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Dissecting telomerase RNA structural heterogeneity in living human cells with DMS-MaPseq

ABSTRACT: Dissecting telomerase RNA structural heterogeneity in living human cells with DMS-MaPseq

PROVIDER: PRJNA1028958 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR26405905_1.fastq.gz	Fastqsanger.gz
	SRR26405905_2.fastq.gz	Fastqsanger.gz
	SRR26405906_1.fastq.gz	Fastqsanger.gz
	SRR26405906_2.fastq.gz	Fastqsanger.gz
	SRR26405907_1.fastq.gz	Fastqsanger.gz

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Dissecting telomerase RNA structural heterogeneity in living human cells with DMS-MaPseq

Project description:Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-containing pseudoknot (t/PK) and the three-way junction (CR4/5). These two hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are thus essential for telomerase catalytic activity. Here, we probe the structure of hTR in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Unexpectedly, approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features required for hTERT binding. Mutagenesis demonstrates that stabilization of the alternative CR4/5 conformation is detrimental to telomerase assembly and activity. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded. Thus, kinetic traps for RNA folding that have been so well-studied in vitro may also present barriers for assembly of ribonucleoprotein complexes in vivo.

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Dissecting telomerase RNA structural heterogeneity in living human cells with DMS-MaPseq.

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Project description:Here we present dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase (TGIRT). DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features for each molecule, and allows genome-wide studies as well as focused investigations of low abundance RNAs. We apply DMS-MaPseq to Drosophila melanogaster ovaries—the first experimental analysis of RNA structure in an animal tissue—and demonstrate its utility in the discovery of a functional RNA structure involved in the non-canonical GUG translation initiation of the human FXR2 mRNA. Additionally, we use DMS-MaPseq to compare the in vivo structure of messages in their pre-mRNA and mature forms. These applications illustrate DMS-MaPseq’s capacity to dramatically expand our ability to monitor RNA structure in vivo.

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Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq.

Project description:Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-pseudoknot (t/PK) and the three-way junction (CR4/5). These hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are essential for telomerase activity. Here, we probe hTR structure in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features required for hTERT binding. The proportion of hTR CR4/5 folded into the primary functional conformation is independent of hTERT expression levels. Mutations that stabilize the alternative CR4/5 conformation are detrimental to telomerase assembly and activity. Moreover, the alternative CR4/5 conformation is not found in purified telomerase RNP complexes, supporting the hypothesis that only the primary CR4/5 conformer is active. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded, suggesting that kinetic RNA folding traps studied in vitro may also hinder ribonucleoprotein assembly in vivo.

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SWI/SNF subunit CHR2 remodels pri-miRNAs via microprocessor component SE to inhibit miRNA production DMS-MaPseq)

Project description:Here we present dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) prepared from leaves of adult plants in Arabidopsis. Chromatin remodeling factor 2 (CHR2) positively regulates transcription of MIR loci whereas repressing microRNA (miRNA) accumulation in vivo. CHR2 can directly bind to and unwind primary miRNAs (pri-miRNAs) and inhibit their processing; and this inhibition entails its remodeling activity in vitro and in vivo.

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