SplicerEX: A tool for the automated detection and classification of mRNA changes from conventional and splice-sensitive microarray expression data

Abstract

The key postulate that one gene encodes one protein has been overhauled with the discovery that one gene can generate multiple RNA transcripts through alternative mRNA processing. In this study, we describe SplicerEX, a novel and uniquely motivated algorithm designed for experimental biologists that (1) detects widespread changes in mRNA isoforms from both conventional and splice sensitive microarray data, (2) automatically categorizes mechanistic changes in mRNA processing, and (3) mitigates known technological artifacts of exon array-based detection of alternative splicing resulting from 59 and 39 signal attenuation, background detection limits, and saturation of probe set signal intensity. In this study, we used SplicerEX to compare conventional and exon-based Affymetrix microarray data in a model of EBV transformation of primary human B cells. We demonstrated superior detection of 39-located changes in mRNA processing by the Affymetrix U133 GeneChip relative to the Human Exon Array. SplicerEX-identified exon-level changes in the EBV infection model were confirmed by RT-PCR and revealed a novel set of EBV-regulated mRNA isoform changes in caspases 6, 7, and 8. Finally, SplicerEX as compared with MiDAS analysis of publicly available microarray data provided more efficiently categorized mRNA isoform changes with a significantly higher proportion of hits supported by previously annotated alternative processing events. Therefore, SplicerEX provides an important tool for the biologist interested in studying changes in mRNA isoform usage from conventional or splicesensitive microarray platforms, especially considering the expansive amount of archival microarray data generated over the past decade. SplicerEX is freely available upon request.

Epstein-Barr Virus Induces Global Changes in Cellular mRNA Isoform Usage That Are Important for the Maintenance of Latency

“…B cells were purified from healthy donor buffy coats through the Carolina Red Cross and Gulf Coast Regional Blood Center and transformed to monoclonal LCLs as described previously (26). BL41 is an EBV-negative Burkitt's lymphoma cell line.…”

“…Importantly, there was substantial overlap in mRNA abundance changes among commonly detected genes on these two platforms (26). EBV infection changed 433 cellular mRNAs at the level of isoform usage, with 271 of these events detected on the HuEx array and 162 detected on the U133 array ( Fig.…”

“…Infection of primary B cells by EBV dramatically reorganizes the cellular transcriptome via multiple mechanisms, ranging from transcriptional activation, effects on RNA stability, and alternative splicing (3,6,26,30). While most studies have focused on EBV-induced mRNA abundance changes, we used the primary B-cell infection system to assay the effects of EBV on mRNA isoform usage.…”

“…Transcript isoform changes were analyzed using two microarray platforms: the U133 Plus 2.0 array and the Human Exon 1.0 ST array. U133 arrays predominantly detect the 3= untranslated regions (UTRs) of mRNAs, while HuEx arrays detect exons throughout the entirety of mRNAs, and the combined use of these two platforms provides better coverage of all events occurring across entire transcripts (26,31). HuEx arrays detect nearly every exon in the genome, with 56,598 probe sets querying 8,006 expressed genes, with an average of 7 exons being interrogated per gene.…”