Quantitative microarray profiling provides evidence against widespread coupling of alternative splicing with nonsense-mediated mRNA decay to control gene expression

Abstract

Sequence-based analyses have predicted that ∼35% of mammalian alternative splicing (AS) events produce premature termination codon (PTC)-containing splice variants that are targeted by the process of nonsense-mediated mRNA decay (NMD). This led to speculation that AS may often regulate gene expression by activating NMD. Using AS microarrays, we show that PTC-containing splice variants are generally produced at uniformly low levels across diverse mammalian cells and tissues, independently of the action of NMD. Our results suggest that most PTC-introducing AS events are not under positive selection pressure and therefore may not contribute important functional roles. Alternative splicing (AS), the process by which exons in transcripts are joined in different combinations to generate multiple mRNA variants, represents an important mechanism for the expression of structurally and functionally distinct proteins from a limited number of genes (Graveley 2001;Black 2003). Regulation of AS plays critical roles in cell growth, differentiation, and cell death, and aberrant AS has been implicated in many human diseases (Smith and Valcarcel 2000;Caceres and Kornblihtt 2002;Cartegni et al. 2002). It has been estimated that at least 74% of human genes contain one or more alternative exons (Johnson et al. 2003), yet it is not known to what extent the resulting splice variants specify functionally relevant transcripts and proteins.Previous studies have shown that the introduction of premature termination codons (PTCs) in spliced transcripts can activate transcript degradation via the process of nonsense-mediated mRNA decay (NMD) (Hillman et al. 2004;Maquat 2004;Alonso 2005). NMD is important for the removal of nonfunctional PTC-containing transcripts (Mendell et al. 2004;Mitrovich and Anderson 2005). It has also been shown to function in the autoregulation of transcript levels of several RNAbinding proteins, including splicing factors (Morrison et al. 1997;Sureau et al. 2001;Wollerton et al. 2004; for review, see Lareau et al. 2004). In addition to a PTC, NMD requires a set of upstream frameshift (UPF) protein factors that associate with the spliced transcripts via interactions with a post-splicing exon junction complex Recent bioinformatics analyses of expressed sequence tag (EST) and cDNA sequence data have predicted that ∼35% of AS events have the potential to introduce PTCs that could elicit NMD of transcripts Lewis et al. 2003). This finding led to speculation that NMD activated by AS may represent a widely used mechanism by which gene expression is down-regulated (Hillman et al. 2004;Neu-Yilik et al. 2004;Alonso 2005;Lejeune and Maquat 2005), and it has also been proposed that the introduction of PTCs by AS serves as a mechanism for the tissue-specific regulation of gene expression (Hillman et al. 2004;Holbrook et al. 2004;Raes and Van de Peer 2005). However, no study has yet examined experimentally the actual levels of PTC-containing mRNA splice variants in normal cells and tissues, and it is not known to what extent AS f...

Intron retention generates ANKRD1 splice variants that are co-regulated with the main transcript in normal and failing myocardium

“…As previously suggested, NMD activated by alternative splicing events can represent a mechanism by which expression of a given gene could be down-regulated in a tissue-restricted manner (Alonso, 2005). Other observations indicate, however, that the majority of PTC-containing transcripts generated by alternative splicing are detected at uniformly low abundance levels across mammalian tissues, independently of the NMD action (Pan et al, 2006). Our data contribute, to some extent, to the current knowledge of this issue.…”

Introns in UTRs: Why we should stop ignoring them

“…Given that mRNAs containing 3UIs are surprisingly common (an estimated 35% of alternatively spliced isoforms [42] plus those with uORFs and constitutive 3UIs; see above), the challenge is to distinguish functional 3UIs from those representing genomic noise or cellular errors [57]. Two potential indicators of function are conservation and tissue-specific expression.…”

“…Among alternative splicing events conserved between human and mouse, approximately 21% introduce termination codons upstream of introns [57,63]. This estimate, based on traditional transcriptomics, was recently re-examined by an in-depth analysis of the 309 protein coding genes within the ENCODE pilot phase regions of the human genome [64].…”

“…To address the question of tissuespecificity of 3UI-containing transcripts, Pan et al [57] undertook a comprehensive analysis of alternatively spliced 3UI-containing isoforms across ten different mouse tissues. Using exon and splice junction arrays to examine inclusion and exclusion of cassette exons, they found little evidence of tissue-specific expression of NMD isoforms.…”

Analysis of Alternatively Spliced Rice Transcripts Using Microarray Data

“…However, the high cost of such experiments is a deterrent to assaying expression in various tissues or under various conditions. As most alternative splice forms are expressed at low levels across various tissues, researchers need to develop an efficient strategy to characterize alternative splicing [26,34].…”