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. 2009 Sep;15(9):1652-60.
doi: 10.1261/rna.1711109. Epub 2009 Jul 17.

Global analysis of alternative splicing uncovers developmental regulation of nonsense-mediated decay in C. elegans

Sergio Barberan-Soler  1 , Nicole J Lambert, Alan M Zahler
Affiliations

Global analysis of alternative splicing uncovers developmental regulation of nonsense-mediated decay in C. elegans

Sergio Barberan-Soler et al. RNA. 2009 Sep.

Abstract

Alternative splicing coupled to nonsense-mediated decay (AS-NMD) is a mechanism for post-transcriptional regulation of gene expression. We analyzed the global effects of mutations in seven genes of the C. elegans NMD pathway on AS isoform ratios. We find that mutations in two NMD factors, smg-6 and smg-7, have weaker global effects relative to mutations in other smg genes. We did an in-depth analysis of 12 pre-mRNA splicing factor genes that are subject to AS-NMD. For four of these, changes in the ratio of alternatively spliced isoforms during development are caused by developmentally regulated inhibition of NMD, and not by changes in alternative splicing. Using sucrose gradient analysis of mRNAs undergoing translation, we find several examples of NMD-dependent enrichment of premature termination codon (PTC) isoforms in the monosome fraction. In contrast, we present evidence of two genes for which the PTC-containing isoforms are found in polysomes and have a translational profile similar to non-PTC-containing transcripts from the same gene. We propose that NMD of certain alternatively spliced isoforms is regulated, and that some stabilized NMD targets may be translated.

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Figures

FIGURE 1.
FIGURE 1.
Hierarchical clustering of AS-NMD targets in embryos. Embryonic isoform ratios (emb-IR) ≥ ±1.0 in at least one NMD mutant were separated according to whether their PTC was generated by cassette exon inclusion or skipping. Genes with any annotation correlating them to splicing are labeled.
FIGURE 2.
FIGURE 2.
RT-PCR validation of embryonic splicing of all splicing factors with high emb-IR in NMD mutants. (A) RT-PCRs for PTC-upon-inclusion splicing factors in N2 and smg-1 samples; (B) RT-PCR for PTC-upon-skipping splicing factors in N2 and smg-1 samples; (C) RT-PCR of two previously reported splicing factors regulated by AS-NMD but not identified in the present microarray analysis in N2 and smg-1 samples; and (D) Graphic display of RT-PCR data for all 11 splicing factors identified as embryonically regulated by AS-NMD. The fraction of steady-state messages from each gene containing the PTC as determined for N2 and seven smg mutant strains are shown.
FIGURE 3.
FIGURE 3.
Developmental regulation of AS-NMD. (A) RT-PCR comparison of wild-type and NMD mutant worms for two developmental time points (embryo and young adults) for 12 splicing factors identified as developmentally regulated by AS-NMD, error bars represent the standard deviation of two independent RNA isolations and RT-PCRs; (B,C) agarose gels of RT-PCRs for swp-1 and asd-1 showing developmental regulation of AS-NMD.
FIGURE 4.
FIGURE 4.
NMD-dependent enrichment of the PTC isoform in monosome fractions. (A–C) The continuous monitoring of the elution profile of sucrose gradient fractions by absorbance at 254 nm. A is the profile for fractionated N2 extract, B is fractionated smg-2 mutant extract, and C is fractionated N2 extract that was pretreated with EDTA to disrupt the ribosomes. (D–F) The relative IR across all nine fraction obtained from the sucrose gradient, in N2, smg-2 and N2+EDTA samples, respectively.
FIGURE 5.
FIGURE 5.
Model for AS-NMD regulation. (1) Strong NMD, PTC-containing isoforms are effectively recognized and degraded with insignificant translation accomplished; (2) inefficient NMD, PTC-containing isoforms are not effectively recognized but a SMG-2-dependent translation repression makes translation of particular transcripts insignificant; and (3) regulated NMD, PTC-containing isoforms are differentially recognized during development, potentially through regulatory cis-elements and trans-factors. The translational profile of these PTC-containing mRNAs is comparable to that of their non-PTC alternative isoforms, leading to the translation of truncated proteins with the potential of acting as dominant negative regulators of splicing.
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