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| 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
Other
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| 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.
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| 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).
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| Contributor(s) |
Reimão-Pinto MM, Manzenreither RA, Burkard TR, Sledz P, Jinek M, Ameres SL |
| Citation(s) |
27729457 |
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| Submission date |
Jul 15, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Thomas Rainer Burkard |
| E-mail(s) |
thomas.burkard@imba.oeaw.ac.at
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| Organization name |
IMBA
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| Street address |
Dr. Bohrgasse 7
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platforms (2) |
| GPL17275 |
Illumina HiSeq 2500 (Drosophila melanogaster) |
| GPL19604 |
Illumina HiSeq 2500 (synthetic construct) |
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| Samples (15)
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| 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 |
| GSM2236746 |
In vitro degradation of randomized RNA substrate_15485 |
| GSM2236747 |
In vitro degradation of randomized RNA substrate_16556 |
| GSM2236748 |
In vitro degradation of randomized RNA substrate_16557 |
| GSM2236749 |
In vitro degradation of randomized RNA substrate_16558 |
| GSM2236750 |
In vitro degradation of randomized RNA substrate_16559 |
| GSM2236751 |
In vitro degradation of randomized RNA substrate_16546 |
| GSM2236752 |
In vitro degradation of randomized RNA substrate_16547 |
| GSM2236753 |
In vitro degradation of randomized RNA substrate_16548 |
| GSM2236754 |
In vitro degradation of randomized RNA substrate_16549 |
| GSM2236755 |
Small RNA cloning of 10-20 nt FLAG-dmDis3l2-CM-associated RNA_32613 |
| GSM2236756 |
Cloning of total RNA_39892 |
| GSM2236757 |
Cloning of FLAG-dmDis3l2-CM-associated RNA_39894 |
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| Relations |
| BioProject |
PRJNA329275 |
| SRA |
SRP078588 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE84466_RAW.tar |
44.7 Mb |
(http)(custom) |
TAR (of TXT, XLSX) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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