhancer C_LIO_LIDLK1 silencing in neuroblastoma cells results in cellular differentiation C_LIO_LIADCT-701, a DLK1-targeting antibody-drug conjugate, shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma preclinical models C_LI

cancer biology↗

bioRxiv2023

H3K27me3 spreading organizes canonical PRC1 chromatin architecture to regulate developmental programs

Polycomb Repressive Complex 2 (PRC2)-mediated histone H3K27 tri-methylation (H3K27me3) recruits canonical PRC1 (cPRC1) to maintain heterochromatin. In early development, polycomb-regulated genes are connected through long-range 3D interactions which resolve upon differentiation. Here, we report that polycomb looping is controlled by H3K27me3 spreading and regulates target gene silencing and cell fate specification. Using glioma-derived H3 Lys-27-Met (H3K27M) mutations as tools to restrict H3K27me3 deposition, we show that H3K27me3 confinement concentrates the chromatin pool of cPRC1, resulting in heightened 3D interactions mirroring chromatin architecture of pluripotency, and stringent gene repression that maintains cells in progenitor states to facilitate tumor development. Conversely, H3K27me3 spread in pluripotent stem cells, following neural differentiation or loss of the H3K36 methyltransferase NSD1, dilutes cPRC1 concentration and dissolves polycomb loops. These results identify the regulatory principles and disease implications of polycomb looping and nominate histone modification-guided distribution of reader complexes as an important mechanism for nuclear compartment organization. Highlights{square} The confinement of H3K27me3 at PRC2 nucleation sites without its spreading correlates with increased 3D chromatin interactions. {square}The H3K27M oncohistone concentrates canonical PRC1 that anchors chromatin loop interactions in gliomas, silencing developmental programs. {square}Stem and progenitor cells require factors promoting H3K27me3 confinement, including H3K36me2, to maintain cPRC1 loop architecture. {square}The cPRC1-H3K27me3 interaction is a targetable driver of aberrant self-renewal in tumor cells.

molecular biology↗

bioRxiv2023

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA

We recently identified glycoRNA--a previously undescribed glycoconjugate--which consists of RNAs modified with secretory N-glycans and presented on the cell surface. While previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging periodate oxidation and aldehyde ligation (rPAL) and Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. The sensitivity and robustness of rPAL provided the first evidence of a direct glycan-RNA linkage, and its flexibility will enable further characterization of glycoRNA biology.

molecular biology↗

bioRxiv2023

Altered chromatin occupancy of patient-associated H4 mutants misregulate neuronal differentiation

Chromatin is a crucial regulator of gene expression and tightly controls development across species. Mutations in only one copy of multiple histone genes were identified in children with developmental disorders characterized by microcephaly, but their mechanistic roles in development remain unclear. Here we focus on dominant mutations affecting histone H4 lysine 91. These H4K91 mutants form aberrant nuclear puncta at specific heterochromatin regions. Mechanistically, H4K91 mutants demonstrate enhanced binding to the histone variant H3.3, and ablation of H3.3 or the H3.3-specific chaperone DAXX diminishes the mutant localization to chromatin. Our functional studies demonstrate that H4K91 mutant expression increases chromatin accessibility, alters developmental gene expression through accelerating pro-neural differentiation, and causes reduced mouse brain size in vivo, reminiscent of the microcephaly phenotypes of patients. Together, our studies unveil a distinct molecular pathogenic mechanism from other known histone mutants, where H4K91 mutants misregulate cell fate during development through abnormal genomic localization.

molecular biology↗

bioRxiv2023

In utero pulse injection of isotopic amino acids quantifies protein turnover rates during murine fetal development

Protein translational control is highly regulated step in the gene expression program during mammalian development that is critical for ensuring that the fetus develops correctly and that all of the necessary organs and tissues are formed and functional. Defects in protein expression during fetal development can lead to severe developmental abnormalities or premature death. Currently, quantitative techniques to monitor protein synthesis rates in a developing fetus (in utero) are limited. Here, we developed a novel in utero stable isotope labeling approach to quantify tissue-specific protein dynamics of the nascent proteome during mouse fetal development. Fetuses of pregnant C57BL/6J mice were injected with isotopically labeled lysine (Lys8) and arginine (Arg10) via the vitelline vein at various gestational days. After treatment, fetal organs/tissues including brain, liver, lung, and heart were harvested for sample preparation and proteomic analysis. We show that the mean incorporation rate for injected amino acids into all organs was 17.50 {+/-} 0.6%. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates (kobs) were calculated between 3.81E-5 and 0.424 hour-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver versus brain), however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates which correlated with known physiological changes during mouse development.

systems biology↗

bioRxiv2023

Comparative analysis of drug-like EP300/CREBBP acetyltransferase inhibitors

The human acetyltransferase paralogs EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potency of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

biochemistry↗

bioRxiv2022

Expanding the epitranscriptomic RNA sequencing and modification mapping mass spectrometry toolbox with field asymmetric waveform ion mobility and electrochemical elution liquid chromatography

Post-transcriptional modifications of RNA strongly influence RNA structure and function. Recent advances in RNA sequencing and mass spectrometry (MS) methods have identified over 140 of these modifications on a wide variety of RNA species. Most next-generation sequencing approaches can only map one RNA modification at a time, and while MS can assign multiple modifications simultaneously in an unbiased manner, MS cannot accurately catalog and assign RNA modifications in complex biological samples due to limitations in fragment length and coverage depth. Thus, a facile method to identify novel RNA modifications while simultaneously locating them in the context of their RNA sequences is still lacking. We combined two orthogonal modes of RNA ion separation before mass-spectrometry identification: high-field asymmetric ion mobility separation (FAIMS) and electrochemically modulated liquid chromatography (EMLC). FAIMS RNA-MS increases both coverage and throughput, while the EMLC LC-MS orthogonally separates RNA of different length and charge. The combination of the two methods offers a broadly applicable platform to improve length and depth of MS-based RNA sequencing while providing contextual access to the analysis of RNA modifications.

biochemistry↗

bioRxiv2022

Post-translational modification by the Pgf glycosylation machinery modulates Streptococcus mutans physiology and virulence

Streptococcus mutans is a keystone pathogen of dental caries, and the ability to form biofilms is essential for its pathogenicity. We identified a glycosylation machinery (Pgf) in S. mutans that post-translationally modifies two surface-associated adhesins, Cnm and WapA. The four pgf genes (pgfS, pgfM1, pgfE, and pgfM2) are part of S. mutans core genome and we hypothesized that the scope of Pgf goes beyond Cnm and WapA. By inactivating each pgf gene individually or creating a quadruple pgf mutant in S. mutans OMZ175, we showed that the Pgf machinery is important for biofilm formation. Compared to OMZ175, differences in surface charge, membrane stability, and genetic competence were also observed for most mutants. Importantly, in silico analyses and tunicamycin MIC assays suggest a functional redundancy between the Pgf machinery and the rhamnose-glucose polysaccharide synthesis pathway. Using a rat oral colonization model, we showed a 10-fold reduction in recovered CFUs for the pgf quadruple mutant compared to OMZ175. Finally, using Cnm as a model, we showed by glycoproteomics analyses that Cnm is heavily modified with N-acetyl hexosamine in OMZ175 whereas phosphorylations were observed for the pgfS mutant. Our findings indicate that the Pgf machinery participates in important aspects of S. mutans pathobiology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/511621v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@146f37eorg.highwire.dtl.DTLVardef@11c0e45org.highwire.dtl.DTLVardef@ece1eaorg.highwire.dtl.DTLVardef@1e3f221_HPS_FORMAT_FIGEXP M_FIG C_FIG Abbreviated summaryIn this study, we demonstrate that the Pgf glycosylation machinery of Streptococcus mutans, a keystone pathogen of dental caries, regulates several aspects of bacterial pathophysiology that ultimately contribute to S. mutans fitness in oral colonization experiments. Using the heavily glycosylated Cnm adhesin as a model, we found that inactivation of the glycosyltransferase PgfS results in loss of Cnm glycosylation, but instead, Cnm became heavily phosphorylated, suggesting a crosstalk/competition between these two post-translational modification mechanisms.

microbiology↗

bioRxiv2022

Targeting acetyl-CoA metabolism attenuates the formation of fear memories through reduced activity-dependent histone acetylation

Histone acetylation is a key component in the consolidation of long-term fear memories. Epigenetic enzymes involved in histone acetylation, including histone acetyltransferases and deacetylases, have been put forward as potential pharmacological targets in the treatment of pathological fear memories, such as those that underlie post-traumatic stress disorder (PTSD). However, these enzymes typically play a ubiquitous role in gene regulation, which precludes the clinical use of systemic manipulations. Recently, we have found that a nuclear-localized metabolic enzyme, Acetyl-coA synthetase 2 (Acss2), modulates histone acetylation during learning and memory. Loss of Acss2 is well-tolerated in mice, with no impact on general health or baseline behavior. Here, we show that an Acss2 null mouse model shows reduced acquisition of long-term fear memories in assays of contextual and cued fear conditioning. We find that loss of Acss2 leads to consolidation-specific reductions in both histone acetylation and the expression of critical learning and memory-related genes in the dorsal hippocampus. Further, we show that systemic administration of blood-brain-barrier (BBB)-permeable Acss2 inhibitors during the consolidation window reduces fear memory formation in mice and rats, and also reduces anxiety in a predator-scent-stress (PSS) paradigm. Our findings suggest that Acss2 plays a critical role in the formation of fear memories, and represents a potential pharmacological target in the treatment of PTSD.

neuroscience↗

bioRxiv2022

Permethylation of ribonucleosides provides enhanced mass spectrometry quantification of post-transcriptional modifications

Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, degradation, as well as human disease states such as cancer. However, the analysis of ribonucleoside modifications is impeded due to the hydrophilicity of the ribonucleoside molecules. In this research, we used solid-phase permethylation to derivatize the ribonucleosides, and the permethylated ribonucleosides, which were then quantitively analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. The solid-phase permethylation efficiently derivatized the ribonucleosides, and more than 60 RNA modifications were simultaneously monitored using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. Because of the increased hydrophobicity of permethylated ribonucleosides, this method enhanced retention, separation, and ionization efficiency, resulting in improved detection and quantification when compared to existing analytical strategies of RNA modifications. We applied this new approach to measure the extent of cytosine methylation and hydroxymethylation in RNA obtained from mouse embryonic stem cells with genetic deficiencies in ten-eleven translocation (TET) enzymes. The results matched previously performed analyses and highlighted the sensitivity, efficacy, and robustness of the new method. The advantage of this method enables comprehensive analysis of RNA modifications in biological samples. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/477959v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@a16649org.highwire.dtl.DTLVardef@424b26org.highwire.dtl.DTLVardef@53d701org.highwire.dtl.DTLVardef@48a9cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

A conserved long intergenic non-coding RNA containing snoRNA sequences, lncCOBRA1, affects Arabidopsis germination and development

Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein-coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system, though we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed gemination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and examine the protein-protein interaction network of lncCOBRA1-interacting proteins.

plant biology↗