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MADS: a New and Improved Method for Analysis of Differential Alternative Splicing by Exon-tiling Microarrays

ABSTRACT: We describe a method, MADS (Microarray Analysis of Differential Splicing), for discovery of differential alternative splicing from exon tiling microarrays. MADS incorporates a series of low-level analysis algorithms motivated by the “probe-rich” design of exon arrays, including background correction, iterative probe selection, and removal of sequence-specific cross-hybridization to off-target transcripts. We used MADS to analyze Affymetrix Exon 1.0 array data on a mouse neuroblastoma cell line after shRNA-mediated knockdown of the splicing factor PTB. From a list of exons with pre-determined inclusion/exclusion profiles in response to PTB depletion, MADS recognized all exons known to have large changes in transcript inclusion levels, and offered improvement over Affymetrix’s analysis procedure. We also identified numerous novel PTB-dependent splicing events. 30 novel events were tested by RT-PCR, and 27 were confirmed. This work demonstrates that the exon tiling microarray design is an efficient and powerful approach for global, unbiased analysis of pre-mRNA splicing. Keywords: control / knockdown comparison

ORGANISM(S): Mus musculus

PROVIDER: GSE11344 | GEO | 2008/05/09

SECONDARY ACCESSION(S): PRJNA106557

REPOSITORIES: GEO

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MADS: a New and Improved Method for Analysis of Differential Alternative Splicing by Exon-tiling Microarrays

Project description:We describe a method, MADS (Microarray Analysis of Differential Splicing), for discovery of differential alternative splicing from exon tiling microarrays. MADS incorporates a series of low-level analysis algorithms motivated by the “probe-rich” design of exon arrays, including background correction, iterative probe selection, and removal of sequence-specific cross-hybridization to off-target transcripts. We used MADS to analyze Affymetrix Exon 1.0 array data on a mouse neuroblastoma cell line after shRNA-mediated knockdown of the splicing factor PTB. From a list of exons with pre-determined inclusion/exclusion profiles in response to PTB depletion, MADS recognized all exons known to have large changes in transcript inclusion levels, and offered improvement over Affymetrix’s analysis procedure. We also identified numerous novel PTB-dependent splicing events. 30 novel events were tested by RT-PCR, and 27 were confirmed. This work demonstrates that the exon tiling microarray design is an efficient and powerful approach for global, unbiased analysis of pre-mRNA splicing. Keywords: control / knockdown comparison Short hairpin knockdown of PTB in mouse N2A neuroblastoma cells was performed as described before (Boutz et al., 2007, Genes Dev 21:1636-1652). The efficiency of the PTB knockdown was monitored by western blot using PTB-NT primary antibody and Cy5 labeled secondary antibody (GE Life Sciences). The blots were imaged using Typhoon 9410 (GE Life Sciences). The band intensities were measured using ImageQuant and normalized to GAPDH. In all cases the efficiency of the knockdown was close to 80% (data not shown). We conducted Exon array profiling on RNAs from three shRNA-PTB treated samples and three mock-treated controls (using empty vectors).

2008-05-09 | E-GEOD-11344 | biostudies-arrayexpress

Quaking is a global regulator of muscle-specific alternative splicing in vertebrates [siRNA data]

Project description:This work provides the first evidence that Qk is a global regulator of splicing in vertebrates, defines a new splicing regulatory network in muscle, and suggests that overlapping splicing networks contribute to the complexity of changes in alternative splicing during differentiation. Alternative splicing contributes to muscle development and differentiation, but the complete set of muscle splicing factors and their combinatorial interactions are not known. Previously work identifies ACUAA (STAR motif) as an enriched sequence near muscle-specific alternative exons such as Capzb exon 9. We did mass spectrometry of proteins selected by wild type and mutant Capzb intron 9 RNA affinity chromatography, and identified Quaking (Qk), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We show that in myoblasts, Qk promotes inclusion of Capzb exon 9 in opposition to repression by PTB. Qk knockdown in myoblasts has little effect on transcript levels, but alters inclusion of 824 cassette exons whose adjacent intron sequences are enriched in ACUAA motifs. During differentiation to myotubes, Qk levels increase 2-3 fold, suggesting a mechanism for Qk-responsive exon regulation. We captured the PTB splicing regulatory network and intersected it with the Qk network, identifying overlap between the functions of Qk and PTB. Approximately 60% of exons whose inclusion is altered during myogenesis appear to be under control of one or both of these splicing factors in myoblasts. This series is the C2C12 Qk and PTB siRNA data. It is 12 arrays: 3 PTB siRNA arrays , 3 Qk siRNA arrays, and 6 mock siRNA arrays.

2013-06-05 | E-GEOD-40961 | biostudies-arrayexpress

Quaking is a global regulator of muscle-specific alternative splicing in vertebrates [siRNA data]

2013-06-05 | GSE40961 | GEO

Quaking is a global regulator of muscle-specific alternative splicing in vertebrates [differentiation data]

2013-06-05 | GSE40956 | GEO

Quaking is a global regulator of muscle-specific alternative splicing in vertebrates [differentiation data]

Project description:This work provides the first evidence that Qk is a global regulator of splicing in vertebrates, defines a new splicing regulatory network in muscle, and suggests that overlapping splicing networks contribute to the complexity of changes in alternative splicing during differentiation. Alternative splicing contributes to muscle development and differentiation, but the complete set of muscle splicing factors and their combinatorial interactions are not known. Previously work identifies ACUAA (STAR motif) as an enriched sequence near muscle-specific alternative exons such as Capzb exon 9. We did mass spectrometry of proteins selected by wild type and mutant Capzb intron 9 RNA affinity chromatography, and identified Quaking (Qk), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We show that in myoblasts, Qk promotes inclusion of Capzb exon 9 in opposition to repression by PTB. Qk knockdown in myoblasts has little effect on transcript levels, but alters inclusion of 824 cassette exons whose adjacent intron sequences are enriched in ACUAA motifs. During differentiation to myotubes, Qk levels increase 2-3 fold, suggesting a mechanism for Qk-responsive exon regulation. We captured the PTB splicing regulatory network and intersected it with the Qk network, identifying overlap between the functions of Qk and PTB. Approximately 60% of exons whose inclusion is altered during myogenesis appear to be under control of one or both of these splicing factors in myoblasts. This series is the C2C12 differentiation data. It is 9 arrays, 3 timepoints, with 3 replicates. The time points are 0 hrs, 24 hrs, and 72hrs.

2013-06-05 | E-GEOD-40956 | biostudies-arrayexpress

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (HJAY)

Project description:To gain global insights into the role of the well-known repressive splicing regulator PTB we analyzed the consequences of PTB knockdown in HeLa cells using high-density oliogonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB repressed and activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons, but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTBactivated to a PTB-repressed exon. Our results demonstrate that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons.

2010-09-01 | GSE23513 | GEO

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (Exon array)

2010-09-01 | GSE23514 | GEO

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (HJAY)

2010-09-01 | E-GEOD-23513 | biostudies-arrayexpress

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (Exon array)

2010-09-01 | E-GEOD-23514 | biostudies-arrayexpress

PTB CLIP-seq

Project description:Recent transcriptome analysis indicates that >90% of human genes undergoes alternative splicing, underscoring the contribution of differential RNA processing to diverse proteomes in higher eukaryotic cells. The polypyrimidine tract binding protein PTB is a well-characterized splicing repressor, but PTB knockdown causes both exon inclusion and skipping. Genome-wide mapping of PTB-RNA interactions and construction of a functional RNA map now revealed that dominant PTB binding near a competing constitutive splice site generally induces exon inclusion whereas prevalent binding close to an alternative site often causes exon skipping. This positional effect was further demonstrated by disrupting or creating a PTB binding site on minigene constructs and testing their responses to PTB knockdown or overexpression. These findings suggest a mechanism for PTB to modulate splice site competition to produce opposite functional consequences, which may be generally applicable to RNA binding splicing factors to positively or negatively regulate alternative splicing in mammalian cells.

2009-12-24 | GSE19323 | GEO

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