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Full-length Sequencing of Non-capped RNAs Reveals the Heterogeneity of RNA Processing and Novel Classes of Noncoding RNAs

ABSTRACT: Purpose:Up to 80% of the human genome gives rise to “dark matter” RNAs that mainly composes of non-capped RNAs (napRNAs). However, determining the functional impacts and metabolisms of the napRNAs requires identification of their full-length sequences. Method:We developed an approach to globally profile the full-length sequences of napRNAs at single-nucleotide resolution Results:We discovered, for the first time, the stable expressed linear intron RNA (sliRNA), a novel class of snoRNA-intron (snotron) RNAs, a new class of RNA embedded in miRNA spacers (misRNA) and thousands of new exceptional structured ncRNAs and repeat-derived ncRNAs (repeatRNA) in humans and mouse. Importantly, these new napRNAs are dynamic changes in response to various stimuli and biological processes, suggesting that they have potential regulatory functions. Furthermore, we show that functional napRNA FS4, discovered by NAP-seq firstly, promotes HepG2 cell proliferation by interacting with DKC1 to maintain the protein stability. Conclusion:Collectively, our approach establishes a paradigm for mapping and annotating novel class of ncRNAs of many living species.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE228168 | GEO | 2024/02/19

REPOSITORIES: GEO

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NAP-seq Reveals Novel Classes of Structured Noncoding RNAs with Regulatory Functions

Project description:Purpose:Up to 80% of the human genome produces “dark matter” RNAs, most of which are noncapped RNAs (napRNAs). However, determining the functional impacts and metabolism of napRNAs requires the identification of their full-length sequences. Method:Here, by developing a novel method, NAP-seq, to globally profile the full-length sequences of napRNAs at single-nucleotide resolution, we revealed several novel classes of exceptionally structured noncoding RNAs (ncRNAs). Results:We discovered stably expressed linear intron RNAs (sliRNAs), a novel class of snoRNA-intron RNAs (snotrons), a new class of RNAs embedded in miRNA spacers (misRNAs) and thousands of new structured ncRNAs in humans and mice. These new napRNAs undergo dynamic changes in response to various stimuli and differentiation stages. Importantly, we showed that the novel napRNA DINAP interacts with dyskerin pseudouridine synthase 1 (DKC1) to promote cell proliferation by maintaining DKC1 protein stability. Conclusion:Our approach establishes a paradigm for discovering novel classes of ncRNAs with regulatory potency.

2024-02-19 | GSE192632 | GEO

RIP-PEN-seq for 15.5K in Hepa1-6 cells

Project description:Here, we developed a RNA ImmunoPrecipitation coupled with sequencing of Paired-Ends of NcRNAs (RIP-PEN-seq) method to identify the full-length sequences of ncRNAs bound by 15.5K protein and discover a novel class of backward K-turn RNAs.

2023-04-10 | GSE182757 | GEO

RIP-PEN-seq for 15.5K in HEK293T cells

2023-04-10 | GSE160970 | GEO

The RNA Atlas expands the catalog of human non-coding RNAs

Project description:The human transcriptome consists of various RNA biotypes including multiple types of non-coding RNAs (ncRNAs). Current ncRNA compendia remain incomplete partially because they are almost exclusively derived from the interrogation of small- and polyadenylated RNAs. Here, we present a more comprehensive atlas of the human transcriptome that is derived from matching polyA-, total-, and small-RNA profiles of a heterogenous collection of nearly 300 human tissues and cell lines. We report thousands of novel RNA species across all major RNA biotypes, including a hitherto poorly-cataloged class of non-polyadenylated single-exon long non-coding RNAs. In addition, we exploit intron abundance estimates from total RNA-sequencing to predict the regulatory potential of various non-coding RNAs. Our study represents a substantial expansion of the current catalogue of human ncRNAs and their regulatory interactions. All data and results are accessible through the R2 webtool and serve as a basis to further explore RNA biology and function.

2021-04-15 | GSE138734 | GEO

Genomic features and regulatory roles of intermediate-size non-coding RNAs in Arabidopsis

Project description:Recent advances in genome-wide techniques allowed the identification of thousands of non-coding RNAs with various sizes in eukaryotes, some of which have further been shown to serve important functions in many biological processes. However, in model plant Arabidopsis, novel intermediate-sized ncRNAs (im-ncRNAs) (50~300nt) have very limited information. By using a modified isolation strategy combined with deep-sequencing technology, we identified 838 im-ncRNAs in Arabidopsis globally. More than half (58%) are new ncRNA species, mostly evolutionary divergent. Interestingly, annotated protein-coding genes with 5’-UTR derived novel im-ncRNAs tend to be highly expressed. For intergenic im-ncRNAs, their average abundances were comparable to mRNAs in seedlings, but subsets exhibited significantly lower expression in senescing leaves. Further, intergenic im-ncRNAs were regulated by similar genetic and epigenetic mechanisms as those of protein-coding genes, and some showed developmentally-regulated expression patterns. Large-scale reverse genetic screening showed that the down-regulation of a number of im-ncRNAs resulted in either obvious molecular changes or abnormal developmental phenotypes in vivo, indicating the functional importance of im-ncRNAs in plant growth and development. Together, our results demonstrate that novel Arabidopsis im-ncRNAs are developmentally-regulated and functional components discovered in the transcriptome. Genome-wide maps of Intermediate-size Non-coding RNAs in Arabidopsis

2014-01-20 | E-GEOD-44235 | biostudies-arrayexpress

CRISPR Display: A modular method for locus-specific targeting of long noncoding RNAs and synthetic RNA devices in vivo [RIP-Seq]

Project description:Noncoding RNAs (ncRNAs) comprise an important class of natural regulators that mediate a vast array of biological processes, including the modulation of chromatin architecture. Moreover, artificial ncRNAs have revealed that the functional capabilities of RNA are extremely broad. To further investigate and harness these capabilities, we developed CRISPR-Display ("CRISP-Disp"), a targeted localization strategy that uses Cas9 to deploy large RNA cargos to specific DNA loci. We demonstrate that exogenous RNA domains can be functionally appended onto the CRISPR scaffold at multiple insertion points, allowing the construction of Cas9 complexes with RNAs nearing one kilobase in length, with structured RNAs, protein-binding cassettes, artificial aptamers and pools of random sequences. CRISP-Disp also allows the simultaneous multiplexing of disparate functions at multiple targets. We anticipate that this technology will provide a powerful method with which to ectopically localize functional RNAs and ribonuceloprotein complexes at specified genomic loci. RNA Immunoprecipitation (RIP) against FLAG-tagged Cas9 protein, coexpressed with a large pool of CRISPR RNAs bearing random internal insertions

2015-05-11 | E-GEOD-62305 | biostudies-arrayexpress

CRISPR Display: A modular method for locus-specific targeting of long noncoding RNAs and synthetic RNA devices in vivo [RNA-Seq]

2015-05-11 | GSE66755 | GEO

CRISPR Display: A modular method for locus-specific targeting of long noncoding RNAs and synthetic RNA devices in vivo [RIP-Seq]

2015-05-11 | GSE62305 | GEO

Sensing self and nonself circular RNAs

Project description:Circular RNAs (circRNAs) are single-stranded RNAs that are joined head to tail. Initially discovered as pathogen genomes such as hepatitis D virus (HDV) and plant viroids, circRNAs are recently recognized as a pervasive class of noncoding RNAs in eukaryotic cells, generated through back splicing. circRNAs have been postulated to function in cell-to-cell information transfer or memory due to their extraordinary stability. Whether and how circRNAs trigger immune recognition is not known. Here we show that exogenous circular RNAs potently stimulate immune signaling, and mammalian cells sense self vs. nonself circRNAs via circRNA biogenesis. Transfection of purified in vitro spliced circRNA into mammalian cells led to potent induction of innate immunity genes. The nucleic acid sensor RIG-I is necessary and sufficient to sense foreign circRNA, and RIG-I and foreign circRNA co-aggregate in cytoplasmic foci. CircRNA activation of innate immunity is independent of 5’ triphosphate, double-stranded RNA structure, or primary sequence of the foreign circRNA. Instead, self-nonself discrimination depends on the intron that programs the circRNA. Use of a human intron to express a foreign circRNA sequence abrogates immune activation, and the mature human circRNA is associated with diverse RNA binding proteins reflecting its endogenous splicing and biogenesis. These results reveal innate immune sensing of circRNA, a prevalent class of host and pathogen RNAs, and highlight introns—the predominant output of mammalian transcription—as unexpected arbiters of self-nonself identity in the RNA world.

2017-06-29 | GSE81345 | GEO

CRISPR Display: A modular method for locus-specific targeting of long noncoding RNAs and synthetic RNA devices in vivo [RNA-Seq]

Project description:Noncoding RNAs (ncRNAs) comprise an important class of natural regulators that mediate a vast array of biological processes, including the modulation of chromatin architecture. Moreover, artificial ncRNAs have revealed that the functional capabilities of RNA are extremely broad. To further investigate and harness these capabilities, we developed CRISPR-Display ("CRISP-Disp"), a targeted localization strategy that uses Cas9 to deploy large RNA cargos to specific DNA loci. We demonstrate that exogenous RNA domains can be functionally appended onto the CRISPR scaffold at multiple insertion points, allowing the construction of Cas9 complexes with RNAs nearing one kilobase in length, with structured RNAs, protein-binding cassettes, artificial aptamers and pools of random sequences. CRISP-Disp also allows the simultaneous multiplexing of disparate functions at multiple targets. We anticipate that this technology will provide a powerful method with which to ectopically localize functional RNAs and ribonuceloprotein complexes at specified genomic loci. Whole cell poly(A) selected RNA seq, from HEK293FT cells bearing lentivirally-integrated Gaussia and Cypridina luciferase reporter loci. Cells were transiently transfected with dCas9~VP64 alone, or with dCas9~VP and one of several modified sgRNAs,each targeting the Gaussia reporter.

2015-05-11 | E-GEOD-66755 | biostudies-arrayexpress

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