Unknown,Transcriptomics,Genomics,Proteomics

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Microprocessor mediates transcription termination in long noncoding microRNA genes

ABSTRACT: MicroRNA (miRNA) play a major role in the post-transcriptional regulation of gene expression. In mammals most miRNA derive from the introns of protein coding genes where they exist as hairpin structures in the primary gene transcript, synthesized by RNA polymerase II (Pol II). These are cleaved co-transcriptionally by the Microprocessor complex, comprising DGCR8 and the RNase III endonuclease Drosha, to release the precursor (pre-)miRNA hairpin, so generating both miRNA and spliced messenger RNA1-4. However, a substantial minority of miRNA originate from Pol II-synthesized long non coding (lnc) RNA where transcript processing is largely uncharacterized5. Here, we show that most lnc-pri-miRNA do not use the canonical cleavage and polyadenylation (CPA) transcription termination pathway6, but instead use Microprocessor cleavage both to release pre-miRNA and terminate transcription. We present a detailed characterization of one such lnc-pri-miRNA that generates the highly expressed liver-specific miR-1227. Genome-wide analysis then reveals that Microprocessor-mediated transcription termination is commonly used by lnc-pri-miRNA but not by protein coding miRNA genes. This identifies a fundamental difference between lncRNA and pre-mRNA processing. Remarkably, inactivation of the Microprocessor can lead to extensive transcriptional readthrough of lnc-pri-miRNA, resulting in inhibition of downstream genes by transcriptional interference. Consequently we define a novel RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells. Chromatin associated RNA-seq from sicntrl,siDrosha,siDGCR8 treated Hela cells. Same for sicntrl and siDGCR8 from Huh7 cells. Nuclear polyA + and polyA- RNA-seq from sicntrl and siDGCR8 in HeLa cells. Chromatin associated RNA-seq from siDicer treated Hela cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Nicholas Proudfoot

PROVIDER: E-GEOD-58838 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Preparation of nuclear extracts D. melanogaster Schneider cells were prepared as previously described by Andrews and Faller (1991). Briefly, in a typical preparation, 8x106 cells (grown in Schneider medium plus 10% fetal calf serum; GIBCO-BRL) were pelleted and resuspended in 1,5 mL cold PBS1X; the cell suspension is then tranferred to a microfuge tube. Cells are pelleted for 10 seconds and resuspended in 400 M-5L cold Buffer A (10mM HEPES-KOH pH 7.9 at 4M-0C, 1.5mM MgCl2, 10mM KCl, 0.5mM duthiothreitol, 0.2mM PMSF, 0.2U/microL RNasin). The cells are allowed to swell on ice for 10 minutes, and then vortexed for 10 seconds. Samples are centrifuged for 10 seconds, and the supernatant fraction is discarded. The pellet is resuspended in 100 M-5L of cold Buffer C (20mM HEPES-KOH pH 7.9, 25% glycerol, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.5mM dithiothreitol, 0.2mM PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris is removed by centrifugation for 2 min at 4M-0C and the supernatant fraction is stored at M-^V70M-0C. All buffers were prepared from double-distilled autoclaved water that had been treated with 0.1% DEPC (Sigma). RNA immunoprecipitation and Reverse Transcription For RNA immunopreciptation, the nuclear extract was incubated whit 50M-5g of monoclonal C1A9 anti HP1 antibody and the mixture was kept at 4M-0C overnight under continuous gentle movement. One-hundred microliters ofprotein G-Sepharose (Sigma) suspension (50% packed Sepharose in Buffer C) was added and the incubation was continued overnight as described.The beads were pelleted by 2 min centrifugation at 240 g at 4M-0C; the pellet was washed briefly three times whit each 1mL of IP Wash Solution (150mM NaCl, 50mM Tris pH 7.5, 0.5% NP40) and the Sepharose was transferred for eluition into a fresh plastic tube , pelleted again and then the supernatant was removed completely. To elute the immunocomplexes for protein analysis, an aliquot of beads were suspended in 50M-5L SDS-PAGE sample buffer and incubated for 10 min at 90M-0C; following centrifugation the supernatant was removed and used for Western blot for testing the presence of HP1 protein. To elute the immunoprecipitated RNAs, the pelleted beads were boiled in 200M-5L of DEPC wather for 5 min, spun, and the supernatant recovered; 1mL of Trizol (Invitrogen) was added to 200 M-5L of supernatant and mixed well, followed by the addition of 200M-5L of chloroform. This mixture was incubated at 4M-0C for 5 min and then centrifuged at 12000 g for 15 min; the RNAs in the aqueous phase was precipitated whit half volume of isopropanol; after precipitation, the RNAs was resuspend in 10M-5L of DEPC wather. Contaminating DNA was digested whit RNase-free DNase I (Sigma). The RNA purified from the previous step is used as a template to synthesize cDNA using oligo dT , random hexamers and SuperScript reverse transcriptase III (Invitrogen) according to the manufacturer's protocol. cDNA amplification and labeling The cDNA was used as template for a two-step random PCR amplification; in Round A, Sequenase is used to extend randomly annealed primers (Primer A) to generate templates for subsequent PCR; during Round B, the specific primer B is used to aplify the templates previously generated and finally round C consist of additional PCR cycles to inorporate the amino allyl dUTP nucleotide. About 25 ng of each cDNA sample was used for two 8 min extension with 2,7 mM Round A primer (5M-^R-GTT TCC CAG TCA CGA TCN NNN NNN NN-3M-^R, N being a mixture of all four nucleotides with 60% A+T and 40% G+C) at 37M-0C with 267U/ml Sequenase version 2.0 (usb). DNA was denatured at 94M-0C for 2 min and cooled to 10M-0C , and Sequenase 2.0 was added between extensions. 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Microarrays Hybridization and washing The dried labeled cDNAs pellet were resuspended in 80M-5l of DIG Easy Hyb (Roche) hybridization buffer containg 0,5 mg/ml yeast tRNa and 0,5 mg/ml salmon sperm DNA. Finally the DNA probe was denaturated by incubation at 65M-0C for 10 min and, after a brief centrifugation to spin down the drops, the mixture was pipetted onto a microarray; a coverslip was applied and the slide was placed in a microarray hybridation chamber (BioRad) and incubated overnight at 37M-0C. After hybridization, the slide was submerged in 0,01% SDS, 1%SSC until the coverslip slipped off the surface; The slide was transferred to a solution of 1%SSC and shaken at 50M-0C for 10 min, then washed once more by shaking in 0,1%SSC. Slide was dried by centrifugation for 3 min at 550 rpm and scanned with an ScanArray Lite Microarray Scanner (Packard Bioscience) with laser intensities chosen to maximize signals while avoiding pixel saturation. ScanArray express softwere was used to quantify hybridation signals; bad spots were flagged automatically by softwere and subsequently each slide was inspected manually.

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Microprocessor mediates transcription termination in long noncoding microRNA genes

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