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| Status |
Public on Jan 01, 2023 |
| Title |
Alternative splicing outcomes across an RNA-binding protein concentration gradient |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Alternative splicing (AS) is a dynamic RNA processing step that produces multiple RNA isoforms from a single pre-mRNA transcript and contributes to the complexity of the cellular transcriptome and proteome. This process is regulated through a network of cis-regulatory sequence elements and trans-acting factors, most-notably RNA binding proteins (RBPs). The muscleblind-like (MBNL) and RNA binding fox-1 homolog (RBFOX) are two well characterized families of RBPs that regulate fetal to adult AS transitions critical for proper muscle, heart, and central nervous system development. To better understand how the concentration of these RBPs influences AS transcriptome wide, we engineered an MBNL1 and RBFOX1 inducible HEK-293 cell line. We found that induction of exogenous RBFOX1 modulated AS outcomes, despite significant levels of endogenous RBFOX1 and RBFOX2 expression. We characterized AS outcomes by RNAseq following dose-dependent MBNL1 induction to generate transcriptome wide dose-response curves of skipped exon events. Analysis of 43 validated splicing events by RT-PCR revealed a spectrum of dose-response splicing curves with a 2-fold range for EC50 values and slope values ranging from 4 to 10. Analysis of this data suggests that exclusion events, when compared to inclusion events, regulated by MBNL1, may require higher protein concentrations to regulate, and that multiple arrangements of YGCY motifs produce similar splicing outcomes. Together these data suggest that rather than a simple association between the configuration of RBP binding sites and a specific splicing outcome, that a complex network governs both AS inclusion and exclusion events that operates in an RBP dose-dependent manner.
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| Overall design |
Alternative splicing analysis of RNAseq data using 6 difference concentrations of induced HA-MBNL1 protein in a HEK293 cell line
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| Contributor(s) |
Ellis JA, Hale MA, Wang ET, Berglund JA |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Dec 28, 2022 |
| Last update date |
Jan 01, 2023 |
| Contact name |
Joseph Anthony Ellis |
| E-mail(s) |
j.ellis@ufl.edu, joeellis.2013@gmail.com, jaellis@albany.edu
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| Organization name |
University of Florida
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| Department |
Biochemistry and Molecular Biology
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| Lab |
JA Berglund
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| Street address |
1200 Newell Drive
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| City |
Gainesville |
| State/province |
FL |
| ZIP/Postal code |
32610 |
| Country |
USA |
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| Platforms (1) |
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| Samples (18)
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| GSM6900476 |
HEK293 cell, Doxycyline 0 ng/ml, rep1 |
| GSM6900477 |
HEK293 cell, Doxycyline 100 ng/ml, rep1 |
| GSM6900478 |
HEK293 cell, Doxycyline 170 ng/ml, rep1 |
| GSM6900479 |
HEK293 cell, Doxycyline 250 ng/ml, rep1 |
| GSM6900480 |
HEK293 cell, Doxycyline 280 ng/ml, rep1 |
| GSM6900481 |
HEK293 cell, Doxycyline 1000 ng/ml, rep1 |
| GSM6900482 |
HEK293 cell, Doxycyline 0 ng/ml, rep2 |
| GSM6900483 |
HEK293 cell, Doxycyline 100 ng/ml, rep2 |
| GSM6900484 |
HEK293 cell, Doxycyline 170 ng/ml, rep2 |
| GSM6900485 |
HEK293 cell, Doxycyline 250 ng/ml, rep2 |
| GSM6900486 |
HEK293 cell, Doxycyline 280 ng/ml, rep2 |
| GSM6900487 |
HEK293 cell, Doxycyline 1000 ng/ml, rep2 |
| GSM6900488 |
HEK293 cell, Doxycyline 0 ng/ml, rep3 |
| GSM6900489 |
HEK293 cell, Doxycyline 100 ng/ml, rep3 |
| GSM6900490 |
HEK293 cell, Doxycyline 170 ng/ml, rep3 |
| GSM6900491 |
HEK293 cell, Doxycyline 250 ng/ml, rep3 |
| GSM6900492 |
HEK293 cell, Doxycyline 280 ng/ml, rep3 |
| GSM6900493 |
HEK293 cell, Doxycyline 1000 ng/ml, rep3 |
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| Relations |
| BioProject |
PRJNA916473 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE221838_d0_v_d1000_SE.MATS.JCEC.txt.gz |
4.2 Mb |
(ftp)(http) |
TXT |
| GSE221838_d0_v_d100_SE.MATS.JCEC.txt.gz |
4.0 Mb |
(ftp)(http) |
TXT |
| GSE221838_d0_v_d170_SE.MATS.JCEC.txt.gz |
4.3 Mb |
(ftp)(http) |
TXT |
| GSE221838_d0_v_d250_SE.MATS.JCEC.txt.gz |
4.1 Mb |
(ftp)(http) |
TXT |
| GSE221838_d0_vs_d280_SE.MATS.JCEC.txt.gz |
4.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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