Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis

Abstract

Alternative splicing (AS) coupled to nonsense-mediated decay (NMD) is a post-transcriptional mechanism for regulating gene expression. We have used a high-resolution AS RT-PCR panel to identify endogenous AS isoforms which increase in abundance when NMD is impaired in the Arabidopsis NMD factor mutants, upf1-5 and upf3-1. Of 270 AS genes (950 transcripts) on the panel, 102 transcripts from 97 genes (32%) were identified as NMD targets. Extrapolating from these data around 13% of intron-containing genes in the Arabidopsis genome are potentially regulated by AS/NMD. This cohort of naturally occurring NMD-sensitive AS transcripts also allowed the analysis of the signals for NMD in plants. We show the importance of AS in introns in 5' or 3'UTRs in modulating NMD-sensitivity of mRNA transcripts. In particular, we identified upstream open reading frames overlapping the main start codon as a new trigger for NMD in plants and determined that NMD is induced if 3'-UTRs were >350 nt. Unexpectedly, although many intron retention transcripts possess NMD features, they are not sensitive to NMD. Finally, we have shown that AS/NMD regulates the abundance of transcripts of many genes important for plant development and adaptation including transcription factors, RNA processing factors and stress response genes.

Figure 1.

Analysis of alternatively spliced NMD substrates. (A) Schematic figure of RT–PCR panel analysis (see ‘Materials and Methods’ section). (B) Venn diagram of the number of transcripts which increase significantly in upf mutants and cycloheximide treatment. (C) Venn diagram of the number of genes with splice isoforms which increase significantly in upf mutants and cycloheximide treatment. (D) General features of transcripts which trigger NMD: (i) long 3′-UTR; (ii) PTC—long 3′-UTR; (iii) splice junction downstream of authentic stop codon (3′-UTR intron); (iv) PTC—downstream splice junctions and long 3′-UTR; (v) uORFs in 5′-UTR. Endogenous transcripts regulated by NMD contain long 3′-UTRs, introns in the 3′-UTR where the splice junction is >50–55 nt from the authentic stop or UORFs (i, iii and v, respectively). Transcripts which contain PTC in the coding region or 5′-UTR (uORF) also generate long 3′-UTRs with or without downstream splice junctions (ii, iv and v, respectively). Exons—open boxes; UTRs—black rectangles; thin lines—introns; diagonal lines—splicing events; stop sign—PTC or authentic termination codon.

Figure 2.

Regulation of GRP7 and GRP8 by alternative splicing and NMD. (A) GRP7 and (B) GRP8 are known to be regulated by AS/NMD. Figures show the GRP7 and GRP8 gene and transcript structures and the alternative splicing events in the introns: alternative 5′ splice sites generate AS isoforms which increase in abundance in the upf1-5 and upf3-1 mutants as illustrated on scans generated from the ABI 3730 data by GeneMapper (transcripts are arrowed). The significant increases in NMD-sensitive transcript abundance are shown in histograms of the ratio of normally spliced and alternatively spliced isoforms (shaded). Significance: ***P< 0.01; **0.01 > P < 0.05. For diagram key see legend to Figure 1.

Figure 3.

Genes with AS isoforms which increase in upf mutants. (A) At4g25500—SR protein gene, At-RS40. (B) At4g33060—cyclophilin 57, CYP57. (C) At4g02200—drought-induced protein gene, At-Di19-1. For all, the gene and transcript structures and relevant splicing events are shown. AS isoforms which increase in the upf mutants are labelled with arrows on ABI3730 scans and the ratios of transcripts are shown in histograms and significant increases are indicated. Significance: ***P < 0.01; **0.01 > P < 0.05. For diagram key see legend to Figure 1.

Figure 4.

Alternative splicing of introns in the 3′-UTR influences turnover of AS isoforms by NMD. Exon–intron structures of genes and transcripts of (A) At2g38880—NF-YB1 transcription factor, NF-YB1/HAP3a and (B) At1g72560—PSD/exportin-t. Alternative splicing of the 3′-UTR introns in these genes generate transcripts with different distances between the authentic stop codon and downstream splice junction consistent with the 50–55 nt rule where distances >50–55 nt trigger NMD. Stop codon to splice junction distances are indicated along with whether the AS isoform is turned over by NMD or not. For diagram key see legend to Figure 1.

Figure 5.

Distribution of stop codon to 3′-end distances. Distribution of frequency of distances in nucleotides (nt) between the first premature termination codon and the 3′-end of the transcript (shaded) compared to the distance between the authentic stop codon and 3′-end of the transcript of cognate genes (unshaded).

Figure 6.

Alternative splicing of introns in the 5′-UTR affects the presence, size and position of uORFs and influences turnover of AS isoforms by NMD. Exon–intron structures of genes and transcripts of (A) At2g02960—zinc finger transcription factor, (B) At3g49430—SR protein gene, At-SR34a, and (C) At3g20270—lipid-binding serum glycoprotein gene. These examples illustrate alternative splicing events in 5′-UTR introns which generate uORFs which overlap the main ORF and correlate with NMD. (A) four of the AS isoforms contain a 26 amino acid uORF (A–D) which overlaps the translation start site of the main ORF and correlates to NMD; (B) an uORF of 61 amino acids overlaps the authentic translation start codon in the AS product; and (C) uORF1 overlaps the translation start of the main coding sequence in the fully spliced transcript while in the alternatively spliced isoform the stop codon of uORF2 lies upstream of the main translation start site. Shaded rectangles below transcripts—uORFs; FS—fully spliced; AS—alternatively spliced. Sequences below the figures show the relationship between the stop codon of uORFs and the translational start AUG of the main ORF. For diagram key see legend to Figure 1.

Figure 7.

Intron retention transcripts are not turned over by NMD. Schematic figures of genes which produce detectable intron retention transcripts and other alternatively spliced transcripts with different NMD phenotypes: (A) At1g76460; (B) At1g49730; (C) At5g37055 and (D) At5g24270. Below each gene, structures of transcripts in the amplified region are shown and display different alternative splicing events. Data for each transcript is shown alongside (−: no change; +, ++ and +++: transcript level increases significantly in upf mutants with P < 0.1; 0.01 > P < 0.05 and P < 0.01, respectively. For diagram key see legend to Figure 1. Grey lines below introns labelled IR—retained introns; FS—fully spliced; AS—alternatively spliced.