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| Status |
Public on Sep 03, 2023 |
| Title |
Regulation of the Drosophila transcriptome by Pumilio and CCR4-NOT deadenylase |
| Organism |
Drosophila melanogaster |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
The sequence-specific RNA-binding protein Pumilio controls development of Drosophila; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of Drosophila cells. We also used an improved RNA co-immunoprecipitation method to identify Pumilio bound mRNAs in Drosophila embryos. Integration of these datasets with the content of Pumilio binding motifs across the transcriptome revealed novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. Additionally, we identified the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observed concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling revealed that Pumilio binding site location, number, density, and context are important determinants of regulation. Moreover, the content of optimal synonymous codons exhibits a striking functional relationship to Pumilio and CCR4-NOT regulation, indicating that the inherent translation efficiency and stability of the mRNA modulates their response to these trans-acting regulatory factors. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.
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| Overall design |
Differential gene expression was measured by performing RNA Seq using ClickSeq library generation on polyA selected RNAs isolated from Drosophila melanogaster DL1 cells. The effect of RNAi induced knockdown of Pumilio, Not1, or Pop2 were compared to negative control samples treated with dsRNA (non-targeting control) coresponding to e. coli LacZ gene. Three biological replicates were sequenced per RNAi condition. Additionally, the Pumilio gene was knocked out in two clonal isolates of DL1 cells and the resulting changes in RNA levels were measured relative to the parental wild type DL1 cell line by ClickSeq.
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| Contributor(s) |
Haugen R, Barnier C, Elrod N, Luo H, Jensen M, Ji P, Smibert C, Lipshitz H, Wagner E, Freddolino PL, Goldstrohm A |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Aug 30, 2023 |
| Last update date |
Sep 03, 2023 |
| Contact name |
Aaron C Goldstrohm |
| E-mail(s) |
agoldstr@umn.edu
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| Phone |
7349450789
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| Organization name |
University of Minnesota
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| Department |
Biochemistry, Molecular Biology, and Biophysics
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| Lab |
6-155 Jackson Hall, 1214A
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| Street address |
321 Church Street S.E.
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| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55455 |
| Country |
USA |
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| Platforms (1) |
| GPL19132 |
Illumina NextSeq 500 (Drosophila melanogaster) |
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| Samples (21)
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| Relations |
| BioProject |
PRJNA1010785 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE241940_Haugen_2023_DeSeq2_Processed_Data.xlsx |
8.2 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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