RNA-DNA DIFFERENCES IN NASCENT RNA
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE39878 | GEO | 2014/02/21
SECONDARY ACCESSION(S): PRJNA171944
REPOSITORIES: GEO
Similar Datasets
Project description:RNA sequences are expected to be identical to their corresponding DNA sequences. Advances in technologies have enabled deep sequencing of nucleic acids that uncovered exceptions to the one-to-one relationship between DNA and RNA sequences. Previously in human cells, post-transcriptional RNA editing was the only known mechanism that changes RNA sequences from the underlying DNA sequences. Here, we sequenced nascent RNA and found all 12 types of RNA-DNA differences. Using various experimental analyses, we validated this finding. Our results showed that sequences of nascent RNAs within 40 nucleotides of the exit channel of RNA polymerase II already differ from the corresponding DNA sequences. These RNA-DNA differences are mediated by RNA processing steps closely coupled with transcription and not by known deaminase-mediated RNA editing mechanisms nor during NTP incorporation by Pol II. This finding identifies sequence substitution as part of co-transcriptional RNA processing. We sequenced nascent RNA using global run-on sequencing, GRO-seq from human B-cells from two individuals and a variant of the GRO-seq procedure, known as precision run-on sequencing, PRO-seq. The RNAs are prepared after a short run-on assay performed with isolated nuclei in the presence of Br-UTP. The isolated RNAs are base hydrolyzed to ~100 nucleotides and affinity purified with anti-BrU beads three times at each successive step of preparing the RNAs for orientation-specific sequencing using Illumina technology. The 5M-bM-^@M-^Y ~half of each sequence represents nascent RNA made in the cell and the 3M-bM-^@M-^Y ~half represents sequences made in vitro during the run-on reaction. The precision variation, PRO-seq, incorporates one or at most a few biotin-labeled nucleoside triphosphates during the run-on, and sequencing from the 3M-bM-^@M-^Y end of this affinity purified, nascent RNA maps the cellular location of engaged polymerases with near single nucleotide precision. We obtained ~ 100 million 100-nucleotide uniquely mapped GRO-seq reads from B-cells of two individuals. For one subject, we also carried out pGRO-seq and obtained 60 million uniquely mapped reads. In addition, we sequenced ~135 million uniquely mapped RNA-seq reads, and the corresponding DNA of the two individuals to 30X and 60X coverage. Additionally, we isolated and sequenced nascent RNA with an alternate method described by Wuarin and Schibler (1994) in order to compare chromatin-bound RNA to the very nascent RNA from PRO-seq. We obtained ~190 million uniquely mapped reads from chormatin-bound RNA-seq.
2014-02-21 | E-GEOD-39878 | biostudies-arrayexpress
Project description:In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that > 90% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3’ acceptor sequences that can alter splicing of the associated exons. Interestingly, these exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites.
2018-04-25 | GSE105773 | GEO
Project description:The RNA editing enzyme ADAR chemically modifies adenosine (A) to inosine (I), which is interpreted by the ribosome as a guanosine. Here we assess cotranscriptional A-to-I editing in Drosophila, by isolating nascent RNA from adult fly heads and subjecting samples to high-throughput sequencing. There are a large number of edited sites within nascent exons. Nascent RNA from an ADAR null mutant strain was also sequenced, indicating that almost all A-to-I events require ADAR. Moreover, mRNA editing levels correlate with editing levels within the cognate nascent RNA sequence, indicating that the extent of editing is set cotranscriptionally. Surprisingly, the nascent data also identify an excess of intronic over exonic editing sites. These intronic sites occur preferentially within introns that are poorly spliced cotranscriptionally, suggesting a link between editing and splicing. We conclude that ADAR-mediated editing is more widespread than previously indicated and largely occurs cotranscriptionally. GSM914095: Fly genomic DNA sequencing. Sequenced on the Illumina GA II. GSM914102-GSM914113: Fly head nascent RNA profiles over 6 time points of a 12hr light:dark cycle in duplicate; sequenced on the Illumina GA II. GSM914114-GSM914119: Fly head nascent RNA profiles of yw, FM7, ADAR0 males in duplicate; sequenced on the HiSeq2000. GSM915213-GSM915214: Fly head mRNA profiles over 2 time points of a 12hr light:dark cycle; sequenced on the Illumina GA II. GSM915215-GSM915220: Fly head mRNA profiles over 6 time points of a 12hr light:dark cycle; paired-end sequenced on the Illumina GA II. GSM915221-GSM91526: Fly head mRNA profiles over 6 time points of a 12hr light:dark cycle; sequenced on the Illumina GA II.
2012-05-31 | E-GEOD-37232 | biostudies-arrayexpress
Project description:Most RNA processing occurs co-transcriptionally. We interrogated nascent pol II transcripts by chemical and enzymatic probing, and determined how the “nascent RNA structureome” relates to splicing, A-I editing and transcription speed. RNA folding within introns and steep structural transitions at splice sites are associated with efficient co-transcriptional splicing. A slow pol II mutant elicits extensive remodeling into more folded conformations with increased A-I editing. Introns that become more structured at their 3’ splice sites get co-transcriptionally excised more efficiently. Slow pol II altered folding of intronic Alu elements where cryptic splicing and intron retention are stimulated, an outcome mimicked by UV which decelerates transcription. Slow transcription also remodeled RNA folding around alternative exons in distinct ways that predict whether skipping or inclusion is favored, even though it occurs post-transcriptionally. Hence co-transcriptional RNA folding modulates post-transcriptional alternative splicing. In summary the plasticity of nascent transcripts has widespread effects on RNA processing.
2021-02-09 | GSE149018 | GEO
Project description:We provide sequences of maize chlroplast RNAs associated with non-PPR editing factors through immunoprecipitation. The accumulated sequences indicate expansive role in RNA processing and potential associations of OZ1-ORRM1 complexes with the ribosome and less efficiently translated mRNAs.
2021-12-31 | GSE151702 | GEO
Project description:Background: Adenosine deaminases that act on RNA (ADARs) bind to double-stranded and structured RNAs and deaminate adenosines to inosines. This A to I editing is widespread and required for normal life and development. Besides mRNAs and repetitive elements, ADARs can target miRNA precursors. Editing of miRNA precursors can affect processing efficiency and alter target specificity. Interestingly, ADARs can also influence miRNA abundance independent of RNA-editing. In mouse embryos where editing levels are low, ADAR2 was found to be the major ADAR protein that affects miRNA abundance. Here we extend our analysis to adult mouse brains where high editing levels are observed. Results: Using Illumina deep sequencing we compare the abundances of mature miRNAs and editing events within them, between wild-type and ADAR2 knockout mice in the adult mouse brain. Reproducible changes in abundance of specific miRNAs are observed in ADAR2 deficient mice. Most of these quantitative changes seem unrelated to A to I editing events. However, many A to G transitions in cDNAs prepared from mature miRNA sequences, reflecting A to I editing events in the RNA, are observed with frequencies reaching up to 80%. About half of these editing events are primarily caused by ADAR2 while a few miRNAs show increased editing in the absence of ADAR2, suggesting preferential editing by ADAR1. Moreover, novel, previously unknown editing events were identified in several miRNAs. In general 64% of all editing events are located within the seed region of mature miRNAs. In one of these cases retargeting of the edited miRNA could be verified in reporter assays. Also, altered processing efficiency upon editing near a processing site could be experimentally verified. Conclusions: ADAR2 can significantly influence the abundance of certain miRNAs in the brain. Only in a few cases changes in miRNA abundance can be explained by miRNA editing. Thus, ADAR2 binding to miRNA precursors, without editing them, may influence their processing and thereby abundance. ADAR1 and ADAR2 have both overlapping and distinct specificities for editing of miRNA editing sites. Over 60% of editing occurs in the seed region possibly changing target specificities for many edited miRNAs. Examination of the effect of ADAR2 on mature miRNA abundance and sequence in adult mouse brain.
2014-10-02 | E-GEOD-61066 | biostudies-arrayexpress
Project description:The RNA editing enzyme ADAR chemically modifies adenosine (A) to inosine (I), which is interpreted by the ribosome as a guanosine. Here we assess cotranscriptional A-to-I editing in Drosophila, by isolating nascent RNA from adult fly heads and subjecting samples to high-throughput sequencing. There are a large number of edited sites within nascent exons. Nascent RNA from an ADAR null mutant strain was also sequenced, indicating that almost all A-to-I events require ADAR. Moreover, mRNA editing levels correlate with editing levels within the cognate nascent RNA sequence, indicating that the extent of editing is set cotranscriptionally. Surprisingly, the nascent data also identify an excess of intronic over exonic editing sites. These intronic sites occur preferentially within introns that are poorly spliced cotranscriptionally, suggesting a link between editing and splicing. We conclude that ADAR-mediated editing is more widespread than previously indicated and largely occurs cotranscriptionally.
2012-05-31 | GSE37232 | GEO
Project description:Background: Adenosine deaminases that act on RNA (ADARs) bind to double-stranded and structured RNAs and deaminate adenosines to inosines. This A to I editing is widespread and required for normal life and development. Besides mRNAs and repetitive elements, ADARs can target miRNA precursors. Editing of miRNA precursors can affect processing efficiency and alter target specificity. Interestingly, ADARs can also influence miRNA abundance independent of RNA-editing. In mouse embryos where editing levels are low, ADAR2 was found to be the major ADAR protein that affects miRNA abundance. Here we extend our analysis to adult mouse brains where high editing levels are observed. Results: Using Illumina deep sequencing we compare the abundances of mature miRNAs and editing events within them, between wild-type and ADAR2 knockout mice in the adult mouse brain. Reproducible changes in abundance of specific miRNAs are observed in ADAR2 deficient mice. Most of these quantitative changes seem unrelated to A to I editing events. However, many A to G transitions in cDNAs prepared from mature miRNA sequences, reflecting A to I editing events in the RNA, are observed with frequencies reaching up to 80%. About half of these editing events are primarily caused by ADAR2 while a few miRNAs show increased editing in the absence of ADAR2, suggesting preferential editing by ADAR1. Moreover, novel, previously unknown editing events were identified in several miRNAs. In general 64% of all editing events are located within the seed region of mature miRNAs. In one of these cases retargeting of the edited miRNA could be verified in reporter assays. Also, altered processing efficiency upon editing near a processing site could be experimentally verified. Conclusions: ADAR2 can significantly influence the abundance of certain miRNAs in the brain. Only in a few cases changes in miRNA abundance can be explained by miRNA editing. Thus, ADAR2 binding to miRNA precursors, without editing them, may influence their processing and thereby abundance. ADAR1 and ADAR2 have both overlapping and distinct specificities for editing of miRNA editing sites. Over 60% of editing occurs in the seed region possibly changing target specificities for many edited miRNAs. Examination of the effect of ADAR2 on gene expression in mature mouse wildtype and ADAR2 knockout brain using AffymetrixM-BM-. GeneChipM-BM-. Whole Transcript (WT) Expression Arrays (Analysis by KFB Regensburg, Germany)
2014-10-02 | E-GEOD-61067 | biostudies-arrayexpress
Project description:Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly due to technical limitations in purifying authentic nascent transcripts. We present a new approach to purify and profile nascent RNA, called Polymerase Intact Nascent Transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5’ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano) and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5’P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that splicing occurs either immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.
2021-03-19 | GSE159326 | GEO
Project description:F. Boonekamp et al., (2019) describe an innocuous watermarking strategy for coding regions that enables the discrimination of DNA and mRNA by sequencing through a k-mer approach and facilitates selective editing of watermarked and non-watermarked sequences. Shot-gun proteomics (LFQ) was performed on the parent and the engineered yeast strain. *Corresponding author: Pascale Daran-Lapujade (p.a.s.daran-lapujade@tudelft.nl). Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ Delft, The Netherlands
2020-09-21 | PXD016914 | Pride