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Expanding the MicroRNA Targeting Code: A Novel Type of Site with Centered Pairing

ABSTRACT: We present “centered sites,” a class of microRNA target sites that lacks both perfect seed pairing and 3'-compensatory pairing and instead has 11–12 contiguous Watson–Crick pairs to the center of the microRNA. In elevated Mg2+, centered sites impart mRNA cleavage, but in cells, centered sites repress protein output without consequential Agronaute-catalyzed cleavage. Our study also identified novel extensively paired sites that are cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavage targets and the identification of the centered site type help explain why central regions of many microRNAs are evolutionarily conserved.

ORGANISM(S): Danio rerio Homo sapiens

PROVIDER: GSE22068 | GEO | 2010/06/25

SECONDARY ACCESSION(S): PRJNA128943

REPOSITORIES: GEO

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Expanding the MicroRNA Targeting Code: A Novel Type of Site with Centered Pairing

Project description:We present “centered sites,” a class of microRNA target sites that lacks both perfect seed pairing and 3'-compensatory pairing and instead has 11–12 contiguous Watson–Crick pairs to the center of the microRNA. In elevated Mg2+, centered sites impart mRNA cleavage, but in cells, centered sites repress protein output without consequential Agronaute-catalyzed cleavage. Our study also identified novel extensively paired sites that are cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavage targets and the identification of the centered site type help explain why central regions of many microRNAs are evolutionarily conserved. To study centered sites and identify miRNA cleavage targets, mRNA degradomes were sequenced from human brain and HeLa cells, and smallRNAs were sequenced from human brain and zebrafish embryo at 24 hours post fertilization (hpf). Replicates were combined before the analysis. Fastq files are not available for GSM548638 and GSM548639.

2010-06-25 | E-GEOD-22068 | biostudies-arrayexpress

Expanding the MicroRNA Targeting Code: A Novel Type of Site with Centered Pairing

Project description:Expanding the MicroRNA Targeting Code: A Novel Type of Site with Centered Pairing

| PRJNA128943 | ENA

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

Enzymatic or in vivo installation of propargyl groups in combination with click chemistry enables enrichment and detection of methyltransferase target sites in RNA

Project description:m6A is the most abundant internal modification in eukaryotic mRNA. It is introduced by METTL3-METTL14 and tunes mRNA metabolism, impacting cell differentiation and development. Precise transcriptome-wide assignment of m6A sites is of utmost importance. However, m6A does not interfere with Watson-Crick base pairing making polymerase-based detection challenging. We developed a chemical biology approach for the precise mapping of methyltransferase (MTase) target sites based on the introduction of a bioorthogonal propargyl group in vitro and in cells. We show that propargyl can be introduced enzymatically by wild-type METTL3-METTL14. Reverse transcription terminated up to 65 % at m6A sites after bioconjugation and purification, hence enabling detection of METTL3-METTL14 target sites by next generation sequencing. Importantly, we implemented metabolic propargyl labeling of RNA MTase target sites in vivo based on propargyl-L-selenohomocysteine and validated different types of known rRNA methylation sites.

2018-02-21 | GSE109298 | GEO

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

Chemical manipulation of m1A mediates its detection in human tRNA

Project description:N1-methyl adenosine (m1A) is a wide-spread RNA modification present in tRNA, rRNA and mRNA. m1A modification sites in tRNAs are evolutionary conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick base pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red‑m1A-seq to sequence human small RNA and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-ATT and 5.8S rRNA.

2024-04-24 | GSE253657 | 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

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

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

2015-10-31 | GSE66224 | GEO

Analysis of gene expression of PC3 human prostate cancer cells incubated with Polypurine reverse Hoogsteen hairpins (PPRH) against survivin

Project description:We had previously shown the effect of PPRHs against the DNA coding for the antiapoptotic protein survivin in PC3 prostate cancer cells. Now, we explored the gene expression profile produced by 24 hours of incubation with specific PPRH against survivin (HpsPr-WT) and with a Watson-Crick PPRH negative control (HpsPr-WC). PC3 cells were incubated with 100 nM of each PPRH for 24 hours. RNA was extracted and Affymetrix Clariom S exon microarray was performed to determine gene expression.

2019-06-04 | GSE107106 | GEO

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