Genome-wide suppression of aberrant mRNA-like noncoding RNAs by NMD in Arabidopsis Yukio Kuriharaa, Akihiro Matsuia, Kousuke Hanadab, Makiko Kawashimaa, Junko Ishidaa, Taeko Morosawaa, Maho Tanakaa, Eli Kaminumac, Yoshiki Mochizukic, Akihiro Matsushimac, Tetsuro Toyodac, Kazuo Shinozakib, and Motoaki Sekia,d,1 aPlant Genomic Network Research Team, Plant Functional Genomics Research Group, bGene Discovery Research Group, RIKEN Plant Science Center, and cBioinformatics and Systems Engineering Division, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan; and dKihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama 244-0813, Japan Edited by Joseph R. Ecker, The Salk Institute for Biological Studies, La Jolla, CA, and approved December 23, 2008 (received for review September 9, 2008) The nonsense-mediated mRNA decay (NMD) pathway is a well- containing PTC were overaccumulated in the upf1 and upf3 known eukaryotic surveillance mechanism that eliminates aber- mutants (17–19). rant mRNAs that contain a premature termination codon (PTC). The In plants, aberrant mRNAs with termination codons located UP-Frameshift (UPF) proteins, UPF1, UPF2, and UPF3, are essential distant (м300 nt) from the 3Ј termini of mRNAs or м50 nt for normal NMD function. Several NMD substrates have been upstream of the last exon–exon junction tend to be recognized identified, but detailed information on NMD substrates is lacking. as substrates for NMD (21–23) (Fig. 1A). Furthermore, NMD in Here, we noticed that, in Arabidopsis, most of the mRNA-like plants also targets mRNAs that do not possess any introns as in nonprotein-coding RNAs (ncRNAs) have the features of an NMD yeast, which is different from NMD in animals (24). substrate. We examined the expression profiles of 2 Arabidopsis We hypothesized that many mlncRNAs are degraded by the mutants, upf1-1 and upf3-1, using a whole-genome tiling array. NMD system, because most of the mlncRNAs have the features The results showed that expression of not only protein-coding of NMD substrates and the potential to be recognized as transcripts but also many mRNA-like ncRNAs (mlncRNAs), including aberrant transcripts by the UPF complex in Arabidopsis. Here, natural antisense transcript RNAs (nat-RNAs) transcribed from we performed whole-genome tiling arrays for upf1-1 and upf3-1 the opposite strands of the coding strands, were up-regulated in mutant plants. The results showed that NMD suppresses not only PLANT BIOLOGY both mutants. The percentage of the up-regulated mlncRNAs to protein-coding transcripts but also many mlncRNAs. Identifica- all expressed mlncRNAs was much higher than that of the up- tion of NMD substrates including many mlncRNAs will aid in regulated protein-coding transcripts to all expressed protein- understanding the regulatory mechanisms of eukaryotic tran- coding transcripts. This finding demonstrates that one of the most scriptomes and elucidating the roles of mlncRNAs. important roles of NMD is the genome-wide suppression of the aberrant mlncRNAs including nat-RNAs. Results Features of mlncRNAs as NMD Substrates. Of the mlncRNAs with ncRNA ͉ tiling array ͉ UPF1 ͉ UPF3 Arabidopsis Genome Initiative (AGI) codes in Arabidopsis The Arabidopsis Information Resource 8 (TAIR8) genome version, n Arabidopsis thaliana, hundreds of nonprotein-coding RNA 74.6% (293/393) have one or more introns. Of the mlncRNAs I(ncRNA) transcripts have been discovered based on the with introns, 39.9% (117/293) have termination codons that are cloning of full-length cDNAs, whole-genome tiling array, and located м50 nt upstream of the last exon–exon junction when the deep-sequencing analysis (1–8). They include microRNA first AUGs near the 5Ј termini are recognized as start codons (miRNA) precursors, transacting siRNA (ta-siRNA) precursors, (Fig. 1B). Notice that the majority of the mlncRNAs with and mRNA-like ncRNAs (mlncRNA). The mlncRNAs were intron(s) have a termination codon located upstream of the last divided into 2 types—natural antisense transcript RNAs (nat- exon–exon junction (Fig. 1B, black bars). However, only 17.0% RNAs) that arise from the strands opposite the coding strands (3,463/20,374) of the protein-coding mRNAs with intron(s) have and other mlncRNAs. the 5Ј-end-closest termination codons that are located м50 nt Nonsense-mediated mRNA decay (NMD) is a eukaryotic upstream of the last exon–exon junction (Fig. 1B, white bars). mRNA quality-control mechanism that eliminates aberrant They include either the termination codons of the protein- mRNAs containing a premature termination codon (PTC) from coding ORFs or the termination codons of the upstream ORFs cells to avoid the production of truncated proteins (9–13). Such (uORFs) that sometimes exist in front of the major ORFs. transcripts can arise by genomic frameshifts, nonsense muta- Furthermore, of the mlncRNAs with ORFs, 87.3% (331/379) tions, inefficiently spliced premRNAs, and so on. Several pro- have termination codons that are located Ͼ300 nt from the 3Ј teins involved in NMD have already been discovered (10). Among them, the UP-Frameshift (UPF) proteins, UPF1, UPF2, and UPF3, are core components of mRNA surveillance com- Author contributions: Y.K. and M.S. designed research; Y.K., M.K., J.I., and M.T. performed plexes and are essential for normal NMD function. In yeasts and research; K.H. contributed new reagents/analytic tools; Y.K., A. Matsui, K.H., T.M., E.K., mammals, aberrant transcripts with nonsense mutations are Y.M., A. Matsushima, and T.T. analyzed data; and Y.K., K.S., and M.S. wrote the paper. recognized by the UPF complex and then degraded from the 5Ј The authors declare no conflict of interest. and 3Ј ends by recruiting-decapping and 5Ј33Ј exonuclease This article is a PNAS Direct Submission. activities and deadenylating and 3Ј35Ј exonuclease activities Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE12101). (14–16). The tiling-array data can also be viewed at http://omicspace.riken.jp/gps/group/psca3. The Plants also have a sophisticated NMD system (10). The detailed dataset of supplementary tables can be downloaded at http://pfgweb.psc.riken.jp/ Arabidopsis genome encodes homologues of 3 UPF genes (UPF1, download.html. UPF2, and UPF3). Some UPF1 and UPF3 mutants were obtained 1To whom correspondence should be addressed. E-mail: [email protected]. in Arabidopsis and analyzed to investigate the NMD system in This article contains supporting information online at www.pnas.org/cgi/content/full/ plants and to identify several mRNA substrates for NMD by 0808902106/DCSupplemental. using microarrays (17–20). For example, the aberrant mRNAs © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808902106 PNAS ͉ February 17, 2009 ͉ vol. 106 ͉ no. 7 ͉ 2453–2458 Downloaded by guest on September 27, 2021 A Compared with the wild-type accumulation of 237 and 167, AGI-annotated transcripts were increased м1.8-fold in upf1-1 and upf3-1, respectively (P initial Ϲ10Ϫ8, FDR ␣ ϭ 0.05, Fig. 2A and supporting information (SI) Table S1 and Table S2). Of the increased AGI-annotated transcripts, 198 (83.5%) and 138 (82.6%) were protein-coding genes in upf1-1 and upf3-1, respec- tively (Fig. 2A and Table S1 and Table S2). We estimated that 55.6% and 60.1% of those in upf1-1 and upf3-1, respectively, were genes whose transcripts have uORFs in their 5ЈUTRs, whereas only 31.8% and 32.0% of the protein-coding genes that were not up-regulated in upf1-1 and upf3-1, respectively, had B uORFs. This result is consistent with previous reports that many transcripts with uORFs were regulated by the NMD system in mammals and yeasts (16, 25, 26). Of the increased AGI-annotated transcripts, 31 (13.1%) and 25 (15.0%) transcripts encoded mlncRNAs in upf1-1 and upf3-1, respectively. Furthermore, of the mlncRNAs that were up- regulated, 25 (80.6%) and 20 (80.0%) were classified as nat- RNAs, and 6 (19.4%) and 5 (20.0%) were classified as other mlncRNAs in upf1-1 and upf3-1 (Fig. 2B). Of the mlncRNAs that were up-regulated in each mutant, 20 were found in both mutants (Fig. 2C). Eighteen (90%) of them were nat-RNAs, and two (10%) were other mlncRNAs. For example, accumulation of a nat-RNA from the gene At3g56408 (Fig. 2D) and an mlncRNA transcribed from the gene At3g26612 (Fig. 2E) are up-regulated in both mutants, but accumulation of the transcripts from the C sense gene At3g56410, encoding an unknown protein, is not up-regulated (Fig. 2D). Importantly, all termination codons of 36 AGI-annotated mlncRNAs up-regulated in upf1-1 and upf3-1 were located Ͼ400 nt upstream (average 1,250 nt upstream) of the 3Ј termini. Furthermore, 30 of the up-regulated mlncRNAs have exon–exon junction(s). Their termination codons are located upstream (average 59 nt upstream) of the last exon–exon junction. These features of the up-regulated mlncRNAs in the upf mutants almost meet the previously proposed consensus of aberrant RNA molecules targeted by the NMD system (21–23). There- fore, our tiling-array analysis indicated that the mlncRNAs are recognized as the aberrant transcripts and suppressed by the NMD system. Of all expressed protein-coding AGI-annotated transcripts Fig. 1. The features of mRNA-like ncRNAs. (A) Illustration of the proposed (16,268 in upf1 and 15,771 in upf3, P initial Ϲ10Ϫ8), the tran- consensus for the NMD target. The mRNAs with termination codons located scripts with м1.8-fold increase (FDR ␣ ϭ 0.05) were 1.2% and distant (м300 nt) from the 3Ј termini of mRNAs or м50 nt upstream of the last exon–exon junction tend to be recognized as substrates for NMD.