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SciMET-cap: High-throughput single-cell methylation analysis with a reduced sequencing burden

ABSTRACT: DNA methylation is a key component of the mammalian epigenome, playing a regulatory role in development, disease, and other processes. Robust, high-throughput single-cell DNA methylation assays are now possible (sciMET); however, the genome-wide nature of DNA methylation results in a high sequencing burden per cell. Here, we leverage target enrichment with sciMET to capture sufficient information per cell for cell type assignment using substantially fewer sequence reads (sciMET-cap). Sufficient off-target coverage further enables the production of near-complete methylomes for individual cell types. We characterize sciMET-cap on human PBMCs and brain (middle frontal gyrus).

ORGANISM(S): Homo sapiens

PROVIDER: GSE250282 | GEO | 2023/12/19

REPOSITORIES: GEO

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mRNA cap methylation in pluripotency and differentiation

Project description:The mRNA cap recruits factors essential for transcript processing and translation initiation. We report that regulated mRNA cap methylation is a feature of embryonic stem cell (ESC) differentiation. Expression of the mRNA cap methyltransferase activating subunit, RAM, is elevated in ESCs resulting in high levels of mRNA cap methylation and expression of Oct4 and Sox2 and other pluripotency-associated factors. During neural differentiation, RAM is suppressed which is required for loss of Oct4 and Sox2 and correct expression of neural markers. Cells were treated with control or RAM siRNA and cytosolic RNA or polysome RNA (actively translated) was sequenced.

2016-06-14 | E-GEOD-81849 | biostudies-arrayexpress

mRNA cap methylation in pluripotency and differentiation

2016-06-14 | GSE81849 | GEO

Influence of CAP-D3 in Arabidopsis thaliana transcription

Project description:Transcriptome analysis of two different mutants of the condensin II subunit CAP-D3. Four week-old plantlets from the T-DNA insertion lines SAIL_826_B06 (cap-d3 SAIL) and SALK_094776 (cap-d3 SALK) were compared to Col-0 wild-type plants.

2020-12-31 | E-MTAB-8969 | biostudies-arrayexpress

2'-O-methylation of the second transcribed nucleotide in eukaryotic RNA 5’ cap impacts protein production levels and susceptibility to DXO-mediated transcript degradation

Project description:Eukaryotic mRNA 5’ end is decorated with cap structure which plays multiple roles in the cell. Most importantly, protects transcript from exonucleolytic degradation and enables translation of encoded protein conducted by cap-dependent machinery. This is also a key feature in the process of recognizing RNA as self or non-self molecule by cellular innate immune system. Cap is composed of 7-methylguanosine linked by a 5′,5′-triphosphate chain to the first transcribed nucleotide, and is formed enzymatically during transcription. This m7GpppN structure, called cap0, can be further modified by CMTR1 methyltransferase to cap1 (m7GpppNm), which predominates in eukaryotic cells. Cap2, with additional 2′-O-methylation at the second transcribed nucleotide (m7GpppNmpNm) added by another methyltransferase CMTR2, also can be found at mRNA 5’ end. We present new tools allowing for in vitro synthesis of RNAs possessing cap2, i.e tetranucleotide cap analogues. Utilizing these for in vitro transcription reactions we obtained RNAs with 2′-O-methylation present at the second transcribed nucleotide. Due to that we investigated the role of cap2 in protein production from reporter mRNA protected with this structure in different conditions, normal or stress ones, or with CMTR1/2 down regulated level. We also assessed the affinity of eIF4E protein to differentially capped RNAs. Furthermore, we verified whether an additional 2′-O-methylation presence in capped RNA makes transcript more resistant to decapping enzyme action.

2022-09-08 | PXD028636 | Pride

MYC Mediates mRNA Cap Methylation of Canonical Wnt/beta-catenin Signaling Transcripts by Recruiting CDK7 and RNA Methyltransferase

Project description:We performed an RNA pulldown against a methyl group on the mRNA cap using a fusion protein, GST-eIF4E, highly specific for a m7G-mRNA cap. We performed RNA-seq on both the pulldowns and inputs and normalized our pulldown to input in order to determine which genes MYC targets for mRNA cap methylation above and beyond any affect at the transcriptional level. We found that MYC targets approximately 30% of transcripts for enhanced mRNA cap methylation above 1.2 fold on a global scale following 24 h of MYC induction, specifically transcripts involved in the canonical Wnt/β-catenin signaling pathway. We used this dataset to validate transcripts targeted by MYC for mRNA cap methylation and to help us determine the mechanism responsible for this phenomenon.

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Part 2 JAWS; 2'-O-methylation of the second transcribed nucleotide in eukaryotic RNA 5’ cap impacts protein production levels and susceptibility to DXO-mediated transcript degradation

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Three Arm Rand Trial of HD Light vs Reveal Cap vs Endocuff Cap for Detection of Colorectal Adenomas (TRACE)

Project description:Protocol Title: Three Arm Prospective Randomized controlled trial of High-Definition White-light colonoscopy versus High-Definition White-light colonoscopy with Reveal Distal Attachment Cap versus High-Definition White-light colonoscopy with Endocuff Vision for the detection of colorectal adenomas Hypothesis: Detection rate of adenomas in patients will be higher in procedures performed with High-Definition White-light (HDWL) colonoscopy with Reveal distal attachment cap and HDWL colonoscopy with Endocuff Vision compared to HDWL colonoscopes alone Design: Multicenter, Prospective, randomized controlled study Primary Aim: To compare the proportion of subjects with at least one adenoma detected during HDWL colonoscopy versus HDWL colonoscopy with Reveal distal attachment cap versus HDWL colonoscopy with Endocuff Vision. Secondary Aims: To compare the number of adenomas detected per subject with HDWL colonoscopy versus HDWL colonoscopy with Reveal distal attachment cap versus HDWL colonoscopy with Endocuff Vision. To compare the detection rates for polyp subtypes (including advanced adenomas, serrated polyps, right sided adenomas, etc.), cecal intubation rate, insertion time, withdrawal time, and complications of HDWL colonoscopy versus HDWL colonoscopy with Reveal distal attachment cap versus HDWL colonoscopy with Endocuff Vision.

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LCL Bio-CAP-seq

Project description:LCL Bio-CAP-seq

| PRJNA795784 | ENA

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Project description:To investigate gene specificity at the level of translation in both the human genome and viruses we devised a high-throughput bicistronic assay to quantify cap-independent translation. We uncover thousands of novel cap-independent translation sequences and provide insights on the landscape of translational regulation in both human and viruses. We find extensive translational elements in the 3â untranslated region (3âUTR) of human transcripts and the polyprotein region of un-capped RNA viruses. Through the characterization of regulatory elements underlying cap-independent translation activity we identify potential mechanisms of secondary structure, short sequence motif and base-pairing with the 18S rRNA. Furthermore, we systematically map the 18S rRNA regions for which reverse complementary enhance translation. Thus we provide insights into the mechanisms of translational control in humans and viruses. high-throughput bicistronic assay for obtaining cap-independent translation measurements of 55,000 fully designed sequences in parallel using fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq).

2015-10-23 | E-GEOD-74277 | biostudies-arrayexpress

Cap-specific terminal N6-methylation of RNA by an RNA polymerase II-associated methyltransferase.

Project description:N6-methyladenosine (m6A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m6A, N6, 2'-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. However, its biogenesis and functional role remain elusive. Using a reverse genetics approach, we identified PCIF1, a factor that interacts with the serine-5-phosphorylated carboxyl-terminal domain of RNA polymerase II, as a cap-specific adenosine methyltransferase (CAPAM) responsible for N6-methylation of m6Am. The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m6A formation. A transcriptome-wide analysis revealed that N6-methylation of m6Am promotes the translation of capped mRNAs. Thus, a cap-specific m6A writer promotes translation of mRNAs starting from m6Am.

2018-11-02 | GSE122071 | GEO

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