Unknown,Transcriptomics,Genomics,Proteomics

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Nuclear-localized tiny RNAs are associated with transcription initiation and splice sites in metazoans

ABSTRACT: We have recently shown that transcription initiation RNAs (tiRNAs) are derived from sequences downstream of transcription start sites. Here we report the identification of a second class of nuclear-specific ~17-18 nucleotide small RNA whose 3M-bM-^@M-^Y ends map precisely to the splice donor site of internal exons in animals. These splice-site RNAs (spliRNAs) are associated with highly expressed genes, and show evidence of developmental stage- and region-specific expression. We also confirm that tiRNAs are nuclear localized, enriched at chromatin marks associated with transcription initiation, and possess a 3M-bM-^@M-^Y nucleotide bias. Additionally, we find that microRNA-offset RNAs (moRNAs), the oncogenic miR-15/16 cluster and most snoRNA-derived small RNAs (sdRNAs) are enriched in the nucleus, whereas most miRNAs and two H/ACA sdRNAs are cytoplasmically enriched. We propose that nuclear localized tiny RNAs are involved in epigenetic regulation of gene expression. Discovery and characterization of small RNA species through high-througput deep sequencing of nuclear, cytoplasmic and total small RNA fractions from THP-1 cells, a monocytic leukemia cell line. Additional files and information are available at http://matticklab.com/index.php?title=NuclearTinyRNAs

ORGANISM(S): Homo sapiens

SUBMITTER: Ryan Taft

PROVIDER: E-GEOD-20664 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Nuclear-localized tiny RNAs are associated with transcription initiation and splice sites in metazoans.

Taft Ryan J RJ Simons Cas C Nahkuri Satu S Oey Harald H Korbie Darren J DJ Mercer Timothy R TR Holst Jeff J Ritchie William W Wong Justin J-L JJ Rasko John E J JE Rokhsar Daniel S DS Degnan Bernard M BM Mattick John S JS

Nature structural & molecular biology 20100711 8

We have recently shown that transcription initiation RNAs (tiRNAs) are derived from sequences immediately downstream of transcription start sites. Here, using cytoplasmic and nuclear small RNA high-throughput sequencing datasets, we report the identification of a second class of nuclear-specific approximately 17- to 18-nucleotide small RNAs whose 3' ends map precisely to the splice donor site of internal exons in animals. These splice-site RNAs (spliRNAs) are associated with highly expressed gen ...[more]

PMID: 20622877

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Project description:To accelerate previous RNA structure probing approaches, which focus on analyzing one RNA sequence at a time, we have developed FragSeq, a high-throughput RNA structure probing method that uses high-throughput RNA sequencing to identify single-stranded RNA (ssRNA) regions from fragments generated by nuclease P1, which is specific for single-stranded nucleic acids. In the accompanying study, we show that we can accurately and simultaneously map ssRNA regions in multiple non-coding RNAs with known structure in experiments probing the entire mouse nuclear transcriptome. We carried out probing in two cell types to assess reproducibility. We also identified and experimentally validated structured regions in ncRNAs never previously probed. We examined mouse nuclear RNA from two cell types: undifferentiated embryonic stem cells (UNDIFF) and cells differentiated into neural precursors (D5NP). For each cell type, nuclear RNA was purified and deproteinized, denatured, and refolded in vitro, from which we prepared three barcoded samples: "nuclease" (RNA partially digested with P1 ssRNA-specific nuclease, yielding 5'-PO4/3'-OH end chemistry at each cleavage site), "control" (control for "nuclease" sample to idenfity endogenous 5'-PO4/3'-OH), and "PNK" (same as "control" but followed by a polynucleotide kinase treatment to convert 5'-OH/3'-cyclic-phosphate ends to clonable 5'-PO4/3'-OH ends). Resulting RNA fragments were cloned using the SOLiD Small RNA Expression Kit (SREK) protocol, which ligates linkers only to 5'-PO4/3'-OH containing RNA, enriching for clones of products resulting from P1 cleavage in "nuclease" sample and selecting against random degradation. Two cell types, three treatments each, thus resulted in six barcoded samples total (barcodes 01, 02, 04, 05, 07, 08). Four other barcoded samples were prepared for separate experiments not used in our study (barcodes 03, 06, 09, 10), so their preparation is not described here. The total run of ten barcodes was done on the ABI SOLiD3 platform and a custom algorithm (FragSeq v0.0.1) was used to compute "cutting scores" (as described in our paper) that show ssRNA regions in hundreds of ncRNAs.

Dataset Information

Nuclear-localized tiny RNAs are associated with transcription initiation and splice sites in metazoans

Publications

Nuclear-localized tiny RNAs are associated with transcription initiation and splice sites in metazoans.

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