Research Article

Empirical Analysis of Transcriptional Activity in the Arabidopsis Genome

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Science  31 Oct 2003:
Vol. 302, Issue 5646, pp. 842-846
DOI: 10.1126/science.1088305

Abstract

Functional analysis of a genome requires accurate gene structure information and a complete gene inventory. A dual experimental strategy was used to verify and correct the initial genome sequence annotation of the reference plant Arabidopsis. Sequencing full-length cDNAs and hybridizations using RNA populations from various tissues to a set of high-density oligonucleotide arrays spanning the entire genome allowed the accurate annotation of thousands of gene structures. We identified 5817 novel transcription units, including a substantial amount of antisense gene transcription, and 40 genes within the genetically defined centromeres. This approach resulted in completion of ∼30% of the Arabidopsis ORFeome as a resource for global functional experimentation of the plant proteome.

The genome sequence of Arabidopsis thaliana serves as a reference for plants (1). The initial identification of transcriptional units in the Arabidopsis genome sequence was carried out largely by ab initio gene predictions, sequence homology, sequence motif analysis, and other nonexperimental methods (27). This led to an estimate of 25,500 protein-coding genes (2). Though gene prediction software has steadily improved, the ability of these programs to precisely determine gene structures in sequenced genomes remains unsatisfactory (810). A recent attempt to verify experimentally the accuracy of the Arabidopsis genome annotation with conventional molecular approaches proved quite inefficient in identifying full-length (fl) open-reading frames (ORFs) (11). Global experimental approaches are expected to greatly improve genome annotation (12, 13).

A genome sequence that is empirically annotated can provide the foundation for the determination of its ORFeome, the complete set of ORF clones for all protein-coding genes. Access to a “gold standard” cDNA/ORF clone collection (1416), representing the entire Arabidopsis proteome, is urgently needed as a common resource for research (17). Here we report the experimental definition of the transcriptional units for all Arabidopsis genes by fl-cDNA discovery and by hybridization of RNA populations to whole-genome arrays (WGAs) (fig. S1).

Annotation and fl cDNAs. Dramatic improvements in genome annotation have been achieved for several organisms by means of sequences of fl-cDNAs (8, 15, 18, 19). For Arabidopsis, three collections of fl-cDNAs have been used for this purpose (8, 2023). Of the 26,828 predicted genes in the Arabidopsis genome, 25,540 were annotated as protein-coding, with the rest being annotated as pseudo- and partial genes (fig. S3). A unique location in the genome can be identified for the majority (99%) of cDNAs (fig. S4). From this analysis, three classes of genes were detected in the annotated genome (fig. S5): (i) annotated expressed (AE) genes; (ii) annotated non-expressed (ANE) genes, and (iii) newly discovered genes located in the intergenic regions (non-annotated expressed; NAE). Seventy percent (18,093) of the 25,540 annotated genes were found to be transcriptionally active on the basis of identification of an expressed sequence tag (EST) or cDNA, and these represent the class of the AE genes. The remaining 7447 genes (30%) are the hypothetical, ANE (24) genes. Only 10,507 (58%) of the AE genes have at least one fl-cDNA clone (Fig. 1). The remaining 7586 genes (42%) have evidence of their expression from At-ESTs and/or non–fl-cDNAs (Fig. 1). Among the 10,507 genes with a fl-cDNA sequence, the annotated structures of 32% of them (3336) were inaccurately annotated. Incorporation of fl-cDNA sequences resulted in the dramatic improvement of the genome annotation (fig. S6). The vast majority of predicted gene structures in the initial release of the Arabidopsis genome annotation (August 2001 V1.0) were not derived from experimental evidence of transcription.

Fig. 1.

Quality of the Arabidopsis genome annotation V1.0. (A) AE and ANE genes (hypothetical). (B) Newly discovered genes. The fl-cDNAs include: community full length (CFL); Ceres (8); Riken Arabidopsis full length [RAFL, (20)] and Chip (C) clones isolated during this study. See (46) for percentage distribution in the two types of annotated genes.

Genome annotation, intergenic regions, and comparative genomics. Alignment of the various At-ESTs and fl-cDNAs on the V1.0 genome annotation reveals that a considerable number of cDNAs mapped within intergenic regions (IGRs) (fig. S5). Clustering analysis (22) reveals that as many as 1347 NAE genes may reside in the IGRs. The density and distribution of the new unpredicted genes are shown in fig. S7 (22). The availability of a substantial number of Brassica, rice, and wheat ESTs allowed us to examine whether any of the aANE genes (Fig. 1) show similarity to these non-Arabidopsis ESTs. This analysis suggests that 45% of the ANE Arabidopsis genes are transcriptionally active [BLAST e-value < 10–25, (22)] (Fig. 1A). Furthermore, alignment of the Brassica, rice, or wheat ESTs within the IGRs identified an additional 1042 transcriptionally active regions (BLAST e-value < 10–25, Fig. 1B). Overall, our analysis revealed that the Arabidopsis genome contains ∼28,000 genes of which 15,033 (59%) annotated genes lack a fl-cDNA clone and, therefore, have not been experimentally verified.

Mapping transcriptional units using high-density oligonucleotide tiling arrays. In the absence of a corresponding fl-cDNA for 15,033 annotated genes (7586 AE and 7447 ANE genes) and for the ∼1350 newly discovered genes located in the IGRs (Fig. 1B), new methods are needed to obtain evidence for the existence of this remaining large number of “untouched” genes. Recently, microarray technology has been used for mapping transcription units in genomes (12, 13, 25). Despite the fact that the E. coli genome is well characterized, nearly 25% of the transcripts were previously unidentified (26). Except for Saccharomyces cerevisiae (27, 28), however, tiling arrays have not been applied on a genome-wide scale for unbiased examination of transcriptional activity in an eukaryote.

To identify transcription units in the Arabidopsis genome, we used custom high-density oligonucleotide arrays that tile the entire genome and RNA samples prepared from a diverse set of tissues and treatments to ensure broad representation of transcriptional activity. Four pilot arrays, identical in design except for the regions of the genome sequence covered, were used to test the feasibility of the approach (fig. S8) (22). In brief, labeled cRNAs were prepared from different plant tissues and hybridized to the four arrays. Figure 2A shows a deconvoluted and merged image of these arrays after hybridization with cRNA from three different plant tissues (flower, etiolated seedlings, and inflorescence). Differential gene expression was revealed across the 5 Mb region. For example, gene At1g12110 (Fig. 2B) is expressed only in seedlings; gene At1g10770 (Fig. 2C), only in flowers; and gene At1g10630, in all three tissues (Fig. 2D). In addition, the hybridization profiles revealed the structures of the individual genes as confirmed by the sequences of fl-cDNAs corresponding to each of these genes (Fig. 2, B to D). Further analysis with mRNAs isolated from light grown (L+), dark grown (L–) and cold-stressed light grown (L+ cold) seedlings (22) representing different growth conditions revealed that 950 (88%) of the AE transcription units present on the four chips could be detected (Fig. 3A, AE). One-half of the ANE transcription units present on these four chips were also detected (Fig. 3A, ANE). Expression was detected for 61% percent of the newly discovered genes present on the four pilot chips (Fig. 3A, NAE).

Fig. 2.

Pilot Affymetrix tiling genome arrays for mapping transcription units. (A) Deconvoluted hybridization image of array 1A hybridized with three different mRNAs from seedlings, flowers, and young inflorescence. The chip images from three independent hybridizations were overlaid electronically and shown on (A). (B) Detection of a transcription unit specifically expressed in a 3-day-old etiolated seedlings treated with ethylene. (C) Detection of a transcription unit specifically expressed in flowers. (D) Detection of a transcription unit expressed in all three tissues.

Fig. 3.

Quantification of total and tissue-specific transcriptional units detected by the pilot (A) and whole genome (B) arrays of AE genes, ANE genes, NAE genes, and IGRs. See (22) for additional details. The number of genes detected for each category present in the arrays are as follows: (A) AE, 950/1083 (88%); ANE, 108/212 (51%); NAE, 38/62 (61%); IGRs, 117/357 (33%). (B) AE, 15,610/18,675 (84%); ANE, 3091/8264 (37%); NAE, 519/953 (54%); IGRs, 2123/9076 (23%).

Validation of tiling chip–detected transcription units was carried out through reverse transcriptase–polymerase chain reaction (RT-PCR) amplification, cloning, and sequencing (fig. S9) (22). RT-PCR products were detected for 78% of the tested transcriptional units (table S1). Fiftyseven percent of the RT-PCR products yielded “perfect” ORF clones from both AE and ANE genes (table S1). The remaining 42% of the clones were without ORFs due to stop codons introduced during RT-PCR amplification (table S1).

Whole-genome arrays. We next designed a set of 12 oligonucleotide arrays representing ∼94% of the Arabidopsis genome sequence [110 Mb (22) (fig. S10)]. Each array contains ∼834,000 25-mer oligos (22). Four RNA populations (22) were hybridized to these arrays, and a transcription map for the entire Arabidopsis genome was determined (figs. S11 to S15) (22). Expression of 84% of the AE genes and 37% of the ANE genes was detected when these four RNA samples were used (Fig. 3B, AE and ANE). In addition, we were also able to detect a large number (∼54%) of transcriptionally active sites corresponding to the chromosomal locations where the newly discovered genes were found (Fig. 3B, NAE). Transcriptional activity was also detected in 2000 intergenic regions (23%) that were thought to be devoid of transcriptional activity (Fig. 3B, IGR).

The five chromosomes were equally transcriptionally active under various growth conditions or developmental stages (fig. S16). Transcriptional activity across the chromosomes using four different messenger RNAs (mRNAs) is quantitatively distinct (Fig. 4). The transcriptional activity across each chromosome was different in various tissues tested (Fig. 4). For example, the right arm of chromosome 2 showed more transcriptionally active sites in suspension cell cultures [Fig. 4, compare (E) with (C) in chr. 2] than in flowers. In addition, in certain cases the transcriptional activity was strand-specific. For example, in roots there was more transcription from the forward-strand than from the reverse complement strand (Fig. 4D, chr. 1). The gene density is approximately the same in both strands. The distribution of tissue-specific transcripts across the five chromosomes is shown in Fig. 5. Functional classification of the tissuespecific transcripts revealed variation in the types of genes expressed in a tissue-specific manner (fig. S17). For example, 16% of the flower-specific AE transcription units encode proteins involved in metabolism, whereas only 6.5% of the same class of genes were detected in suspension cell cultures (fig. S17C, compare flower with suspension cell culture).

Fig. 4.

Global gene expression detected by whole genome arrays in the Arabidopsis chromosomes. (A) Predicted gene density on each strand of the five chromosomes. (B) Percentage of expressed genes on each strand of the five chromosomes in light-grown seedlings. (C) Flowers. (D) Roots. (E) Suspension cell culture. Forward strand is indicated as top (T), whereas reverse strand as bottom (B). The genetically defined centromeres were indicated as red dots.

Fig. 5.

Chromosomal locations of tissue-specific expressed transcription units. (A) AE genes. (B) ANE genes.

More detailed analyses of the whole-genome transcription map focused on examination of regions of the genome, such as pseudogenes and centromeres, thought to be relatively transcriptionally silent and also on genomic locations showing antisense RNA expression. (Fig. 6A). We detected transcription for ∼20% of the 1332 ANE “pseudogenes” (Fig. 6A), suggesting that these ORFs may be misannotated. Alternatively, as recently described for mouse (29), such actively transcribed “pseudogenes” may function to regulate gene activity. In total, ∼7600 annotated genes (∼30% of all annotated genes) were identified that showed significant antisense RNA expression (Fig. 6B), suggesting that double-strand RNA formation may be a general phenomenon in plant cells (30). We found complementary patterns of tissue-specific expression of sense and antisense RNAs for many genes, indicating a possible biological role for these transcripts (31). Lastly, we surveyed regions located within the genetically defined centromeres for transcriptional activity [(2, 32); Fig. 6C]. Transcriptional activity was confirmed for ∼90% of the AE genes. Transcriptional activity was also observed for ∼45% of the “unexpressed” genes, and we identified 29 new transcribed genes within the five centromere regions. Surprisingly, two unusually highly expressed sites, “hot spots,” were identified within the genetically defined centromeres of two chromosomes (2, 22, 32) (Fig. 6D). The centromere region of chromosome 2 (CEN2) contains a large (∼600 kb) insertion of a rearranged copy of the mitochondrial genome (2). This entire genomic location showed high level of transcriptional activity. In addition, several unannotated regions within the centromere region of chromosome 3 (CEN3) were identified that displayed a significantly higher level of transcriptional activity from both forward and reverse complement DNA strands (Fig. 6D). These highly transcribed sequences within CEN3 were largely composed of transposon or retrotransposon-like sequences but also contained some unique sequences.

Fig. 6.

WGA analysis of transcription of pseudogenes, antisense RNAs, and centromere-located genes. Proportion of AE and ANE genes for (A) predicted pseudogenes, (B) antisense genes, or (C) centromere regions. (D) Transcriptionally active “hot spots” with the genetically defined centeromeres for chromosomes 2 and 3. See SOM text for further description.

Overall, evidence from both cDNA sequencing and WGA expression studies allowed detection of transcriptional activity for 5817 previously ANE (hypothetical) genes (Fig. 3B, ANE; figs. S6, A and C).

Determination of the Arabidopsis ORFeome. Large-scale ORFeome projects not only aid in the improvement of whole genome annotation, but also provide valuable resources to the community for functional genomic studies and proteomics (33, 34). We constructed 8750 error-free ORF clones [Table 1 (22, 35)] representing approximately 32% of the annotated genes. Functional classification of the constructed ORFeome revealed a similar distribution of the various functional classes compared with the complete set of annotated genes (fig. S19). Figure S20 shows the distribution of the ORF clones constructed among the various available fl-cDNAs.

Table 1.

ORF clones constructed.

Class of genes Type of ORF cloneView inline
U C
Annotated expressed 7685 486
Annotated non-expressed N/A 275
Newly discovered 171 27
Annotated as pseudogenes 19 87
Total 7875 875
  • View inline* See (22) for clone terminology.

  • Utility of WGAs for genomic analysis. We used a strategy for mapping transcription units that included both a traditional cDNA cloning and DNA sequencing approach and a novel high-throughput approach that utilizes tiling array technology (12, 13, 25). Sequencing ESTs and fl-cDNAs verified 40% of the annotated genes, dramatically improved genome annotation, and allowed the construction of 30% of the ORFeome. As with other large-scale cDNA-based gene collection projects (8, 15, 18), the traditional approach of sequencing fl-cDNAs reaches a point of diminishing returns: two-thirds of the total annotated Arabidopsis genes still have no corresponding fl-cDNA. The WGA technology allowed us to identify transcripts for the ANE genes and enabled construction of the remaining 30% of the Arabidopsis ORFeome. We were able to detect ∼60% of the total annotated genes (AE and ANE) using only a limited number of RNA samples (four). A more comprehensive survey of tissues and cell types should enable a more complete description of the transcriptome.

    WGAs serve as a “molecular roadmap” for detecting transcription units and their structures across the chromosomes. This capability arrives at a convenient time, because several major efforts to generate fl-cDNAs for identification of a full set of ORF clones of expressed genes are under way [the Mammalian Gene Collection (15, 16) and FANTOM (36)]. As these collections become more complete, the marginal utility of each added EST decreases, and the prospect for discovering new genes diminishes. Use of full-genome tiling arrays is a natural way to complete the identification of transcription units. In addition, the data presented here raise the prospect that novel, tissue-specific transcription units can be identified in Arabidopsis by means of WGAs, cloned, and functionally studied with biochemical and reverse genetic approaches (37). The WGAs allowed us to detect transcription from many of the “pseudogenes,” providing additional evidence that this class of genes may function in the regulation of their paralogous counterparts (29). Our studies also revealed that a large fraction of Arabidopsis genes showed expression of an antisense RNA transcript that, in many cases, was tissue-specific. These results suggest that the production of double-stranded RNAs may play a greater role in the regulation of normal plant development than is currently understood.

    The use of genome tiling array technology is not restricted to transcription unit mapping of RNA polymerase II transcripts. WGAs can also be used for detecting mitochondrial- or plastid-derived RNAs and potentially could be used for the identification of a variety of small noncoding RNAs (nc-RNAs) (38). By design, WGAs contain DNA sequences absent from traditional gene expression arrays, such as the promoters, introns, and intergenic “dark matter.” Thus, they are well suited for global mapping of DNA binding sites by location analysis (39, 40) and provide an “unbiased view” of DNA binding sites that recent studies suggest may lie within coding regions at a greater frequency than previously suspected (41). Genome tiling arrays can also be used to detect DNA sequence polymorphisms and potentially to detect differences in cytosine methylation patterns among Arabidopsis ecotypes, which would be useful for rapid genome-wide mapping of quantitative trait loci (QTL) (42). Finally, WGAs might also be used for direct mapping of point mutations without the use of segregated populations.

    The impact of these findings and of the experimental resources developed is not restricted to Arabidopsis. For example, 85% of Arabidopsis genes have close homologs in the rice genome (43). Therefore, this resource will aid in the elucidation of the function(s) of the vast majority of genes in plant genomes (43, 44), providing fundamental knowledge that we hope will eventually lead to the engineering of new plant species.

    Supporting Online Material

    www.sciencemag.org/cgi/content/full/302/5646/842/DC1

    Materials and Methods

    SOM Text

    Figs. S1 to S27

    Tables S1 to S6

    References

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