ABSTRACT
BACKGROUND: In recent years, covalent modifications on RNA nucleotides have emerged as pivotal moieties influencing the structure, function, and regulatory processes of RNA Polymerase II transcripts such as mRNAs and lncRNAs. However, our understanding of their biological roles and whether these roles are conserved across eukaryotes remains limited. RESULTS: In this study, we leveraged standard polyadenylation-enriched RNA-sequencing data to identify and characterize RNA modifications that introduce base-pairing errors into cDNA reads. Our investigation incorporated data from three Poaceae (Zea mays, Sorghum bicolor, and Setaria italica), as well as publicly available data from a range of stress and genetic contexts in Sorghum and Arabidopsis thaliana. We uncovered a strong enrichment of RNA covalent modifications (RCMs) deposited on a conserved core set of nuclear mRNAs involved in photosynthesis and translation across these species. However, the cohort of modified transcripts changed based on environmental context and developmental program, a pattern that was also conserved across flowering plants. We determined that RCMs can partly explain accession-level differences in drought tolerance in Sorghum, with stress-associated genes receiving a higher level of RCMs in a drought tolerant accession. To address function, we determined that RCMs are significantly enriched near exon junctions within coding regions, suggesting an association with splicing. Intriguingly, we found that these base-pair disrupting RCMs are associated with stable mRNAs, are highly correlated with protein abundance, and thus likely associated with facilitating translation. CONCLUSIONS: Our data point to a conserved role for RCMs in mRNA stability and translation across the flowering plant lineage.
Subject(s)
Arabidopsis , RNA Splicing , Arabidopsis/genetics , Arabidopsis/metabolism , Sorghum/genetics , RNA Stability/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , Zea mays/genetics , Setaria Plant/genetics , Setaria Plant/metabolism , Gene Expression Regulation, Plant , Magnoliopsida/genetics , RNA Processing, Post-TranscriptionalABSTRACT
Nanopore direct RNA sequencing (DRS) has emerged as a powerful tool for RNA modification identification. However, concurrently detecting multiple types of modifications in a single DRS sample remains a challenge. Here, we develop TandemMod, a transferable deep learning framework capable of detecting multiple types of RNA modifications in single DRS data. To train high-performance TandemMod models, we generate in vitro epitranscriptome datasets from cDNA libraries, containing thousands of transcripts labeled with various types of RNA modifications. We validate the performance of TandemMod on both in vitro transcripts and in vivo human cell lines, confirming its high accuracy for profiling m6A and m5C modification sites. Furthermore, we perform transfer learning for identifying other modifications such as m7G, Ψ, and inosine, significantly reducing training data size and running time without compromising performance. Finally, we apply TandemMod to identify 3 types of RNA modifications in rice grown in different environments, demonstrating its applicability across species and conditions. In summary, we provide a resource with ground-truth labels that can serve as benchmark datasets for nanopore-based modification identification methods, and TandemMod for identifying diverse RNA modifications using a single DRS sample.
Subject(s)
Oryza , Sequence Analysis, RNA , Humans , Sequence Analysis, RNA/methods , Oryza/genetics , RNA Processing, Post-Transcriptional , Nanopores , RNA/genetics , RNA/metabolism , Nanopore Sequencing/methods , Deep Learning , Inosine/metabolism , Inosine/genetics , Transcriptome/geneticsABSTRACT
Recently, post-transcriptional regulation of mRNA mediated by N6-methyladenosine (m6A) has been found to have profound effects on transcriptome regulation during plant responses to various abiotic stresses. However, whether this RNA modification can affect an oxidative stress response in plants has not been studied. To assess the role of m6A modifications during copper-induced oxidative stress responses, m6A-IP-seq was performed in Arabidopsis seedlings exposed to high levels of copper sulfate. This analysis revealed large-scale shifts in this modification on the transcripts most relevant for oxidative stress. This altered epitranscriptomic mark is known to influence transcript abundance and translation; therefore we scrutinized these possibilities. We found an increased abundance of copper-enriched m6A-containing transcripts. Similarly, we also found increased ribosome occupancy of copper-enriched m6A-containing transcripts, specifically those encoding proteins involved with stress responses relevant to oxidative stressors. Furthermore, the significance of the m6A epitranscriptome on plant oxidative stress tolerance was uncovered by assessing germination and seedling development of the mta (N6-methyladenosine RNA methyltransferase A mutant complemented with ABI3:MTA) mutant exposed to high copper treatment. These analyses suggested hypersensitivity of the mta mutant compared to the wild-type plants in response to copper-induced oxidative stress. Overall, our findings suggest an important role for m6A in the oxidative stress response of Arabidopsis.
ABSTRACT
Post-transcriptional modifications of RNA (PRMs) and post-translational modifications of proteins (PTMs) are important regulatory mechanisms in biological processes and have many commonalities. However, the integration of these research areas is lacking. A recent discussion identified the priorities, areas of emphasis, and necessary technologies to advance and integrate these areas of study.
Subject(s)
Protein Processing, Post-Translational , Proteins , RNAABSTRACT
Hematopoietic stem cell (HSC) transplantation using umbilical cord blood (UCB) is a potentially life-saving treatment for leukemia and bone marrow failure but is limited by the low number of HSCs in UCB. The loss of HSCs after ex vivo manipulation is also a major obstacle to gene editing for inherited blood disorders. HSCs require a low rate of translation to maintain their capacity for self-renewal, but hematopoietic cytokines used to expand HSCs stimulate protein synthesis and impair long-term self-renewal. We previously described cytokine-free conditions that maintain but do not expand human and mouse HSCs ex vivo. Here we performed a high throughput screen and identified translation inhibitors that allow ex vivo expansion of human HSCs while minimizing cytokine exposure. Transplantation assays show a ~5-fold expansion of long-term HSCs from UCB after one week of culture in low cytokine conditions. Single cell transcriptomic analysis demonstrates maintenance of HSCs expressing mediators of the unfolded protein stress response, further supporting the importance of regulated proteostasis in HSC maintenance and expansion. This expansion method maintains and expands human HSCs after CRISPR/Cas9 editing of the BCL11A+58 enhancer, overcoming a major obstacle to ex vivo gene correction for human hemoglobinopathies.
ABSTRACT
Various messenger RNA (mRNA) decay mechanisms play major roles in controlling mRNA quality and quantity in eukaryotic organisms under different conditions. While it is known that the recently discovered co-translational mRNA decay (CTRD), the mechanism that allows mRNAs to be degraded while still being actively translated, is prevalent in yeast, humans, and various angiosperms, the regulation of this decay mechanism is less well studied. Moreover, it is still unclear whether this decay mechanism plays any role in the regulation of specific physiological processes in eukaryotes. Here, by re-analyzing the publicly available polysome profiling or ribosome footprinting and degradome sequencing datasets, we discovered that highly translated mRNAs tend to have lower co-translational decay levels. Based on this finding, we then identified Pelota and Hbs1, the translation-related ribosome rescue factors, as suppressors of co-translational mRNA decay in Arabidopsis. Furthermore, we found that Pelota and Hbs1 null mutants have lower germination rates compared to the wild-type plants, implying that proper regulation of co-translational mRNA decay is essential for normal developmental processes. In total, our study provides further insights into the regulation of CTRD in Arabidopsis and demonstrates that this decay mechanism does play important roles in Arabidopsis physiological processes.
ABSTRACT
Posttranscriptional regulation of mRNA mediated by methylation at the N6 position of adenine (N6-methyladenosine [m6A]) has profound effects on transcriptome regulation in plants. Focused studies across eukaryotes offer glimpses into the processes governed by m6A throughout developmental and disease states. However, we lack an understanding of the dynamics and the regulatory potential of m6A during biotic stress in plants. Here, we provide a comprehensive look into the effects of m6A on both the short-term and long-term responses to pathogen signaling in Arabidopsis (Arabidopsis thaliana). We demonstrate that m6A-deficient plants are more resistant to bacterial and fungal pathogen infections and have altered immune responses. Furthermore, m6A deposition is specifically coordinated on transcripts involved in defense and immunity prior to and proceeding the pathogen signal flagellin. Consequently, the dynamic modulation of m6A on specific stress-responsive transcripts is correlated with changes in abundance and cleavage of these transcripts. Overall, we show that the m6A methylome is regulated prior to and during simulated and active pathogen stress and functions in the coordination and balancing of normal growth and pathogen responses.
Subject(s)
Arabidopsis , Gene Expression Regulation , Plant Immunity , Arabidopsis/genetics , Arabidopsis/metabolism , Methylation , Plant Immunity/genetics , Plants/genetics , RNA, Messenger/genetics , Transcriptome/geneticsABSTRACT
As part of the National Institutes of Health Human BioMolecular Atlas Program to develop a global platform to map the 37 trillion cells in the adult human body, we are generating a comprehensive molecular characterization of the female reproductive system. Data gathered from multiple single-cell/single-nucleus and spatial molecular assays will be used to build a 3D molecular atlas. Herein, we describe our multistep protocol, beginning with an optimized organ procurement workflow that maintains functional characteristics of the uterus, ovaries, and fallopian tubes by perfusing these organs with preservation solution. We have also developed a structured tissue sampling procedure that retains information on individual-level anatomic, physiologic, and individual diversity of the female reproductive system, toward full exploration of the function and structure of female reproductive cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation and preservation of the female reproductive system (ovaries, fallopian tubes, and uterus) prior to procurement Basic Protocol 2: Removal of the female reproductive system en bloc Basic Protocol 3: Postsurgical dissection of ovaries Basic Protocol 4: Postsurgical dissection of fallopian tubes Basic Protocol 5: Postsurgical dissection of cervix Basic Protocol 6: Postsurgical dissection of uterine body Support Protocol 1: OCT-embedded tissue protocol Support Protocol 2: Tissue fixation protocol Support Protocol 3: Snap-frozen tissue protocol Basic Protocol 7: Tissue slice preparation for Visium analysis Support Protocol 4: Hematoxylin and eosin staining for 10X Visium imaging Basic Protocol 8: Manual tissue dissociation for Multiome analysis Basic Protocol 9: Tissue dissociation for Multiome analysis using S2 Singulator.
Subject(s)
Genitalia, Female , Uterus , United States , Adult , Female , Humans , Cervix Uteri , Ovary , Fallopian TubesABSTRACT
Since the discovery of the first ribonucleic acid (RNA) modifications in transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), scientists have been on a quest to decipher the identities and functions of RNA modifications in biological systems. The last decade has seen monumental growth in the number of studies that have characterized and assessed the functionalities of RNA modifications in the field of plant biology. Owing to these studies, we now categorize RNA modifications based on their chemical nature and the RNA on which they are found, as well as the array of proteins that are involved in the processes that add, read, and remove them from an RNA molecule. Beyond their identity, another key piece of the puzzle is the functional significance of the various types of RNA modifications. Here, we shed light on recent studies that help establish our current understanding of the diversity of RNA modifications found in plant transcriptomes and the functions they play at both the molecular (e.g., RNA stability, translation, and transport) and organismal (e.g., stress response and development) levels. Finally, we consider the key research questions related to plant gene expression and biology in general and highlight developments in various technologies that are driving our insights forward in this research area.
Subject(s)
RNA, Transfer , RNA , RNA/genetics , RNA/chemistry , RNA/metabolism , RNA, Transfer/chemistry , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , RNA Processing, Post-Transcriptional , RNA, Plant/genetics , RNA, Plant/metabolismABSTRACT
Although covalent nucleotide modifications were first identified on the bases of transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), a number of these epitranscriptome marks have also been found to occur on the bases of messenger RNAs (mRNAs). These covalent mRNA features have been demonstrated to have various and significant effects on the processing (e.g. splicing, polyadenylation, etc.) and functionality (e.g. translation, transport, etc.) of these protein-encoding molecules. Here, we focus our attention on the current understanding of the collection of covalent nucleotide modifications known to occur on mRNAs in plants, how they are detected and studied, and the most outstanding future questions of each of these important epitranscriptomic regulatory signals.
Subject(s)
Nucleotides , Polyadenylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Nucleotides/genetics , RNA Processing, Post-Transcriptional/geneticsABSTRACT
RNA turnover is essential in maintaining messenger RNA (mRNA) homeostasis during various developmental stages and stress responses. Co-translational mRNA decay (CTRD), a process in which mRNAs are degraded while still associated with translating ribosomes, has recently been discovered to function in yeast and three angiosperm transcriptomes. However, it is still unclear how prevalent CTRD across the plant lineage. Moreover, the sequence features of co-translationally decayed mRNAs have not been well-studied. Here, utilizing a collection of publicly available degradome sequencing datasets for another seven angiosperm transcriptomes, we have confirmed that CTRD is functioning in at least 10 angiosperms and likely throughout the plant lineage. Additionally, we have identified sequence features shared by the co-translationally decayed mRNAs in these species, implying a possible conserved triggering mechanism for this pathway. Given that degradome sequencing datasets can also be used to identify actively translating upstream open reading frames (uORFs), which are quite understudied in plants, we have identified numerous actively translating uORFs in the same 10 angiosperms. These findings reveal that actively translating uORFs are prevalent in plant transcriptomes, some of which are conserved across this lineage. We have also observed conserved sequence features in the regions flanking these uORFs' stop codons that might contribute to ribosome stalling at these sequences. Finally, we discovered that there were very few overlaps between the mRNAs harboring actively translating uORFs and those sorted into the co-translational decay pathway in the majority of the studied angiosperms, suggesting that these two processes might be nearly mutually exclusive in those species. In total, our findings provide the identification of CTRD and actively translating uORFs across a broad collection of plants and provide novel insights into the important sequence features associated with these collections of mRNAs and regulatory elements, respectively.
ABSTRACT
Sex differences exist for many lung pathologies, including COVID-19 and pulmonary fibrosis, but the mechanistic basis for this remains unclear. Alveolar type 2 cells (AT2s), which play a key role in alveolar lung regeneration, express the X-linked Ace2 gene that has roles in lung repair and SARS-CoV-2 pathogenesis, suggesting that X chromosome inactivation (XCI) in AT2s might impact sex-biased lung pathology. Here we investigate XCI maintenance and sex-specific gene expression profiles using male and female AT2s. Remarkably, the inactive X chromosome (Xi) lacks robust canonical Xist RNA "clouds" and less enrichment of heterochromatic modifications in human and mouse AT2s. We demonstrate that about 68% of expressed X-linked genes in mouse AT2s, including Ace2, escape XCI. There are genome-wide expression differences between male and female AT2s, likely influencing both lung physiology and pathophysiologic responses. These studies support a renewed focus on AT2s as a potential contributor to sex-biased differences in lung disease.
Subject(s)
COVID-19 , RNA, Long Noncoding , Female , Male , Humans , Mice , Animals , X Chromosome Inactivation/genetics , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Genes, X-Linked , COVID-19/genetics , SARS-CoV-2/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , TranscriptomeABSTRACT
The secondary structure of an RNA molecule affects its regulation and function, but whether transcript isoforms adopt specific arrangements affecting their functionality is unclear. In a recent issue of Nature, Yang et al. reveal that the non-coding RNA COOLAIR can adopt isoform specific secondary structures that differentially regulate flowering.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Flowers/metabolism , Gene Expression Regulation, Plant , MADS Domain Proteins/genetics , RNA , RNA, UntranslatedABSTRACT
Our recent cognizance of diverse RNA classes undergoing dynamic covalent chemical modifications (or epitranscriptomic marks) in plants has provided fresh insight into the underlying molecular mechanisms of gene expression regulation. Comparatively, epigenetic marks comprising heritable modifications of DNA and histones have been extensively studied in plants and their impact on plant gene expression is quite established. Based on our growing knowledge of the plant epitranscriptome and epigenome, it is logical to explore how the two regulatory layers intermingle to intricately determine gene expression levels underlying key biological processes such as development and response to stress. Herein, we focus on the emerging evidence of crosstalk between the plant epitranscriptome with epigenetic regulation involving DNA modification, histone modification, and non-coding RNAs.
Subject(s)
Epigenesis, Genetic , Histones , DNA Methylation , Histones/metabolism , Plants/genetics , Plants/metabolism , RNA/metabolismABSTRACT
Long intergenic noncoding RNAs (lincRNAs) are a large yet enigmatic class of eukaryotic transcripts that can have critical biological functions. The wealth of RNA-sequencing (RNA-seq) data available for plants provides the opportunity to implement a harmonized identification and annotation effort for lincRNAs that enables cross-species functional and genomic comparisons as well as prioritization of functional candidates. In this study, we processed >24 Tera base pairs of RNA-seq data from >16,000 experiments to identify â¼130,000 lincRNAs in four Brassicaceae: Arabidopsis thaliana, Camelina sativa, Brassica rapa, and Eutrema salsugineum. We used nanopore RNA-seq, transcriptome-wide structural information, peptide data, and epigenomic data to characterize these lincRNAs and identify conserved motifs. We then used comparative genomic and transcriptomic approaches to highlight lincRNAs in our data set with sequence or transcriptional conservation. Finally, we used guilt-by-association analyses to assign putative functions to lincRNAs within our data set. We tested this approach on a subset of lincRNAs associated with germination and seed development, observing germination defects for Arabidopsis lines harboring T-DNA insertions at these loci. LincRNAs with Brassicaceae-conserved putative miRNA binding motifs, small open reading frames, or abiotic-stress modulated expression are a few of the annotations that will guide functional analyses into this cryptic portion of the transcriptome.
Subject(s)
Arabidopsis , Brassicaceae , RNA, Long Noncoding , Genomics , Sequence Analysis, RNA , TranscriptomeABSTRACT
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. However, we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed germination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and propose a novel mechanism of lincRNA action in which it may act as a scaffold with the RACK1A protein to regulate germination and development, possibly through a role in ribosome biogenesis.
ABSTRACT
Plants respond to low temperatures by altering the mRNA abundance of thousands of genes contributing to numerous physiological and metabolic processes that allow them to adapt. At the post-transcriptional level, these cold stress-responsive transcripts undergo alternative splicing, microRNA-mediated regulation and alternative polyadenylation, amongst others. Recently, m6 A, m5 C and other mRNA modifications that can affect the regulation and stability of RNA were discovered, thus revealing another layer of post-transcriptional regulation that plays an important role in modulating gene expression. The importance of m6 A in plant growth and development has been appreciated, although its significance under stress conditions is still underexplored. To assess the role of m6 A modifications during cold stress responses, methylated RNA immunoprecipitation sequencing was performed in Arabidopsis seedlings esposed to low temperature stress (4°C) for 24 h. This transcriptome-wide m6 A analysis revealed large-scale shifts in this modification in response to low temperature stress. Because m6 A is known to affect transcript stability/degradation and translation, we investigated these possibilities. Interestingly, we found that cold-enriched m6 A-containing transcripts demonstrated the largest increases in transcript abundance coupled with increased ribosome occupancy under cold stress. The significance of the m6 A epitranscriptome on plant cold tolerance was further assessed using the mta mutant in which the major m6 A methyltransferase gene was mutated. Compared to the wild-type, along with the differences in CBFs and COR gene expression levels, the mta mutant exhibited hypersensitivity to cold treatment as determined by primary root growth, biomass, and reactive oxygen species accumulation. Furthermore, and most importantly, both non-acclimated and cold-acclimated mta mutant demonstrated hypersensitivity to freezing tolerance. Taken together, these findings suggest a critical role for the epitranscriptome in cold tolerance of Arabidopsis.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cold Temperature , Freezing , Gene Expression Regulation, Plant/genetics , RNA, Messenger/geneticsABSTRACT
RNA is dynamically modified in cells by a plethora of chemical moieties to modulate molecular functions and processes. Over 140 modifications have been identified across species and RNA types, with the highest density and diversity of modifications found in tRNA (tRNA). The methods used to identify and quantify these modifications have developed over recent years and continue to advance, primarily in the fields of next-generation sequencing (NGS) and mass spectrometry (MS). Most current NGS methods are limited to antibody-recognized or chemically derivatized modifications and have limitations in identifying multiple modifications simultaneously. Mass spectrometry can overcome both of these issues, accurately identifying a large number of modifications in a single run. Here, we present advances in MS data acquisition for the purpose of RNA modification identification and quantitation. Using this approach, we identified multiple tRNA wobble position modifications in Arabidopsis thaliana that are upregulated in salt-stressed growth conditions and may stabilize translation of salt stress induced proteins. This work presents improvements in methods for studying RNA modifications and introduces a possible regulatory role of wobble position modifications in A. thaliana translation.
Subject(s)
RNA Processing, Post-Transcriptional , RNA, Transfer , Mass Spectrometry/methods , RNA, Transfer/chemistryABSTRACT
Cerebral malaria (CM), coma caused by Plasmodium falciparum-infected red blood cells (iRBCs), is the deadliest complication of malaria. The mechanisms that lead to CM development are incompletely understood. Here we report on the identification of activation and inhibition pathways leading to mouse CM with supporting evidence from the analysis of human specimens. We find that CM suppression can be induced by vascular injury when sporozoites exit the circulation to infect the liver and that CM suppression is mediated by the release of soluble factors into the circulation. Among these factors is insulin like growth factor 1 (IGF1), administration of which inhibits CM development in mice. IMPORTANCE Liver infection by Plasmodium sporozoites is a required step for infection of the organism. We found that alternate pathways of sporozoite liver infection differentially influence cerebral malaria (CM) development. CM is one of the primary causes of death following malaria infection. To date, CM research has focused on how CM phenotypes develop but no successful therapeutic treatment or prognostic biomarkers are available. Here we show for the first time that sporozoite liver invasion can trigger CM-inhibitory immune responses. Importantly, we identified a number of early-stage prognostic CM inhibitory biomarkers, many of which had never been associated with CM development. Serological markers identified using a mouse model are directly relevant to human CM.