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| Status |
Public on Oct 01, 2018 |
| Title |
Comparison of PRPF4 knockdown in HEK293T cells compared to scrambled shRNA control |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
HEK293T cells transduced with two shRNAs from MISSION library (TRCN0000364755 and TRCN0000074769) using lentiviral delivery system. HEK293T cells transduced with scrambled shRNA, gifted from Dr. Mauricio Reginato. Sequence is 5′-CCTAAGGTTAAGTCGCCCTCGCTCTAGCGAGGGCGACTTAACCTT-3′. Primary contributions to creation of cell lines, preparation of RNA/cDNA for microarray, and validation of results were from Tara L Davis, S. RaElle Jackson, Beth Adams, Anh Trinh, Nicholas Fox, Jessica Kopenhaver, Alyson Hurlock, and Achraf Jardaly, all Drexel University College of Medicine, Philadephia PA, 19102. Hetty Rodriguez and John Tobias, affiliated with the Molecular Profiling Facility and Genomic Analysis Core Bioinformatics Group at the University of Pennsylvania, Philadelphia PA, performed Bioanalyzer and microarray expreriments and initial data processing.
pre-mRNA Processing Factor 4 (PRPF4) is a scaffolding protein, consisting of two N-terminal intrinsically disordered motifs and a C-terminal WD-40 domain. PRPF4 associates with the human spliceosome, the complex and dynamic machinery that removes intronic sequence from pre-messenger RNA (pre-mRNA). Although PRPF4 affects the ability of the spliceosome to assemble and catalyze splicing in vitro through de-stabilization of the tri-snRNP complex, little is known about what PRPF4 does to regulate transcription and splicing in vivo. To understand the function of PRPF4 in the nucleus, we knocked down PRPF4 in human cells. We characterized a set of alternative splicing and transcriptional events that are PRPF4-responsive. We used these splicing and transcriptional bioassays to show that PRPF4-responsive events are largely specific. The development of a bioassay for PRPF4 function can be used to answer fundamental questions about the role of spliceosomal proteins in regulating splicing and other nuclear functions.
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| Overall design |
3 replicates of PRPF4 knockdown cell lines in HEK293T cells, stably integrated, selected using puro, shRNAs TRCN0000364755 sequence 5'-CCGGCCACGAACTGTGTAGACATTGCTCGAGCAATGTCTACACAGTTCGTGGTTTTTG-3' and TRCN0000074769 sequence 5'-CCGGGCTCTCTCTAAGGAGCTGTTTCTCGAGAAACAGCTCCTTAGAGAGAGCTTTTTG-3'. 3 replicates of SCR control cell lines in HEK293T cells, stably integrated, selected using puro, sequence 5′-CCTAAGGTTAAGTCGCCCTCGCTCTAGCGAGGGCGACTTAACCTT-3′. Total RNA purified from ~1 million cells, cDNA converted using random hexamer primers, quantity measured using NanoDrop, quality quantified using BioAnaylzer). Affymatrix HTA2.0 array
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| Contributor(s) |
Davis TL, Jackson SR, Adams B, Trinh A, Fox N, Kopenhaver J, Hurlock A, Jardaly A, Rodriguez H, Tobias J |
| Citation missing |
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| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R00 GM094293 |
Functional and structural characterization of spliceosomal cyclophilins |
DREXEL UNIVERSITY |
Tara L Davis |
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| Submission date |
Jul 19, 2018 |
| Last update date |
Oct 29, 2018 |
| Contact name |
Tara Davis |
| E-mail(s) |
Tara.Davis@DrexelMed.edu
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| Organization name |
Drexel University College of Medicine
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| Department |
Biochemistry and Molecular Biology
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| Lab |
Lab 10127
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| Street address |
245 N. 15th St. MS 497
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19102 |
| Country |
USA |
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| Platforms (2) |
| GPL17585 |
[HTA-2_0] Affymetrix Human Transcriptome Array 2.0 [probe set (exon) version] |
| GPL17586 |
[HTA-2_0] Affymetrix Human Transcriptome Array 2.0 [transcript (gene) version] |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA481964 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE117384_RAW.tar |
392.5 Mb |
(http)(custom) |
TAR (of CEL, CHP) |
| Processed data included within Sample table |
| Processed data provided as supplementary file |
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