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, 106 (7), 2453-8

Genome-wide Suppression of Aberrant mRNA-like Noncoding RNAs by NMD in Arabidopsis

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Genome-wide Suppression of Aberrant mRNA-like Noncoding RNAs by NMD in Arabidopsis

Yukio Kurihara et al. Proc Natl Acad Sci U S A.

Abstract

The nonsense-mediated mRNA decay (NMD) pathway is a well-known eukaryotic surveillance mechanism that eliminates aberrant mRNAs that contain a premature termination codon (PTC). The UP-Frameshift (UPF) proteins, UPF1, UPF2, and UPF3, are essential for normal NMD function. Several NMD substrates have been identified, but detailed information on NMD substrates is lacking. Here, we noticed that, in Arabidopsis, most of the mRNA-like nonprotein-coding RNAs (ncRNAs) have the features of an NMD substrate. We examined the expression profiles of 2 Arabidopsis mutants, upf1-1 and upf3-1, using a whole-genome tiling array. The results showed that expression of not only protein-coding transcripts but also many mRNA-like ncRNAs (mlncRNAs), including natural antisense transcript RNAs (nat-RNAs) transcribed from the opposite strands of the coding strands, were up-regulated in both mutants. The percentage of the up-regulated mlncRNAs to all expressed mlncRNAs was much higher than that of the up-regulated protein-coding transcripts to all expressed protein- coding transcripts. This finding demonstrates that one of the most important roles of NMD is the genome-wide suppression of the aberrant mlncRNAs including nat-RNAs.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The features of mRNA-like ncRNAs. (A) Illustration of the proposed consensus for the NMD target. The mRNAs with termination codons located distant (≧300 nt) from the 3′ termini of mRNAs or ≧50 nt upstream of the last exon–exon junction tend to be recognized as substrates for NMD. Arrows (−) and (+) indicate upstream and downstream from the last exon–exon junction, respectively. (B) Distribution of the distances from the last exon–exon junctions to the 5′-end-closest termination codons of protein-coding mRNAs or mRNA-like ncRNAs (mlncRNAs). (C) Distribution of the distances from the 3′termini of RNAs to the 5′-end-closest termination codons of protein-coding mRNAs or mlncRNAs. Ter, termination codon. CDS, coding sequence.
Fig. 2.
Fig. 2.
AGI-annotated transcripts up-regulated in upf1-1 and upf3-1. (A) Classification of AGI-annotated transcripts up-regulated ≧1.8-fold in upf1-1 and upf3-1, respectively (P initial ≦10−8, FDR α = 0.05). (B) Classification of AGI-annotated mlncRNAs up-regulated >1.8-fold (P initial ≦10−8, FDR α = 0.05) into 2 types, nat-RNA and other mlncRNA. (C) Overlap of AGI-annotated mlncRNAs up-regulated between upf1-1 and upf3-1 (P initial ≦10−8, FDR α = 0.05). (D and E) Detection of a nat-RNA (At3g56408) (D) and another mlncRNA (At3g26612) (E) up-regulated ≧1.8-fold in both upf1-1 and upf3-1 (P initial ≦10−8, FDR α = 0.05). Black arrows indicate the gene structures of nat-RNA or other mlncRNA in TAIR8. The deep-blue regions are exons and the light-blue regions are introns. The red and green bars indicate the relative signal intensity of probes (red ≧400, green <400). The sense gene, At3g56410, is also shown. The tiling-array expression data are available at http://omicspace.riken.jp/gps/group/psca3. (F) Percentages of the protein-coding transcripts and mlncRNAs up-regulated ≧1.8-fold to the expressed protein-coding transcripts and the expressed mlncRNAs (P initial ≦10−8, FDR α = 0.05).
Fig. 3.
Fig. 3.
Predicted non-AGI TUs up-regulated in upf1-1 and upf3-1. (A) Classification of predicted mlncRNAs up-regulated ≧1.8-fold in upf1-1 and upf3-1 into 2 types, nat-RNA and incRNA (P initial ≦10−8, FDR α = 0.05). (B) Overlap of non-AGI TUs up-regulated ≧1.8-fold (P initial ≦10−8, FDR α = 0.05) in upf1-1 and upf3-1. (C and D) Detection of a putative nat-RNA (G2116) (C) and a putative incRNA (G2870) (D) up-regulated >1.8-fold in both upf1-1 and upf3-1 (P initial ≦10−8, FDR α = 0.05). Black arrows indicate the predicted gene structures. The deep-orange regions are putative exons and the light-orange regions are putative introns. The red and green bars indicate the relative signal intensity of probes (Red ≧400, Green <400). Asterisks show the group representatives in G2116 and G2870, respectively. The sense gene, At3g52430 (PAD4), is also shown as a blue bar.
Fig. 4.
Fig. 4.
Quantitative RT-PCR analysis of AGI and non-AGI mlncRNAs. (A) Illustration of 5′RACE-based RT-PCR used for nat-RNA detection. The sample RNA was subjected to dephosphorylation and then decapping reaction. The RNA adapter was ligated to the 5′ end of the RNA. After the RT reaction by using a specific oligo(dT) primer, first PCR was performed by using F1 and R1 primer set. When specific signals were not detectable in the first PCR, additional second (nested) PCR was performed by using the first PCR product as a template and F2 and R1 primer set. (B) Detection of selected nat-RNAs, incRNAs, and other mlncRNAs with ≧1.8-fold increase (P initial ≦10−8). ACT2 mRNA was used as an internal control. (n) indicates the result of the second (nested) PCR.
Fig. 5.
Fig. 5.
Quantitative RT-PCR analysis of some mlncRNAs in the cycloheximide (CHX)-treated plants. Two independent samples from CHX-untreated (CHX-) and CHX-treated (CHX+) plants, respectively, were loaded. ACT2 mRNA was used as an internal control.

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