Co-regulation of alternative splicing by diverse splicing factors in Caenorhabditis elegans

Abstract

Regulation of alternative splicing is controlled by pre-mRNA sequences (cis-elements) and trans-acting protein factors that bind them. The combinatorial interactions of multiple protein factors with the cis-elements surrounding a given alternative splicing event lead to an integrated splicing decision. The mechanism of multifactorial splicing regulation is poorly understood. Using a splicing-sensitive DNA microarray, we assayed 352 Caenorhabditis elegans alternative cassette exons for changes in embryonic splicing patterns between wild-type and 12 different strains carrying mutations in a splicing factor. We identified many alternative splicing events that are regulated by multiple splicing factors. Many splicing factors have the ability to behave as splicing repressors for some alternative cassette exons and as splicing activators for others. Unexpectedly, we found that the ability of a given alternative splicing factor to behave as an enhancer or repressor of a specific splicing event can change during development. Our observations that splicing factors can change their effects on a substrate during development support a model in which combinatorial effects of multiple factors, both constitutive and developmentally regulated ones, contribute to the overall splicing decision.

Figure 1.

Hierarchical clustering of splicing factors mutant strains. Isoform ratios for all 352 alternative splicing events included in the microarray were used to cluster the different mutant strains.

Figure 2.

RT–PCR validations of splicing changes for alternative splicing events with high co-regulation values; (A) F42G9.6, (B) pqn-52, (C) C06A6.4, (D) lec-3 and (E) hrpf-1. An Agilent Bioanalyzer 2100 was used to quantitate the experimental results, error bars are shown representing results from at least two different RT–PCR reactions, and an electropherogram for each experiment is shown.

Figure 3.

Developmental changes in splicing regulation. RT–PCRs with embryo and adult samples were performed for C06A6.4 (A) and F42G9.6 (B) using mRNA samples from splicing factor mutant strains identified as regulators of embryonic splicing.

Figure 4.

Developmental changes in expression levels for splicing factors. Detection by quantitative RT–PCR of expression changes during development for five splicing factors. Housekeeping genes gpd-2 and rps-1 levels were used to normalize RNA levels between embryos and adults, and the log₂ ratio of the comparison for each factor is displayed.