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Series GSE84466 Query DataSets for GSE84466
Status Public on Feb 10, 2017
Title Molecular basis for cytoplasmic RNA surveillance by uridylation-triggered decay in Drosophila
Organisms Drosophila melanogaster; synthetic construct
Experiment type Expression profiling by high throughput sequencing
Non-coding RNA profiling by high throughput sequencing
Summary The post-transcriptional addition of nucleotides to the 3´ end of RNA regulates the maturation, function, and stability of RNA species in all domains of life. Here, we show that, in flies, 3´ terminal RNA uridylation triggers the processive, 3´-to-5´ exoribonucleolytic decay via the RNase II/R enzyme CG16940, a homolog of the human Perlman syndrome exoribonuclease Dis3l2. Together with the TUTase Tailor, dmDis3l2 forms the cytoplasmic, uridylation-triggered RNA processing (TRUMP) complex, that functionally cooperates in the degradation of structured RNA. RNA-immunoprecipitation and high-throughput sequencing reveals a variety of TRUMP complex substrates, including abundant non-coding RNA, such as 5S rRNA, tRNA, snRNA, snoRNA, and the essential RNase MRP, uncovering a key function of the TRUMP complex in the cytoplasmic quality control of RNA polymerase III transcripts. Together with high-throughput biochemical characterization of dmDis3l2 and bacterial RNase R our results imply a conserved molecular function of RNase II/R enzymes as 'readers' of destabilizing post-transcriptional marks - uridylation in eukaryotes and adenylation in prokaryotes - that play important roles in RNA surveillance.
Overall design For biochemical high-throughput analysis of dmDis3l2 (Figure 2), input-libraries (15482) served as control and three timepoints after treatment (0.5 min = 15483, 2 min = 15484, 10 min = 15485) have been analyzed as described in detail in the manuscript. The libraries 16546 (Input), 16547 (0.5 min), 16547 (2 min) and 16549 (10 min) serve as replicates and have been used to generate Figure 7. For biochemical high-throughput analysis of ecoRNaseR (Figure 7), input-libraries (16556) served as control and three timepoints after treatment (5 sec = 16557, 10 sec = 16558, 30 sec = 16559) have been analyzed as described in detail in the manuscript. For sample 32613, RNA co-immunoprecipitating with FLAG-dmDis3l2-CM have been subjected to size-selection (10-20 nt) and subjected to small RNA cloning (for details see manuscript, Figure S5). For sample 39892, RNA co-immunoprecipitating with FLAG-dmeDis3l2-CM under optimized conditions (shorter incubation time and addition of EDTA) was subjected to 3 adapter ligation and library preparation using Lexogen FLEX kit (Lexogen, Vienna, Austria). Libraries were subjected to PE50 sequencing but only reverse run was analyzed, since it provided information on post-transcriptional modification status and seuqence identity, by mapping to the Drosophila genome (for details see manuscript, Figure 5). For sample 39894, total RNA was subjected to the same cloning protocol as sample 39892, hence serves as control regarding post-transcriptional modification status and 3 end accumulation in cellular total RNA (Figure 5).
Contributor(s) Reimão-Pinto MM, Manzenreither RA, Burkard TR, Sledz P, Jinek M, Ameres SL
Citation(s) 27729457
Submission date Jul 15, 2016
Last update date May 15, 2019
Contact name Thomas Rainer Burkard
Organization name IMBA
Street address Dr. Bohrgasse 7
City Vienna
ZIP/Postal code 1030
Country Austria
Platforms (2)
GPL17275 Illumina HiSeq 2500 (Drosophila melanogaster)
GPL19604 Illumina HiSeq 2500 (synthetic construct)
Samples (15)
GSM2236743 In vitro degradation of randomized RNA substrate_15482
GSM2236744 In vitro degradation of randomized RNA substrate_15483
GSM2236745 In vitro degradation of randomized RNA substrate_15484
BioProject PRJNA329275
SRA SRP078588

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Supplementary file Size Download File type/resource
GSE84466_RAW.tar 44.7 Mb (http)(custom) TAR (of TXT, XLSX)
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Raw data are available in SRA
Processed data provided as supplementary file

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