Transcript function is determined by sequence elements arranged on an individual RNA molecule. Variation in transcripts can affect messenger RNA stability, localization and translation1, or produce truncated proteins that differ in localization2 or function3. Given the existence of overlapping, variable transcript isoforms, determining the functional impact of the transcriptome requires identification of full-length transcripts, rather than just the genomic regions that are transcribed4, 5. Here, by jointly determining both transcript ends for millions of RNA molecules, we reveal an extensive layer of isoform diversity previously hidden among overlapping RNA molecules. Variation in transcript boundaries seems to be the rule rather than the exception, even within a single population of yeast cells. Over 26 major transcript isoforms per protein-coding gene were expressed in yeast. Hundreds of short coding RNAs and truncated versions of proteins are concomitantly encoded by alternative transcript isoforms, increasing protein diversity. In addition, approximately 70% of genes express alternative isoforms that vary in post-transcriptional regulatory elements, and tandem genes frequently produce overlapping or even bicistronic transcripts. This extensive transcript diversity is generated by a relatively simple eukaryotic genome with limited splicing, and within a genetically homogeneous population of cells. Our findings have implications for genome compaction, evolution and phenotypic diversity between single cells. These data also indicate that isoform diversity as well as RNA abundance should be considered when assessing the functional repertoire of genomes.
At a glance
Transcript isoform variation and its functional relevance have been studied in detail for several single genes. For example, pluripotent cells express a dominant, truncated version of p53 that inhibits the function of the full protein, thereby promoting cell proliferation3. However, the genome-wide characterization of isoform variation has been limited. Identifying either 5′ or 3′ transcript boundaries individually6, 7, 8 cannot determine the respective co-occurrence of start and end sites, which is essential for ascertaining the functional potential of a transcript. Thus, most studies have attributed variations at either transcript end to changes in the full-length messages. This interpretation is inaccurate in general, owing to transcripts that could arise from neighbouring genes, short abortive transcripts, bicistronic messages, and transcripts with differing lengths that overlap a gene. Thus, an important dimension of transcriptome complexity has remained largely unexplored. Here, we characterized the heterogeneity of transcript isoforms in Saccharomyces cerevisiae by jointly sequencing the 5′ and 3′ ends of each RNA molecule using an approach we term transcript isoform sequencing (TIF-Seq).
To capture S. cerevisiae transcript isoforms, capped and polyadenylated RNAs were converted into full-length complementary DNA molecules that were subjected to intramolecular ligation, fragmentation and capture of the 5′–3′ junctions through a biotin tag (Fig. 1a, Supplementary Fig. 1 and Supplementary Tables 1 and 2). The start and end sites of individual RNA molecules were then identified at single-nucleotide resolution by paired-end sequencing of the tagged fragments. We applied TIF-Seq to wild-type yeast grown in two conditions (with glucose (YPD) or galactose (YPGal) as the carbon source). We identified the exact 5′ cap and 3′ polyadenylation sites of more than 19 million individual RNA molecules (Fig. 1b, c). These transcripts are arranged in a remarkably complex, overlapping pattern across the genome (Fig. 1d). In addition to genes with variations in their untranslated regions (UTRs) (for example, CBK1), we discerned overlapping tandem genes (for example, GIM3–YCK2) and bicistronic transcripts (for example, PGA1–IGO1) (Fig. 1d). A comparison of our data with separate 5′ and 3′ end maps illustrates that the former cannot distinguish mono- from bicistronic transcripts, and the latter cannot distinguish 3′ UTR variation from short, overlapping 3′ end transcripts (for example, YNL155W and the antisense transcript of YCK2 in Fig. 1d).
Altogether, in a genome containing only ~6,000 open reading frames (ORFs)9, we detected over 1.88 million unique transcript isoforms (TIFs) (or 776,874 supported by at least two sequencing reads, Supplementary Data 1) that are defined by a unique combination of 5′ and 3′ end sites at single-nucleotide resolution. To enable analysis of major differences, we clustered the transcripts with each of their 5′ and 3′ end sites co-occurring within 5 nucleotides, and selected the highest expressed TIF per cluster as the representative mTIF (major transcript isoform, see Methods), yielding 371,087 mTIFs genome-wide (Supplementary Fig. 2 and Supplementary Data 2). This total corresponds to about half of all TIFs supported by two or more sequencing reads, demonstrating that there are both minor and substantial variations in transcript boundaries. Our further analysis uses TIFs and mTIFs supported by at least two sequencing reads. These numbers represent conservative estimates for transcript diversity, as our detection of isoforms is limited by sequencing depth, RNA abundance and length (Supplementary Information). We verified the accuracy of our transcript isoform mapping with extensive controls and independent confirmations (Supplementary Information, Supplementary Figs 3–8 and Supplementary Table 3).
Our data set reveals the extent to which different classes of transcripts are affected by isoform variation (Fig. 2a and Supplementary Fig. 9). We detected a median of 26 mTIFs (48 TIFs) that cover the coding region per verified or uncharacterized ORF in glucose and galactose (Fig. 2b–e). These mTIFs display a median positional variation of 75 nucleotides (26 for the 5′ start and 36 for the 3′ end, when considered independently) (Supplementary Fig. 10). Notably, this diversity is not dominated by a few highly abundant isoforms: a median of 10 mTIFs (or 29 TIFs) per gene is required to explain 80% of the mRNA population (Supplementary Fig. 11). Isoform heterogeneity is also found in non-coding genes, including an average of 7 mTIFs per stable unannotated transcript (SUT4) (Fig. 2b and Supplementary Fig. 12). In addition, we detected thousands of multicistronic mTIFs that cover two or more ORFs (Fig. 2a). Although the number of TIFs is probably higher than what we observed, we estimate a maximum of ~100 mTIFs (or 500 TIFs) per gene (Supplementary Fig. 13). Altogether, 5,211 ORFs were covered by at least one mTIF, including 86% of verified or uncharacterized ORFs and 223 dubious ORFs9 (Supplementary Data 3).
Most TIFs begin as expected: downstream of annotated transcription preinitiation complex (PIC) sites10 and within the +1 nucleosome (Fig. 2c, Supplementary Discussion and Supplementary Figs 14–17). Notably, some interdependence between transcript start and end sites was observed in 382 protein-coding genes (false discovery rate (FDR) < 10%, Supplementary Fig. 18, Supplementary Data 4 and Supplementary Discussion), supporting the existence of interactions between promoters and terminators11.
Although it is unclear how much of the isoform variation is functional, we discovered several cases where phenotypic consequences would be expected. First, we observed considerable variation in post-transcriptional regulatory elements. Most genes (66.9% in glucose, 71.9% across both conditions) with putative RNA-binding protein (RBP) sites12 express mTIFs with different combinations of binding sites (Supplementary Fig. 19). Furthermore, RBP sites are enriched in regions that vary between isoforms (P < 2.2 × 10−16, Supplementary Information). Second, we observed significant variation in upstream ORFs (uORFs), short coding regions in the 5′ UTR that modulate translation efficiency of the downstream gene13. The standard understanding is that uORFs are transcribed along with the downstream gene, as both elements must be on the same RNA to interact. Yet over half of the genes with annotated uORFs14 (703, 59% in Fig. 3a) expressed alternative mTIFs both with and without the uORF (for example, ICY1 in Fig. 3a, b, Supplementary Data 5). This previously undetected occurrence, in addition to the variation in RNA binding sites, exemplifies transcriptional control of post-transcriptional regulatory potential: the precise isoform transcribed dictates the regulation that can be imposed on the gene.
Notably, our data set reveals that uORFs are not only translational regulators, but can also have an independent identity. Isoforms containing only the uORF were detected for 48% (567) of genes with known uORFs (Fig. 3c and Supplementary Fig. 20). Using ribosome profiling data15, we found that genes containing genuine uORFs (where the mTIFs always span both the uORF and the main ORF) are significantly less translated than those where the uORFs are in fact independent, misannotated transcripts (P < 2 × 10−4) (Fig. 3d). This is consistent with the expected absence of translational repression by uORFs in the latter case. In addition to re-annotating uORFs, we detected the first downstream ORFs (dORFs), defined as short coding sequences within TIFs that also cover the upstream coding gene (for example, COX19, Supplementary Fig. 7 and Supplementary Data 6).
We confirmed the existence of several short transcripts previously misannotated as uORFs by northern blot (for example, PCL7, Fig. 3e and Supplementary Fig. 8). The fact that these transcripts have a canonical mRNA structure (5′ capped and polyadenylated), are bound by ribosomes14, and are evolutionarily conserved (Supplementary Fig. 21) suggests that they are new short coding RNAs (scRNAs, Supplementary Data 5). Short peptides can perform crucial functions, as has recently been described in cellular differentiation16. The capacity of TIF-Seq to detect potentially peptide-encoding scRNAs opens new avenues for studying their function and regulation.
We also analysed the impact of transcript variation on protein diversity. Previous studies have identified alternative transcript isoforms that skip the first start codon, leading to loss of amino-terminal signal peptides and to alternative protein localization (for example, SUC2 (ref. 2), whose protein product can be either cytosolic or secreted, and VAS1 (ref. 17)). We identified 153 additional genes in which at least half of the TIFs with coding potential skipped the first start codon (Fig. 4a and Supplementary Data 7). The translation of these truncated isoforms is supported by recent ribosome profiling data15 (Fig. 4b and Supplementary Fig. 22), which along with recent proteomics data18 indicate that N-terminal truncation via alternative start codon usage is a common phenomenon. This phenomenon can be transcriptionally regulated: we detected 9 genes with significantly differential truncation between glucose and galactose (P < 10−3, FDR < 0.1, Fig. 4c and Supplementary Table 4). These findings indicate a more common production of truncated 5′ transcripts than previously appreciated, which can lead directly to increased protein diversity even without post-transcriptional regulation.
Our data set also provides evidence for the production of carboxyl-terminal protein truncation via alternative polyadenylation sites. We identified 33 genes enriched for internal polyadenylation that introduces early stop codons into the RNA that are not encoded by the DNA19 (FDR < 10%, Supplementary Discussion, Supplementary Fig. 23, Supplementary Table 5 and Supplementary Data 8). Among them is GAL10 (P < 1.2 × 10−9, Fig. 4d), which encodes a bifunctional enzyme in S. cerevisiae with two enzymatic domains that are encoded by two separate genes in other organisms20. In galactose media, such early stop codons result in additional transcripts encoding proteins with only one of these domains (Fig. 4d). Our evidence of protein truncation via alternative isoform usage represents a plausible means for organisms such as yeast, in which alternative splicing is uncommon, to increase protein diversity by selective domain truncation.
Our genome-wide map of transcript boundaries enabled us to measure the extent of transcriptional compaction on each strand of the genome. Most tandem TIFs are separated by approximately 150 base pairs (bp; Supplementary Fig. 24). However, chained arrangements between adjacent TIFs are common, where the end of one TIF coincides with the start of the TIF for the downstream gene. In fact, of 2,747 tandem ORF pairs in the genome, 27% (743) express overlapping mTIFs (for example, GIM3–YCK2, Fig. 1d and Supplementary Data 9) and 6.7% (185) produce bicistronic transcripts (Supplementary Data 10). Most overlapping transcripts stop within the first 100–200 bp of the downstream gene (Supplementary Discussion and Supplementary Fig. 24), indicating that upstream elongating and downstream initiating RNA polymerases are distinguished within this window21. This common overlap facilitates crosstalk between transcriptional units, wherein the expression of a gene can depend not only on its own promoter, but also on the expression of its neighbours22.
Our data set reveals the extent of transcript isoform diversity in the yeast genome at unprecedented resolution. Since most yeast genes have fewer than one mRNA molecule per cell23, the sheer number of isoforms detected here, even within a single environmental condition, indicates that every cell in a clonal population has a unique transcriptome in terms of RNA abundance, sequence and thus regulatory potential. Such cell-to-cell heterogeneity may confer evolutionary advantages, enabling more rapid adaptation of the species to unforeseen environmental challenges. The variation in transcript isoforms has functional consequences through its impact on post-transcriptional regulatory potential, as well as on protein length and localization. In addition, we discovered hundreds of short coding RNAs whose function can now be investigated. Further applications of the TIF-Seq method, or of alternative paired-end strategies such as RNA-PET24, to additional environmental conditions, genetic backgrounds and organisms will deepen our understanding of transcriptional complexity. In multicellular organisms, the combination of transcript boundary variation and alternative splicing is expected to amplify the diversity of transcript isoforms generated from a single genomic sequence, thereby expanding the functional repertoire of the genome.
S. cerevisiae strain SLS045 (MATa/α GAL2/GAL2, S288c background) was grown to mid-log phase (D600 nm ≈ 1) using either YPD (1% yeast extract, 2% peptone, 2% glucose) or YPGal (1% yeast extract, 2% peptone, 2% galactose). Total RNA was isolated by the standard hot phenol method and contaminant DNA was removed by DNase I treatment. Sequenced samples are shown in Supplementary Table 1.
For the construction of the TIF-Seq libraries, we used 60 µg of DNA-free total RNA as input. As an internal control, we added capped and polyadenylated in vitro transcripts (ATCC 87482, 87483 and 87484). The 5′ end of the non-capped RNA molecules was dephosphorylated by treatment with 6 units of shrimp alkaline phosphatase (SAP, Fermentas) for 30 min at 37 °C in the presence of RNase inhibitor (RNasin +, Promega). The RNA was then purified by a double phenol extraction and ethanol precipitation. CAP was removed by a 1-h incubation at 37 °C with 5 units of tobacco acid pyrophosphatase (TAP, Epicentre) in the presence of RNase inhibitor. The sample was phenol:chloroform-purified and ethanol-precipitated. Finally, for oligonucleotide ligation to the 5′ end of the formerly capped molecules, the treated RNA sample was incubated overnight at 16 °C with 20 units of T4 RNA ligase 1 (NEB) in the presence of 10 mM DNA/RNA ‘5oligocap’ oligonucleotide (Supplementary Table 2), 10% dimethylsulphoxide (DMSO) and RNase inhibitor. The RNA was column-purified (RNeasy, Qiagen) and its integrity was checked (Bioanalyzer, Agilent).
To control for the presence of chimaeras, each ligated RNA sample was divided into two aliquots and independently processed to generate two fractions of full-length cDNA (FlcDNA) with different terminal barcoding (see Supplementary information for details). Specifically, each fraction (11.2 µl) was mixed with 1 µl RT priming oligonucleotide, 1 µM either 3cDNANotI_A or 3cDNANotI_B (Supplementary Table 2), and 1 µl 10mM dNTPs. The sample was incubated at 65 °C for 5 min and transferred to ice. 4 µl of 5× First-Strand buffer (Invitrogen), 2 µl DTT 0.1 M and 0.5 µl RNase inhibitor were added to each sample, which was incubated at 42 °C for 2 min to minimize possible mispriming. 2 µl of Superscript III reverse transcriptase (200 U μl−1, Invitrogen) and high temperature (55 °C) incubation was then used for the retrotranscription to minimize the effects of RNA secondary structure. The reaction was incubated for 20 min at 42 °C, 40 min at 50 °C, and 20 min at 55 °C with a final 15 min enzyme inactivation at 70 °C. RNA removal was performed by adding 0.5 µl RNase cocktail (Ambion) and 2.5 units of RNase H (NEB) to each sample and incubating at 37 °C for 30 min. Samples were purified using Agencourt AMPure XP beads (Beckman Coulter Genomics) according to the manufacturer’s instructions and eluted in 19 µl EB (10 mM TrisHCl pH 8.0). 19 µl of resulting FlcDNA samples were PCR-amplified using 20 µl 2× HF Phusion MasterMix (Finnzymes), 0.5 µl biotinylated oligonucleotide 5 µM (either 5BioNotI_A or 5BioNotI_B) and 0.5 µl oligonucleotide 5 µM (either 3AmpNotI_A or 3AmpNotI_B) (Supplementary Table 2). The following thermocycler program was used: 30 s for initial denaturation at 98 °C, 10 cycles (20 s of denaturation at 98 °C, 30 s of annealing at 50 °C (+1 °C per cycle) and 5 min elongation at 72 °C (+10 s per cycle)) and a final elongation of 5 min at 72 °C. Samples were purified using AMPure XP beads. The two independently barcoded aliquots were ultimately pooled together.
To generate cohesive ends, FlcDNA samples were digested for 1 h at 37 °C with 100 units of NotI (NEB) and heat-inactivated for 20 min at 65 °C. The samples were AMPure XP-purified and DNA yield was quantified. Between 300 and 600 ng of digested FlcDNA was circularized for 16 h at 16 °C by intramolecular ligation with 20 µl of T4 DNA ligase (2,000 units μl−1, NEB) in 600 µl final volume. Non-circularized molecules were degraded by incubating the samples for 20 min at 37 °C with 20 units each of exonuclease III (NEB) and exonuclease I (NEB). Enzymes were inactivated by adding 12 µl of 0.5 M EDTA and incubating the samples at 70 °C for 30 min. Circularized FlcDNAs were then phenol:chloroform-purified and ethanol-precipitated.
Purified, circularized FlcDNAs were resuspended in 130 µl EB and sonicated with a Covaris S220 (4 min, 20% duty cycle, intensity 5, 200 cycles per burst). The fragmented DNA was purified with AMPure XP beads and eluted with 20 µl EB. Biotin-containing fragments were captured by incubating the samples for 30 min at room temperature with 20 µl of Streptavidin-conjugated Dynabeads M-280 (Invitrogen) and washed according to the manufacturer’s instructions.
Addition of forked barcoded adapters to the captured molecules was performed using the standard Illumina DNA-Seq library generation protocol with some minor modifications. Specifically, purifications using AMPure beads were replaced with separation on magnetic Dynabeads and NEBNext Master Mixes (NEB) were used. A 20-cycle PCR enrichment was performed using Phusion polymerase (Finnzymes). 300-bp libraries were isolated using e-Gel 2% SizeSelect (Invitrogen) and sequenced with a HiSeq 2000 (Illumina) using paired-end sequencing of 105 bp reads.
TIF-Seq method for long mRNA molecules
We used the same method as described above, but introduced an additional size selection step. Specifically, after the initial PCR amplification, the FlcDNA samples were size-selected on a 1.5% agarose gel, and fragments over 2 kb were purified using QIAquick Gel Extraction Kit (Qiagen). The recovered FlcDNA samples were reamplified using 10 cycles of PCR before the NotI digestion.
TIF-Seq method for non-capped mRNA molecules (mono- and triphosphorylated mRNAs)
We used the same method as described above, but modified steps before the ssRNA ligation. RNA was dephosphorylated using shrimp alkaline phosphatase as described above, but instead of proceeding to treatment with tobacco acid pyrophosphatase, RNA was rephosphorylated for 1 h at 37 °C using T4 polynucleotide kinase (NEB).
RNA circularization and targeted sequencing
Sixty micrograms of DNA-free RNA samples were SAP-and TAP-treated as described above to obtain full-length RNA molecules with 5′ phosphate ends. RNA circularization was performed in the presence of RNase inhibitor, 10% DMSO, T4 RNA ligase buffer, and 50 units of T4 RNA ligase 1 (ssRNA ligase, NEB) for 16 h at 16 °C. The circularized RNA was purified with RNeasy columns (Qiagen) and subjected to random hexamer retrotranscription. The resulting cDNAs were used as a template for standard PCR amplification with divergent oligonucleotides (Supplementary Table 2). The PCR products were cloned using the TOPO TA cloning system (Invitrogen). Individual clones were bidirectionally sequenced with Sanger sequencing to determine both 5′ and 3′ ends. Only clone sequences spanning a poly(A)-tail were taken into consideration.
The DIG Starter Northern kit (Roche) was used according to the manufacturer’s instructions. Strand-specific RNA-DIG probes were generated by in vitro transcription.
Sequencing read processing and alignment
Sequencing reads were de-multiplexed and barcode sequences were removed. The presence of internal chimaera control barcodes was assessed by the Needleman–Wunsch global alignment method provided by the R Biostrings package from Bioconductor (http://www.bioconductor.org/). Samples were classified into 4 groups (putative intermolecular (A-A and B-B) or intramolecular events (A-B and B-A)). Only high-confidence reads with both chimaera control barcodes and a poly(A)-tail were considered for further analysis.
Pairs of 5′ sequences and 3′ sequences were trimmed and then separately aligned to the reference genome using Novoalign V2.07.10 (http://www.novocraft.com) using default parameters. The S288c S. cerevisiae genome (SGD R64, http://www.yeastgenome.org), along with the sequences of the in vitro transcripts that were included as spike-in controls, were used as reference sequences. Only sequences where both ends mapped to the reference were further analysed. Intermolecular pairs (A-B and B-A) were discarded. To exclude any other possible intermolecular cDNA species, only TIFs with both ends mapping to the same chromosome with a length ranging from 40 to 5,000 bp were considered for further analysis.
TIF clustering and mTIF definition
We clustered the transcripts with 5′ and 3′ end sites co-occurring within 5 bp (Supplementary Fig. 2a). Specifically, we defined TSS and TTS clusters separately. Each cluster was defined by both a window and a mTSS/TTS (the most abundant within that window). Clusters of TSS/TTSs were assigned iteratively in decreasing order of expression. In this process, each TSS/TTS site was compared to previously defined clusters, and the site was: (1) defined as a new mTSS/TTS with a 5-bp window (±2 bp up/downstream) if the window did not overlap with previous clusters; (2) defined as a new mTSS/TTS with a smaller window (<5 bp) to avoid overlap with previously defined clusters, if the TSS/TTS was ≥ 5 bp away from the closest previously defined mTSS/TTS but ≤ 2 bp from the closest cluster; and (3) merged with a previously defined cluster if the TSS/TTS was < 5 bp away from the closest mTSS/TTS, in which case the original cluster window was extended to include the newly assigned TSS/TTS (thus the maximum window size of one cluster would be 9 bp); if the TSS/TTS overlapped with 2 previously defined mTSS/TTS in < 5 bp, it was merged with the cluster with the closer (or higher expressed) mTSS/TTS. After this assignment process, only clusters defined by mTSS/TTSs with at least 3 supporting reads were considered. mTIFs were defined as connections between mTSS and mTTS supported by at least 2 reads connecting the associated clusters. All TIFs that shared a given TSS/TTS cluster were assigned to the corresponding mTIF cluster.
The TIFs were aligned to genome annotation features and classified as: (1) ORFs, if they covered an intact ORF coding region; (2) bicistronic TIFs, if they covered 2 or more ORFs; (3) SUTs, if the common region between the TIF and SUT included more than 80% of the length of both the TIF and the SUT; (4) CUTs/XUTs, same as SUTs; (5) overlapping 2 ORFs, if they overlapped two ORFs but did not entirely cover either; (6) overlapping 5′ of one ORF, if they overlapped only the 5′ of one ORF; (7) overlapping 3′ of one ORF, if they overlapped only the 3′ of one ORF; and (8) intergenic TIF, if they did not overlap with any annotated ORFs or overlapped with less than 80% of annotated SUTs, CUTs or XUTs. Annotated transcripts (ORF-Ts, SUTs and CUTs) with TSSs and TTSs are from our previous study that used tiling arrays4.
Comparing TSSs and TTSs to nucleosome data
Nucleosome raw data are derived from ref. 27. Normalized nucleosome occupancy values for the regions flanking the TSSs or TTSs (±500 bp) were extracted and the median values in each position were calculated and plotted. TSSs or TTSs from mTIFs were used.
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We thank R. Aiyar for help in editing and refining the manuscript. We thank W. Huber, C. Zhu, A. I. Järvelin, S. Clauder-Münster, J. Zaugg, S. Adjalley, G. Lin and the members of the Steinmetz laboratory for helpful discussions and critical comments on the manuscript. We thank V. N. Gladyshev and C. Pineau for sharing published data. This study was technically supported by the EMBL Genomics Core Facility. This study was financially supported by the National Institutes of Health (to L.M.S.). V.P. was supported by an EMBO fellowship.
- Supplementary Information (2.7 MB)
This file contains Supplementary Figures 1-24, Supplementary Methods, a Supplementary Discussion, Supplementary Tables 1-5, legends for the Supplementary Data (see separate file) and Supplementary References.
- Supplementary Data (15 MB)
This zipped file contains Supplementary Data files 1-10 – see Supplementary Information for full legends. Supplementary Data 10 was updated on 4 July 2013 to correct a misannotation.