“Public archives of next-generation sequencing data are growing exponentially, but the difficulty of marshaling this data has led to its underutilization by scientists. Here we present ASCOT, a resource that allows researchers to summarize, visualize, and query alternative splicing patterns in public RNA-Seq data. ASCOT enables rapid identification of splice-variants across tens of thousands of bulk and single-cell RNA-Seq datasets in human and mouse. To demonstrate the utility of ASCOT, we first focused on the nervous system and identified many alternative exons used only by a single neuronal subtype. We then leveraged datasets from the ENCODE and GTEx consortiums to study the unique splicing patterns of rod photoreceptors and found that PTBP1 knockdown combined with overexpression of MSI1 and PCBP2 activates rod-specific exons in HepG2 liver cancer cells. Furthermore, we observed that MSI1targets intronic UAG motifs proximal to the 5’ splice site and interacts synergistically with PTBP1 downregulation. Finally, we show that knockdown of MSI1 in the retina abolishes rod-specific splicing. This work demonstrates how large-scale analysis of public RNA-Seq datasets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events.”
ASCOT splicing and gene expression data tables, software, and interactive browser are available at http://ascot.cs.jhu.edu.
A) Mouse RNA-Seq datasets were manually curated from the SRA, covering a broad range of cell types and organs. Cell type datasets were generated from various independent labs using FACS or affinity isolation.
B-D) Our splicing analysis method reliably identifies alternative exons that are unique to specific cell types.
A) Importantly, we can identify alternative exons that are only spliced in retina.
B) Bigwig visualizations of raw GTEx RNA-Seq data.
C) Comparing rod-specific exons in mouse with retina-specific exons in human yields a set of 31 syntenic exons that are likely important for photoreceptor function.
A) UCSC visualization of rod-specific exons, both conserved (BSG, IMPDH1, MAN2A2, PPP3CC, TNRC6A) and nonconserved (RDX, GDAP1, MTRR)
B) Electroporation of Msi1 shRNA in mouse retina abolishes nearly all rod-specific splicing (Atp1b2, Efr3a, Cask).
C) Model for the regulation of photoreceptor-specific splicing
Jonathan P Ling, Christopher Wilks, Rone Charles, Devlina Ghosh, Lizhi Jiang, Clayton P Santiago, Bo Pang, Anand Venkataraman, Brian S Clark, Abhinav Nellore, Ben Langmead, Seth Blackshaw. ASCOT identifies key regulators of neuronal subtype-specific splicing.
doi: https://doi.org/10.1101/501882
This article is a preprint and has not been peer-reviewed