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. 2008 Feb 29;4(2):e1000001.
doi: 10.1371/journal.pgen.1000001.

Alternative splicing regulation during C. elegans development: splicing factors as regulated targets

Sergio Barberan-Soler  1 , Alan M Zahler
Affiliations

Alternative splicing regulation during C. elegans development: splicing factors as regulated targets

Sergio Barberan-Soler et al. PLoS Genet. .

Abstract

Alternative splicing generates protein diversity and allows for post-transcriptional gene regulation. Estimates suggest that 10% of the genes in Caenorhabditis elegans undergo alternative splicing. We constructed a splicing-sensitive microarray to detect alternative splicing for 352 cassette exons and tested for changes in alternative splicing of these genes during development. We found that the microarray data predicted that 62/352 (approximately 18%) of the alternative splicing events studied show a strong change in the relative levels of the spliced isoforms (>4-fold) during development. Confirmation of the microarray data by RT-PCR was obtained for 70% of randomly selected genes tested. Among the genes with the most developmentally regulated alternatively splicing was the hnRNP F/H splicing factor homolog, W02D3.11 - now named hrpf-1. For the cassette exon of hrpf-1, the inclusion isoform comprises 65% of hrpf-1 steady state messages in embryos but only 0.1% in the first larval stage. This dramatic change in the alternative splicing of an alternative splicing factor suggests a complex cascade of splicing regulation during development. We analyzed splicing in embryos from a strain with a mutation in the splicing factor sym-2, another hnRNP F/H homolog. We found that approximately half of the genes with large alternative splicing changes between the embryo and L1 stages are regulated by sym-2 in embryos. An analysis of the role of nonsense-mediated decay in regulating steady-state alternative mRNA isoforms was performed. We found that 8% of the 352 events studied have alternative isoforms whose relative steady-state levels in embryos change more than 4-fold in a nonsense-mediated decay mutant, including hrpf-1. Strikingly, 53% of these alternative splicing events that are affected by NMD in our experiment are not obvious substrates for NMD based on the presence of premature termination codons. This suggests that the targeting of splicing factors by NMD may have downstream effects on alternative splicing regulation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Splicing sensitive microarray design.
(A) general outline of the probe design used to detect AS of cassette exons; probes were designed as follows: “alt probe” to detect the cassette exon and thus the inclusion isoform, “junction probe” to detect the splice junction formed in the skipping isoform, and at least two different constitutive probes, c1 and c2, to detect levels of gene expression. (B) AS ratio calculation; (C) loop design to compare all six different time points, each arrow correspond to a direct comparison including the dye-swap experiment (for example, embryo vs. L1 and L1 vs. embryo hybridizations were performed), for a total of 12 microarray hybridization experiments.
Figure 2
Figure 2. Cluster of top changes.
AS ratios showing greater than four-fold differences during development were clustered using hierarchical clustering and TreeView ,. Positive AS ratios correspond to cassette exons with higher inclusion in embryos, negative AS ratios correspond to higher inclusion in any of the other stages. A and B are two clusters of events with lower and higher inclusion in embryos respectively.
Figure 3
Figure 3. RT-PCR validations of microarray results.
From the group of 32 events with AS ratios changes greater than four-fold in development when compared to embryos, 14 events were randomly selected for further confirmation by RT-PCR. AS ratios obtained with the microarray (dark grey), are compared to AS ratios obtained by RT-PCR (light grey).
Figure 4
Figure 4. hrpf-1 alternative splicing.
(A) Gene model for AS of hrpf-1, two isoforms (a and b) are generated by either inclusion or skipping of exon 5; (B) Virtual gel of RT-PCR of hrpf-1 during C. elegans development as demonstrated on a Bioanalyzer (Agilent). The percentage of hrpf-1 messages that are spliced to include exon 5 are plotted; (C) RT-PCR of the hrpf-1 mRNA from total embryonic RNA from NMD defective worms (smg-1(r861)) compared to wild type embryo RNA. This experiment demonstrates that hrpf-1b is degraded by NMD; (D) regulation of alternative splicing of the C. briggsae hrpf-1 homolog (CBG04052) during development.
Figure 5
Figure 5. Alternative splicing regulation by hnRNP F/H splicing factors.
(A) Graphical representation of the structure of hnRNP H/F proteins in C. elegans; (B) RT-PCR of hrpf-1 and rnp-6 in N2 and sym-2(mn617) embryos. The fraction of mRNA showing inclusion of the alternative exon is indicated below each lane; (C) RT-PCR of hrpf-1 and lin-10 under hrpf-1(RNAi) and control RNAi conditions. Molar ratios of hrpf-1 and lin-10 mRNA levels were calculated using a Bioanalyzer 2100; (D) RT-PCR of top-1 under hrpf-1(RNAi) and control RNAi conditions. The fraction of mRNA showing inclusion of the alternative exon is indicated below each lane.
Figure 6
Figure 6. Correlation between steady-state mRNA levels and AS for hrpf-1.
Relative levels of hrpf-1 mRNA during development were derived from published microarray data and alternative splicing levels were determined from our microarray data in Table 1. By regulating the AS of hrpf-1, C. elegans is able to further down-regulate the functional transcripts of this gene encoding the full-length, three RRM isoform of this protein in L1 animals.
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