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Chemical modifications mark alternatively spliced and actively degrading messenger RNAs in Arabidopsis and humans

ABSTRACT: Post-transcriptional chemical modification of RNA bases is a widespread and physiologically relevant regulator of RNA maturation, stability, and function. While modifications are best characterized in short, noncoding RNAs such as transfer RNAs (tRNAs), growing evidence indicates that messenger RNAs (mRNAs) and long noncoding RNAs (lncRNAs) are likewise modified. Here, we apply our High-throughput Annotation of Modified Ribonucleotides (HAMR) pipeline to identify and classify modifications that affect Watson-Crick base-pairing at three different levels of the human and Arabidopsis thaliana transcriptomes (polyadenylated, small, and degrading RNAs). We find modifications primarily within actively degrading mRNAs and lncRNAs, suggesting they can act as a potent signal for RNA turnover. Additionally, modifications within stable mRNAs mark alternatively spliced introns, suggesting they regulate splicing. Furthermore, these modifications target mRNAs with coherent functions, including stress responses. Thus, our comprehensive analysis of RNA modification across multiple RNA classes yields new functional insights into these covalent RNA additions.

ORGANISM(S): Arabidopsis thaliana Homo sapiens

PROVIDER: GSE66224 | GEO | 2015/10/31

SECONDARY ACCESSION(S): PRJNA276134

REPOSITORIES: GEO

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Chemical modifications mark alternatively spliced and actively degrading messenger RNAs in Arabidopsis and humans

Project description:Post-transcriptional chemical modification of RNA bases is a widespread and physiologically relevant regulator of RNA maturation, stability, and function. While modifications are best characterized in short, noncoding RNAs such as transfer RNAs (tRNAs), growing evidence indicates that messenger RNAs (mRNAs) and long noncoding RNAs (lncRNAs) are likewise modified. Here, we apply our High-throughput Annotation of Modified Ribonucleotides (HAMR) pipeline to identify and classify modifications that affect Watson-Crick base-pairing at three different levels of the human and Arabidopsis thaliana transcriptomes (polyadenylated, small, and degrading RNAs). We find modifications primarily within actively degrading mRNAs and lncRNAs, suggesting they can act as a potent signal for RNA turnover. Additionally, modifications within stable mRNAs mark alternatively spliced introns, suggesting they regulate splicing. Furthermore, these modifications target mRNAs with coherent functions, including stress responses. Thus, our comprehensive analysis of RNA modification across multiple RNA classes yields new functional insights into these covalent RNA additions. polyA-selected RNA-seq, smRNA-seq, and polyA-selected global mapping of uncapped transcripts (GMUCT) in Arabidopsis thaliana; smRNA-seq in HEK293T and HeLa human cell lines.

2015-10-31 | E-GEOD-66224 | biostudies-arrayexpress

tRNA Modification Identification and Quantification through High Throughput Sequencing

Project description:We report the identification and quantification of Watson-Crick modifications in tRNA and rRNA through the use of high throughput sequencing. We apply the recently published DM-tRNA-Seq method to generate demethylase treated and untreated 293T samples, and using computational methods we are able to flag sites using a modification index. This index allows us to generate site-resolved information about modification that we can use to identify and quantify Watson-Crick face modifications in tRNA and rRNA. With the demethylase treated samples, we are able to validate numerous nucleotide modifications from demethylase substrates, and the absence of demethylase action also serves to aid in identification. We find numerous novel modification sites in tRNA as well as striking comparisons between tissues cultures lines. Our study reports a comprehensive analysis of the tRNA modification landscape by identifying sites of modification as well as quantifying modification levels.

2017-02-10 | GSE76434 | GEO

Carbodiimide reagents for the chemical probing of RNA structure in cells

Project description:Deciphering the conformations of RNAs in their cellular environment allows identification of RNA elements with potentially functional roles within biological contexts. Insight into the conformation of RNA in cells has been achieved using chemical probes that were developed to react specifically with flexible RNA nucleotides, or the Watson-Crick face of single-stranded nucleotides. The most widely used probes are either selective SHAPE (2'-hydroxyl acylation and primer extension) reagents that probe nucleotide flexibility, or dimethyl sulfate (DMS), which probes the base-pairing at adenine and cytosine but is unable to interrogate guanine or uracil. The constitutively charged carbodiimide N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) is widely used for probing G and U nucleotides, but has not been established for probing RNA in cells. Here, we report the use of a smaller and conditionally charged reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), as a chemical probe of RNA conformation, and the first reagent validated for structure probing of unpaired G and U nucleotides in intact cells. We showed that EDC demonstrates similar reactivity to CMC when probing transcripts in vitro. We found that EDC specifically reacted with accessible nucleotides in the 7SK noncoding RNA in intact cells. We probed structured regions within the Xist lncRNA with EDC and integrated these data with DMS probing data. Together, EDC and DMS allowed us to refine predicted structure models for the 3’ extension of repeat C within Xist. These results highlight how complementing DMS probing experiments with EDC allows the analysis of Watson-Crick base-pairing at all four nucleotides of RNAs in their cellular context.

2018-12-26 | GSE118309 | GEO

Dicer-microRNA-Myc circuit promotes transcription of hundreds of long noncoding RNAs

Project description:Long noncoding RNAs (lncRNAs) are important regulators of cell fate, and their mis-expression has been implicated in many diseases. While distinct polymerases generate messenger vs. noncoding ribosomal or tRNAs3, little is known about distinct mechanisms controlling lncRNA expression. Here we show that transcription of lncRNAs is quantitatively different from that of messenger RNAs (mRNAs)--as revealed by deficiency of Dicer (Dcr), a key ribonuclease that generates microRNAs (miRNAs). Loss of Dcr in mouse embryonic stem cells (mESCs) led surprisingly to decreased level of the majority of lncRNAs. The canonical Dgcr8-Dcr-miRNA pathway is required for robust lncRNA expression, at the level of transcriptional initiation and elongation of lncRNA genes rather than at the level of their stability. cMyc, an oncogenic transcription factor, whose expression is indirectly regulated by Dcr-miRNA in mESCs, is partly responsible for lncRNA transcription. Loss of cMyc led to a more dramatic decrease of lncRNAs than mRNAs, and cMyc overexpression rescues lncRNA expression in Dcr KO cells. A quantitative metric of “mRNA-lncRNA decoupling” revealed that Dcr and cMyc differentially regulate lncRNAs vs. mRNAs in diverse cell types and in vivo, as evidenced by hundreds of microarray experiments. Thus, Dcr and cMyc may allow numerous lncRNAs to be activated or deactivated as a class, implicating lncRNAs to potential regulatory roles in development and disease states where Dcr and cMyc have been associated with. RNA was sequenced from WT and Dcr KO mESCs and the expression of lncRNAs and mRNAs are compared between WT and Dcr KO mESCs.

2014-06-15 | E-GEOD-55338 | biostudies-arrayexpress

Affymetrix custom RNA microarray for WT and jhd2-delete strains in terminal spores (20h)

Project description:RNA transcript signals were profiled in WT (MMY718) and jhd2M-bM-^HM-^F (MMY1879) terminally sporulated cultures (20h of sporulation) using Affymetrix high resolution tiling microarrays. Total RNA was isolated, biotin labelled, and hybridized to custom Affymetrix Scerevisiae_tlg tiling arrays. The array contains 25nt probes covering the Watson strand of the yeast genome with 8nt tiling resolution, and another set of 25nt probes covering the Crick strand with 8nt resolution, with a 4nt offset from the Watson probes. This gives an overall tiling resolution of 4bp with a total of 6.5 million probes, and also allows detection of strand-specific RNA signals.

2012-10-01 | E-GEOD-41002 | biostudies-arrayexpress

Functional classification of chromatin associated lncRNAs via histone modification specific PIRCh-seq analysis

Project description:Many long noncoding RNAs (lncRNAs) regulate gene transcription through binding to histone modifying complexes, therefore a comprehensive study of chromatin associated RNA in a histone modification specific manner is critical to understand their regulatory mechanisms. In this study, we developed a new method named Profiling Interacting RNAs on Chromatin followed by deep sequencing (PIRCh-seq), and profiled chromatin associated transcriptome on 3 cell types, mouse v6.5 embryonic stem cells (mESC), mouse embryonic fibroblasts (MEF) and mouse neuronal precursor cells (NPC), with antibodies attached to histone H3 and 6 active and repressive histone modifications. Compared with existing methods, PIRCh-seq identifies chromatin associated RNAs with significant lower nascent transcription, and classifies chromatin enriched lncRNAs into 6 functional groups based on the patterns of their attachment to nucleosome with specific chemical modifications. Further analysis showed that lncRNAs were also enriched with different modified chromatin in different cell types, suggesting lncRNAs’ regulation may also be cell type specific. By integrating profiles of RNA secondary structure from icSHAPE and RNA m(6)A modification, we noticed that RNA bases attached with chromatin tend to be more single stranded. These results provide a unique resource to globally study the functions of chromatin associated lncRNAs and elucidates the basic mechanisms of chromatin-RNA association.

2019-10-30 | GSE119006 | GEO

The long noncoding RNA expression profile of human intervertebral disc degeneration identified by microarray analysis

Project description:In addition to the well-known short noncoding RNAs as miRNAs, increasing evidence suggests that long noncoding RNAs (lncRNAs) act as key regulators in a wide aspect of biologic processes. Dysregulated expression of lncRNAs has been demonstrated being implicated in a variety of human diseases. However, there is relative paucity of information regarding the role of lncRNAs in intervertebral disc degeneration (IDD) hitherto. We have addressed the expression profiles of miRNAs in IDD previously. To determine whether lncRNAs is differentially expressed in IDD, we employed a lncRNA-mRNA microarray analysis of human nucleus pulposus (five degenerative as IDD and five normal specimens as control). With abundant probes accounting for 33,045 lncRNAs and 30,215 coding transcripts in our microarray, microarray data profiling indicated that 18995 lncRNAs and 14635 mRNAs were detected in all samples with 2309 lncRNAs and 3017 mRNAs differentially expressed in degenerated samples. Amongst them, 164 lncRNAs (97 up and 67 down) and 265 mRNAs were highly differentially expressed with an absolute fold change greater than ten. The upregulated mRNAs included the extracellular matrix components as COL1A2, LUM, FN1ACAN, DCN and ITFG2. 1100 lncRNAs and 1379 mRNAs were altered in the same direction in at least 4 of the 5 degenerative samples with fold change greater than 2 respectively. Three lncRNAs were chosen for the real-time quantitative PCR (qRT-PCR) analysis. qRT-PCR results showed a high consistency with the microarray data. In this study, to explore the potential involvement of lncRNAs in IDD, we conducted lncRNA and mRNA profiling in 5 human control nucleus pulposus tissue and 5 degenerative nucleus pulposus tissue by microarray.Biological replicates: 5 control, 5 degenerated, independently harvested.

2014-11-17 | E-GEOD-56081 | biostudies-arrayexpress

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