Extensive Transcript Diversity and Novel Upstream
Open Reading Frame Regulation in Yeast
Karl Waern*,†and Michael Snyder*,†,1
*Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, and†Department of
Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520
ABSTRACT To understand the diversity of transcripts in yeast (Saccharomyces cerevisiae) we analyzed the
transcriptional landscapes for cells grown under 18 different environmental conditions. Each sample was
analyzed using RNA-sequencing, and a total of 670,446,084 uniquely mapped reads and 377,263 poly-
adenylated end tags were produced. Consistent with previous studies, we find that the majority of yeast
genes are expressed under one or more different conditions. By directly comparing the 59 and 39 ends of
the transcribed regions, we find extensive differences in transcript ends across many conditions, especially
those of stationary phase, growth in grape juice, and salt stimulation, suggesting differential choice of
transcription start and stop sites is pervasive in yeast. Relative to the exponential growth condition (i.e.,
YPAD), transcripts differing at the 59 ends and 39 ends are predicted to differ in their annotated start codon
in 21 genes and their annotated stop codon in 63 genes. Many (431) upstream open reading frames (uORFs)
are found in alternate 59 ends and are significantly enriched in transcripts produced during the salt re-
sponse. Mutational analysis of five genes with uORFs revealed that two sets of uORFs increase the expres-
sion of a reporter construct, indicating a role in activation which had not been reported previously, whereas
two other uORFs decreased expression. In addition, RNA binding protein motifs are statistically enriched for
alternate ends under many conditions. Overall, these results demonstrate enormous diversity of transcript
ends, and that this heterogeneity is regulated under different environmental conditions. Moreover, tran-
script end diversity has important biological implications for the regulation of gene expression. In addition,
our data also serve as a valuable resource for the scientific community.
Regulation of gene expression occurs on many levels, both transcrip-
protein levels and protein function is inadequate. In yeast, RNA levels
and protein levels do not perfectly correlate, instead having an r2value
of 0.77 (Lee et al. 2011). Thereby, there must be features of messenger
RNAs (mRNAs) or the RNA-handling machinery that affects mRNA
levels, translational efficiency, and/or protein turnover.
It has been known for some time that untranslated regions (UTRs)
play a significant role in the regulation of gene expression (e.g., van
der Velden and Thomas 1999). Sequences in UTRs may control trans-
of mechanisms (e.g., Mignone et al. 2002). The UTR of a transcript
often changes through the use of differential transcription start sites
[e.g., (Law et al. 2005). These differential transcript ends—referred to
as differential ends in this article—may thus have a role in posttran-
scriptional regulation of gene expression.
The yeast Saccharomyces cerevisiae provides an ideal organism in
which to study this phenomenon. A wealth of molecular, genetic, and
genomic information exists for S. cerevisiae, much of it assembled and
conveniently available at the Saccharomyces Genome Database
(Cherry et al. 1998). Although the complete genome sequence has
been known since 1996 (Goffeau et al. 1996), information on the exact
length of mRNAs has historically been scarce. Several investigators
have attempted to provide this information by using cDNA sequenc-
ing (Miura et al. 2006), microarrays (David et al. 2006), Illumina-
based RNA sequencing (Nagalakshmi et al. 2008), Illumina-based 39
Copyright © 2013 Waern, Snyder
Manuscript received July 2, 2012; accepted for publication December 15, 2012
This is an open-access article distributed under the terms of the Creative
Commons Attribution Unported License (http://creativecommons.org/licenses/
by/3.0/), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Supporting information is available online at http://www.g3journal.org/lookup/
1Corresponding author: Department of Genetics, 300 Pasteur Drive, Lane Building
Rm. 329, Stanford, CA 94305-5120. E-mail: firstname.lastname@example.org
Volume 3|February 2013|
RNA-Seq (Yoon 2010), and Helicos-based RNA sequencing for 39
ends (Ozsolak et al. 2010). Furthermore, previous studies in which
authors examined alternative UTRs in yeast have only considered cells
growing in one condition, with the exception of the studies by Miura
et al. and Yoon et al., whereby analysis was performed under two or
three different conditions, respectively (Miura et al. 2006; Yoon 2010).
Miura et al. found that most genes had multiple 59 and 39 ends, but
they did not examine how these were regulated under different in-
cubation conditions in the same strain. A large-scale systematic analysis
of differential transcript ends under varying environmental conditions
and stressors has thus not previously been performed. Our study ex-
amined not only which and how many transcripts have alternate forms
but also how differential ends vary under a large set of different con-
ditions and how these differential ends affect gene expression.
There has long been evidence that alternative 59 UTR sequences in
mRNAs can have biological relevance by affecting protein synthesis or
protein function. Previous authors have observed that transcripts in
yeast may vary in length and result in (1) a relative increase in protein
level (Law et al. 2005), (2) a decrease in protein level (Law et al. 2005),
(3) secretion of a particular protein (Carlson and Botstein 1982), and
(4) an entirely different protein from an overlapping out-of-frame
open reading fame (ORF) (Guittaut et al. 2001; Coelho et al. 2002;
Kumar et al. 2002). Existing knowledge on alternative UTRs is com-
piled in several reviews, such as (van der Velden and Thomas 1999;
Mignone et al. 2002; Landry et al. 2003; Kochetov 2006).
RNA sequencing (RNA-Seq) provides a powerful tool to study
alternative UTRs. Previous studies (Nagalakshmi et al. 2008; Yoon
2010; Ozsolak et al. 2010) have used RNA-Seq to study the yeast
transcriptome in a few conditions and identified many genes with
multiple 39 transcript boundaries. RNA-Seq provides much better
resolution than microarrays (Wang et al. 2009), and differential tran-
script ends have been identified in RNA-Seq data by examining
expression drop-offs (Nagalakshmi et al. 2008); in addition, poly-
adenylation in eukaryotic transcripts can be identified in RNA-Seq
data as “end tags,” runs of adenines that do not map back to the
genome that lie as the ends of “mappable sequences” (see e.g., Wang
et al. 2009) for an explanation).
This study generated deep RNA-Seq data from yeast incubated
under many different environmental conditions and stresses, selected
to activate a broad variety of gene expression patterns and regulatory
networks. We focused on differential transcript ends and found that
alternative UTRs are pervasive in yeast. Importantly, we also found
that the uORFs introduced in at least two genes with longer 59 ends
enhance gene expression.
MATERIALS AND METHODS
The BY4741 strain used in (Nagalakshmi et al. 2008) was used in this
study. RNA-Seq was performed using the protocol developed in
(Nagalakshmi et al. 2008), further described in (Nagalakshmi et al.
2010) and (Waern et al. 2011), and using the modifications developed
by (Parkhomchuk et al. 2009) to generate strand-specific reads.
Analysis was performed on custom software developed in-house
using BowTie (Langmead et al. 2009) to map reads to the S288C
reference genome available on SGD, downloaded on May 17, 2010.
Python, NumPy, SciPy, and matplotlib were used to further process
the data. The software’s source code is available (Saccharomyces Ge-
nome Database). Of note, to maximize the information gleaned,
unmappable reads were trimmed by four bases from the 39 end and
remapping was attempted; this was done iteratively until only 28 bp
remained, at which point the read was considered unmappable. This
end trimming typically doubled or more the number of mappable reads.
Expression levels were calculated by adding, for each base pair, the
number of times that base pair was encountered in the RNA-Seq data.
For example, if 14 reads overlapped a particular base pair, that base
pair would have an expression level of 14. The expression level of
a gene was calculated as the mean expression level across the
annotated ORF; these values were quantile normalized for compar-
isons between multiple conditions.
The mappability of genes was calculated because if a gene is in or
near an area of the genome with high homology to another genomic
area, it becomes impossible to assign the genomic origin of an RNA-
Seq read stemming from one of the homologous areas. Mappability of
genes was determined by creating, for the plus and minus strands,
simulated 76-mer and 28-mer reads starting at each base pair in the
annotated genome and processing these reads in the pipeline. (Note
from above that reads may have been 28, 32, 36, ..., 76-mers.) Per-
fectly mappable genes would thus have an expression level of 104, as
all 76 of the 76-mer reads and all 28 of the 28-mer reads would have
intersected every base pair of the gene. Genes were considered map-
pable if the mean expression level across the gene was 90% or more of
that (94 reads).
Calling 39 end tags was done as in Nagalakshmi et al. 2008, which
is further described in Wang et al. 2009; three or more nongenomic A
bases were required for each tag.
For a full explanation of the end-calling algorithm, refer to the
software source code. In summary, for each annotated ORF the log2
expression levels for YPAD exponential growth and the other
condition were retrieved and median normalized. The standard
deviation of the expression level difference over the annotated ORF
was calculated as the measure of signal noise, here called n, since the
median-normalized expression of the annotated ORF should have
been the same in both conditions. Ends were called as the first re-
gion—determined by both a 10-bp and 80-bp sliding window—which
was expressed at a level of 3.5 times n or more away from (i.e., above
or below) the expression level in YPAD exponential growth, although
not less than a fourfold difference in expression. This cutoff was
chosen by manual inspection and represents a conservative cutoff
level. Tests were then applied to make sure that the called end was
sufficiently expressed, was not an annotated intron, and was in a map-
pable region of the yeast genome. Each differential end also had to be
at least 40 bp long.
RNA-binding protein (RBP) motifs from (Riordan et al. 2011)
were called by using the consensus motifs in Supporting Information,
Table S1 as a standard text search algorithm, for example, their
motif AAACACAW could be matched to either AAACACAA or
AAACACAU; no probabilistic weighting of nucleotide combinations
Differential expression was determined using the DESeq software
package (Anders and Huber 2010). Subsequent Gene Ontology (GO)
(Ashburner et al. 2000) analyses were conducted using the GO::Term-
Finder software (Boyle et al. 2004).
39 RACE on the CDC19 gene was carried out using the RLM-
RACE kit from Ambion, following all instructions therein. The 39
RACE outer primer had sequence CACCGAAACCGTCGCTGCCT
and the 39 RACE inner primer had sequence TTTTCGAACAAAAG
Luciferase assays were performed using firefly luciferase with
Renilla luciferase as a control, as described previously (McNabb and
Reed 2005). Instead of integrating the luciferase constructs, however,
they were used on a plasmid, as described in (Chu et al. 2011).
Plasmid inserts were produced by DNA 2.0, and sequences are pro-
vided in Table S8.
|K. Waern and M. Snyder
Mapping expressed regions of the yeast genome in 18
In this study, we used strand-specific RNA-Seq (Parkhomchuk et al.
2009) to analyze the transcriptional landscapes of yeast under 18
different environmental conditions, detailed in Table 1. Each condi-
tion was analyzed using two biological replicates, and one additional
technical replicate was performed for exponential growth in YPAD
medium. In total, 850,570,328 reads of 76-bp length were sequenced
on an Illumina GA IIx [see (Shendure and Ji 2008) for platform
details], of which 670,446,084 reads and 377,263 poly-adenylated
end tags (a total of 41,663,340,382 bases) were uniquely mapped to
the yeast genome using Bowtie (Langmead et al. 2009).
The number of uniquely mapped reads per condition ranged from
28 to 55 million (hydroxyurea and sorbitol, respectively), and the
biological replicates showed high correlation. The Pearson r values of
gene expression levels, calculated as described in the Methods section,
ranged from 0.981 to 0.999 (alpha factor and high calcium, respec-
tively). Figure 1 shows the number of reads sequenced for each con-
dition, and Table S1 shows the Pearson correlations (r values) of the
biological replicates for a given condition.
Approximately 93% of the yeast genome can be uniquely mapped
with 76-bp reads. Most of the yeast genome is expressed, consistent
with previous studies (e.g., David et al. 2006; Nagalakshmi et al. 2008),
and the data in this study demonstrate that approximately 80% (range
77–87%) of the base pairs in the yeast genome are expressed from one
or both strands (i.e., as a percentage of the 12-Mb genome) at a depth
of five sequencing reads of coverage or more per base (Figure S1). At
the same level of coverage, approximately 45% (range 40–51%) of
the base pairs are expressed as a percentage of the 24-Mb genome, i.
e., separating out the Watson and Crick strands. Although we
attempted to minimize potential artifacts from second-strand syn-
thesis from reverse transcriptase, it is still possible that some of the
observed transcription might be due to reverse transcription. The
RNA-Seq method used in this study did not yield lower levels of
antisense transcription when actinomycin D was used (Parkhomchuk
et al. 2009), unlike that found in previous studies (Perocchi et al. 2007;
Levin et al. 2010). Examination of annotated genes, however, revealed
that transcription from one strand was accompanied by only a fraction
of that amount of transcription occurring on the opposite strand. The
median gene’s ratio of sense to anti-sense transcription was 5.1 to 6.4
log-two power (Figure S3). These results indicate that most of the
yeast genome is truly expressed under different conditions and that
some true antisense transcription is present, as expected (Xu et al.
Most yeast genes are differentially expressed
We analyzed gene expression across the 18 conditions. Of 6058
mappable genes (i.e., not in repetitive regions), we found that 5958
genes were expressed in at least one condition (with a threshold of
each ORF base detected five or more times on average); 100 genes fell
below this threshold, and 85 of these were classified as “dubious.”
Interestingly, 611 dubious genes are expressed at or above our
Analysis of differential expression for the 5362 genes which are
both mappable and nondubious was done using DESeq (Anders and
Huber 2010). Using a binomial test (P # 0.01), the majority (5245;
97.8%) was differentially expressed relative to exponential growth in
YPAD media in at least one condition. Adding a criterion of 1.5-fold
enrichment reduced this number to 4800 (89.5%), and this figure
further reduces to 3421 (63.8%) when a 2-fold threshold is used. In-
terestingly, only 117 genes never differ in their overall RNA levels
when we use the 1.5-efold enrichment threshold, listed in Table S9.
These are enriched for the GO categories “biological process,” “intron
homing,” “cellular component,” “molecular function,” and “endonu-
clease activity” (Ashburner et al. 2000) at a P-value of 0.05 (corrected)
against a background set of mappable genes; the GO::TermFinder
software suite (Boyle et al. 2004) was used to calculate enrichment.
GO-enrichment analysis of the genes that are differentially
expressed using the binomial test revealed largely expected results,
e.g., alpha factor2stimulated cells had as their top five categories
“conjugation,” “conjugation with cellular fusion,” “sexual reproduction,”
“interaction between organisms,” and “response to pheromone.” Simi-
larly, the top five categories for Congo red stimulation, which damages
cell walls, were “cell wall organization and biogenesis,” “external encap-
sulating structure organization and biogenesis,” “cell morphogenesis,”
“morphogenesis,” and “anatomical structure development”; one of the
n Table 1 The 18 conditions under which RNA-Seq was performed
SC glycerol media
Description No. of Sequenced Reads
1 M NaCl for 45 min
1 mM MMS for 1 hr
2.5 mM for 45 min; add another 50 mL to 25 mL yeast for another 30 min
1 M sorbitol for 45 min
0.4 M H2O2for 45 min
in 37? shaker for 1 hr
18 d in 30? incubator
Synthetic complete medium
4% glycerol instead of glucose in SC medium
10 mM calcium chloride medium
1/5 the normal amount of Yeast Nitrogen Base in YPAD
see File S1
0.1% for 1 hr
0.075M for 1 hr
Walgreen’s brand grape juice, filtered
5 mg/mL for 1 hr
30 mg/mL of Congo red for 1 hr
Unless otherwise noted, the conditions consist of YPAD medium with the indicated reagents added for the indicated incubation time. SC, synthetic complete.
Volume 3February 2013|Transcript Diversity in Yeast|
top categories for heat shocked cells was “protein folding” (see Table S2
for complete results).
Interestingly, cells grown in commercial grape juice had as their
top GO category “vitamin metabolism,” and the 41 genes in this
category contained many vitamin biosynthetic genes related to thia-
mine, nicotinamide adenine dinucleotide/nicotinamide adenine dinu-
cleotide phosphate, flavin, and carnitine, and are aimed at rapidly
processing sugar. Two genes among these, PYC1 and PYC2, are asso-
ciated with lactic acidosis when their human homolog is mutated
[gene annotations taken from the Saccharomyces Genome Database
(Cherry et al. 1998)].
Extensive differences in 59 ends in stress conditions
We were particularly interested in examining differential transcript
end diversity in yeast. A custom software pipeline was developed for
the identification of differentially expressed transcript ends. This pipeline
compared signal tracks for the exponential growth condition to every
other condition, one at a time, and called differential ends when the
difference in signal between the two conditions exceeded a stringent
threshold calculated for each gene based on internal signal noise (see
Materials and Methods; Figure S4). As transcriptional landscapes were
directly compared, the term “topology comparisons”was used to describe
this approach. Table S3 contains the complete list of alternate ends.
Extensive differences were observed in 59 and 39 ends, and Figure 2
summarizes the number of genes with differential ends. Relative to
cells grown in rich medium, we find 613 cases of longer 59 ends and
598 instances of shorter 59 ends across the conditions, affecting 471
and 364 unique ORFs, respectively. For some conditions (e.g., alpha
factor) cells exhibit minimal use of alternative 59 ends, and, of those
that do, the differential 59 ends most often are shorter relative to cells
grown in rich medium, that is, the transcription start site is closer to
the start codon for translation for the nonrich medium condition.
Two conditions, salt stimulation and stationary phase, show an
abundance of transcripts with increased 59 UTR size, with more than
200 genes each expressing a longer variant of its transcript relative to
growth in rich medium. Of 15 transcripts with an alternative 59 UTR
longer than 300 base pairs, seven were found in stationary phase and
four under salt stimulation. The longest alternative 59 UTR, for the
gene CDC8, was 853 base pairs longer in cells in stationary phase.
One interesting effect is when a differential transcript is shortened
to the point that the annotated start codon is not transcribed. This is
observed in 21 instances and 18 unique genes across 10 conditions;
the affected genes are AAC1, ATG16, BRL1, CCM1, COS6, COX23,
EMI2, FYV10, GEP7, GLR1, LAP2, MAD1, MRN1, MSN4, PRP39,
RDH54, SUC2, and UBP7 (Table S1). Of these, only SUC2 has been
characterized previously as a gene that encodes two transcript forms
that produce proteins with differential amino termini (Carlson and
Upstream ORFs lie in many differential 59 ends
Examining differential 59 ends identified across all conditions revealed
a total of 614 instances of uORFs in the differential transcribed region
(see Materials and Methods). These affect 446 transcripts and a total of
543 unique uORFs (47 of which consist of solely a start and stop
codon, with no intervening codon). These results are presented in
We next compared the number of uORFs found in differential
ends to the number expected by chance in differential ends of the
same sizes, using random sampling of promoter sequences within
1000 bp of annotated start codons in the yeast genome. We found that
for cells incubated in the presence of high salt, transcripts were
statistically enriched for uORFs in the differential 59 ends (z-value . 3).
Interestingly, there was no statistical enrichment of uORFs in
shortened 59 UTRs in salt, but only in longer 59 UTRs. Of the 222
Figure 1 Sequencing done for each condition. Shown is the number of sequence tags generated for each condition (i.e., two biological
replicates, pooled). Of the reads labeled as “unaligned,” some still provide useful information as poly-A tags.
| K. Waern and M. Snyder
genes with a longer alternative 59 UTR in the salt response, 104 had
one or more uORFs whereas a total of 148 uORFs were introduced in
the longer-form UTR, where only 107 (std. dev.: 9.7) would have been
expected by chance alone (Table S5). Overall, these results indicate
that the differential 59 ends, particularly long ones, are found under
cell growth in extreme conditions such as high salt.
Diverse roles of differential uORFs in salt response
To further analyze the role of differential uORFs in gene expression,
we selected five genes with uORFs for further study, and fused their
upstream sequences from the gene’s start codon to the nearest neigh-
boring gene upstream of a firefly luciferase reporter. A Renilla lucif-
erase gene fused to a constitutive promoter was used as a loading
control for these same cells (McNabb and Reed 2005; plasmid pTH650
described in Chu et al. 2011). A version of the upstream sequence in
which the “in register uORFs” had been removed also was produced
based on sequential AUGs, where the AUG start codon was changed
to UUG (see Figure 3 for an explanation). In each case, the longest
uORFs and the first accessible uORFs were removed. Results of the
luciferase assays are presented in Figure 4.
Of note, HEM3 and ATP18 were expressed less in salt relative to
YPAD according to the RNA-Seq data (0.6· and 0.5·, respectively),
and the uORFs were found to play a repressive role, i.e., the mutated,
uORF-less construct was expressed greater. GID7 and TID3, however,
were expressed greater in salt (2.5· more in both cases), and the
plasmids with uORF knockouts express less reporter indicating that
the uORFs for these genes played an activating role in protein
expressed. RPH1 also was examined, although mutations analyses
revealed no change indicating that its uORFs did not appear to play
a significant role controlling gene expression.
As a further control that the uORFs themselves were responsible
for the translation changes and not a condition-specific splicing event
(e.g., Cheah et al. 2007). TopHat (Trapnell et al. 2009) was used to
analyze the transcripts for splicing events. No evidence of alternative
splicing was found. Thus, we conclude that most uORFs affect gene
expression; for some genes they activate gene expression and in other
instances they repress gene expression
Extensive differences in mRNA 39 ends
Analysis of 39 ends revealed a number of genes with differential 39
ends in each of the various conditions as determined by topology
comparisons (Figure 2). Relative to exponential growth in YPAD,
there were 451 instances of longer 39 ends, and 1991 instances of
shorter 39 ends across all conditions, representing 328 and 993 unique
ORFs, respectively. Table S3 contains the complete list of altered 39
lengths. Yeast grown in grape juice was clearly distinct, with 764 genes
Figure 2 Number of differen-
tial ends. Shown is the number
of genes with differential longer
and shorter ends on either the
59 or 39 side. All comparisons
Volume 3 February 2013| Transcript Diversity in Yeast|
having a shorter transcript. Interestingly, one gene, REE1, was short-
ened in every condition relative to rich medium.
In addition to topology comparisons, analysis of 377,263 poly-
adenylated end tags in our data set can be used to analyze 39 ends.
These are sequencing reads with a nongenomic run of A bases at the
ends of mappable sequences are presumed to be a poly-adenylation
event and thus mark the end of a transcript. These reads are recovered
from the population of reads that did not initially map to the yeast
genome, as the poly-adenylated tail will prevent alignment. Determin-
ing differential ends using this approach from our sequencing method
is sensitive to thresholding and vulnerable to noise from low sampling
levels; with 377,263 such reads across 18 conditions and a 6000 gene
organism, only 3.5 reads per gene would be expected in each condi-
tion, assuming a uniform distribution of gene expression levels. In
addition, poly-adenylated tags may be hampered by artifacts due to
oligo-dT primer ligation to A-rich genomic sequences. Topology com-
parisons are thus relied upon as the main approach in this study.
Ozsolak et al. (Ozsolak et al. 2010) used poly-adenylation to call tran-
script ends because their protocol only sequenced poly-adenylated regions
of the transcriptome and does not suffer from undersampling. They found
91,891 poly-adenylation sites (File S1), and reported that 72.1% of yeast
genes have at least two poly-adenylation sites further than 50 bp apart.
Pooling the results for all conditions in our RNA-Seq data, we
found that 171 genes had more than 10 end tags. Of those, 128 (74.9%)
had two or more discrete poly-adenylation start sites, indicating
variability in 39 end structure. Among our interesting results, we found
one gene, CDC19/PYK1 (pyruvate kinase), which has an alternative
39 end (Figure 5). Rapid amplification of cDNA ends polymerase chain
reaction (RACE PCR) confirmed the existence of three distinct ends,
one of which is visible only as an expression drop, another only from
poly-adenylation tags, and a third visible from both. In a study of
39 UTRs in Caenorhabditis elegans using polyA capture, 39 RACE,
cDNA sequencing, and RNA-Seq, 27% of called ends were only called
using one technique (Mangone et al. 2010); thus, the use of multiple
methods for calling ends is generally useful for 39 end mapping.
Although the effects were often less dramatic than those seen at the
59 end, expression at the 39 end can decrease to levels where the main
stop codon is not part of the dominant form of the transcript, or not
present at all in the data. Figure S2B shows the 39 end of the VMA5 gene,
which does not appear to have a stop codon at all in synthetic complete
media. Under exponential growth, there are 32 poly-adenylated end tags
near VMA5 between genomic co-ordinates 285,831 and 285,837 on chro-
mosome XI, although there is clearly some expression past that point,
whichwouldexposetheannotated stop codoninaminorityoftranscripts.
In another example, PPZ2, which encodes a phosphatase involved
in regulating cell cycle progression and shown in Figure S2A, the
transcript level for the gene as a whole increases by 2.2-fold in the
shift from exponential growth to stationary phase, but the level of the
differential end, called algorithmically from genomic co-ordinates
1,336,923 to 1,336,988 on chromosome IV, increases by 15.8-fold
(after we adjusted for sequencing library size). A total of 97 other
instances of a shortened 39 end were found using topology compar-
isons, represented in 63 unique ORFs in examples from all 17 non-
exponential growth conditions. They are listed in the Table S6. Thus,
many yeast transcripts may lack a translation termination codon.
The differential ends called by topology comparisons were exam-
ined for RBP motifs using the motifs published in (Riordan et al. 2011).
Most conditions were statistically enriched for RBP motifs among
the 39 sequences that were shortened relative to rich YPD medium, with
the exception of the oxidative stress and sorbitol conditions (Table S7).
However, only five conditions were enriched for RBPs among the
lengthened 39 sequences (grape juice, oxidative stress, heat shock, sorbi-
tol, and stationary phase). Statistical enrichment was calculated, similarly
to that of uORFs, based on random sampling from the yeast genome of
sequences of equal length to the differential ends, and a z-value cutoff
greater than 3. Thus, transcripts with differential ends are likely to affect
differences in RNA binding proteins and presumably gene regulation.
Prematurely terminated transcripts
One particularly surprising finding of our study is that 63 ORFs were
found which, under at least one condition, showed a 39 end that likely
terminated before the annotated stop codon. These transcripts would
Figure 3 uORFs knockouts. Illustration of the manner in which uORFs were mutated. The upper track shows expression, the dotted line under
exponential growth in YPAD, and the dashed line under salt stimulation conditions. The lower track shows the main ORF in solid black and the
knocked-out uORFs in light gray. Out-of-register ORFs, which were not knocked out, are shown in dark gray. (A) ATP18; (B) GID7. In the case of
GID7, the shorter of those two out-of-register ORFs is conserved across Saccharomyces paradoxus, Saccharomyces mikatae, and Saccharomyces
bayanus (see sequence data from Cliften et al. 2003). Please also note that the exact insert placed into the plasmid ran from the far left of each
panel to the beginning of the main ORF; the latter was replaced with luciferase.
| K. Waern and M. Snyder
be left without a stop codon, potentially triggering a nonstop decay of
the mRNA (Frischmeyer et al. 2002) and/or production of a polylysine
tail on the protein that is produced. PPZ2, whose transcript exhibits
this property, is a serine/threonine phosphatase involved in regulating
cell-cycle progression, so its appearance in the stationary phase con-
dition is of interest. The phenomenon of premature poly-adenylation
has been noted before and described as an attenuating regulator of
human mobile elements (Perepelitsa-Belancio and Deininger 2003),
and the nonstop decay pathwayhas been described in yeast (Frischmeyer
et al. 2002). Although the pathway is understood, there is little un-
derstanding of how prevalent it is (Garneau et al. 2007). This study
indicates the particular genes that may be controlled in this fashion
and that the phenomenon made be more widespread that previously
59 ORF truncations
Of the 18 genes found to have 59 ORF truncations, i.e., the dominant
form of the transcript did not contain the annotated start codon, one,
Figure 4 Luciferase analysis of uORFs. Luciferase assays confirming the relevance of uORFs in four genes with longer alternative 59 UTRs in the
salt stress condition. (A) ATP18; (B) HEM3; (C) GID7; and (D) TID3. The left two bars show results in SC–ura media and the right two bars show
results in SC–ura media with 1 M salt for 1 hr. White bars are the results using the leader sequences, and gray bars have had uORF start codons
Volume 3 February 2013| Transcript Diversity in Yeast|
SUC2, has been extensively characterized previously. For SUC2 the
long form contains the signal sequence that causes the sucrose in-
vertase to be secreted (Carlson and Botstein 1982). Carlson and Bot-
stein found that glucose-repressed cells produce a longer form of
SUC2, which is consistent with the results of our study, in which
the RNA-Seq data show a longer transcript present in media using
glycerol as the carbon source. In addition to SUC2, 17 other genes
produce shorter proteins. Only SUC2, however, is predicted to encode
a protein that contains a signal sequence that is missing in the shorter
transcript [performed using SignalP 4.0 (Petersen et al. 2011)]. The
alternative amino terminal sequence in these 17 instances may affect
protein function in other ways.
In 2442 differential 39 ends (1991 shortened and 451 lengthened)
across 17 non-YPAD exponential growth conditions, 6420 RBP motifs
were present (motifs from Riordan et al. 2011). RBP motifs are, to
date, quite degenerate in the method they are defined, and this likely
leads to many false positives. Nevertheless, statistical enrichment was
more often seen in shortened 39 ends, suggesting that cells may
remove the layer of control offered by RBPs under stress conditions.
Upstream ORFs are a known regulatory feature. The availability of
genomic sequences for many organisms, yeast since 1996 (Goffeau
et al. 1996), has allowed for a theoretical search for uORFs, and a large
abundance has been found. Whether they have meaning as a method
for translational control depends first and foremost on whether they
are transcribed; these data have been harder to produce. Using a ran-
dom lacZ insertion method, Burns et al. and Ross-Macdonald et al.
revealed extensive translation of short and out of frame ORF expres-
sion through the yeast genome (Burns et al. 1994; Ross-Macdonald
et al. 1999). In a recent RNA-Seq study, Nagalakshmi et al. (2008)
concluded that 6% of genes had uORFs. Estimates vary and range up
to 20% (Hood et al. 2009). Our data indicate that many of these lie in
Two broad mechanisms of uORF action are known (see Vilela and
MCcarthy 2003): control by uORFs modulating posttermination be-
havior of ribosomes and control modulated by the actual peptide
produced by a uORF. The canonical example of the first is GCN4,
characterized in (Mueller and Hinnebusch 1986), and of the second
the arginine attenuator peptide found before the CPA1 gene (Gaba
et al. 2001).
Even if transcribed, whether uORFs have regulatory function
further depends on their interaction with the translational machinery.
Genes such as GCN4 (Mueller and Hinnebusch 1986), CPA1 (Gaba
et al. 2001), and YAP1/YAP2 (Vilela et al. 1998) are well characterized,
and further examples do exist. Some, however, show no effect [see
(Zhang and Dietrich 2005) where not all validate], and some show an
effect dependent on RNA processing [see (Zhang and Dietrich 2005),
for an example involving riboswitches in N. crassa]. Most studies to
date have used bioinformatic approaches solely and analyze sequence
context and/or conservation, which does not offer proof of uORF
relevance [see e.g., (Cvijovi? c et al. 2007; Lawless et al. 2009; Selpi
et al. 2009), all in S. cerevisiae]. In our study, in which we performed
luciferase validation experiments, we examine five leader sequences
with uORFs, only four of which had a direct impact on luciferase
levels (Zhang and Dietrich 2005). In another study, authors analyzed
the ribosome footprint to mRNA ratios in meiosis and concluded that
uORFs in differential ends could alter translational efficiency of
mRNA. They also reported that in the case of uORFs with an AUG
start codon, the translational efficiency and ribosome occupancy
showed a “strong negative correlation” (Brar et al. 2012). A subse-
quent microarray-based study of meiotic yeast confirmed the presence
Figure 5 Pyruvate kinase differential end validation. (A)
The top track shows the expression profile for CDC19/
PYK1 (pyruvate kinase) under exponential growth in
YPAD and grape juice conditions. The middle track
shows the number of sequenced end tags from the
study as a whole. The bottom track shows the annotated
ORF. The numbers 1, 2, and 3 show Sanger sequencing
results from the 39 RACE bands in part B. (B) Gel show-
ing the results of 39 RACE PCR for CDC19. The ladder
shows the 600- and 500-bp markers. The center lane is
the result for RNA from the exponential growth condi-
tion, and the right lane is from grape juice. The bands
labeled 1, 2, and 3 were sequenced and correspond to
the marked locations in part A.
| K. Waern and M. Snyder
of abundant transcript architecture changes in meiotic and sporulating
yeast (Kim Guisbert et al. 2012).
In this study, there was only a statistical enrichment for uORFs
relative to promoter sequences in longer 59 ends in the salt condition.
Under no conditions was a statistically significant enrichment of
uORFs present in shortened 59 UTRs, implying that normally used
uORFs are seldom removed by alternative promoter choices.
In each of the four cases of a uORF affecting translation reported
in (Zhang and Dietrich 2005), removing the uORFs led to increased
luciferase activity. In this study, a new phenomenon is reported:
uORFs which increase luciferase activity (GID7 and TID3; see Figure
4). That uORFs might increase translational efficiency has been hy-
pothesized (Neafsey and Galagan 2007; see also Hood et al. 2009), but
no previous examples have been found or directed demonstrated
using mutational analyses. One possibly is that these uORFs have
RNA binding sites or ribosome entry sites that attracting ribosomes
to those mRNAs.
Overall, this study demonstrates pervasive differential transcript
ends occur in yeast and that in a least several cases these differences
likely affect gene expression. The mechanisms by which differential
ends are selected remains to be determined, and such information will
be important to obtain a details understanding of how eukaryotic gene
expression is controlled.
Jennifer Li-Pook-Than and Jennifer E.G. Gallagher provided com-
ments on the manuscript. This research was supported by National
Institute of Health grants NIH P50 HG 002357 and NIH/CA077808.
Anders, S., and W. Huber, 2010
quence count data. Genome Biol. 11: R106.
Ashburner, M., C. A. Ball, J. Blake, D. Botstein, H. Butler et al., 2000
Ontology: tool for the unification of biology. Nat. Genet. 25: 25.
Boyle, E., S. Weng, J. Gollub, H. Jin, D. Botstein et al., 2004
Finder—open source software for accessing Gene Ontology information
and finding significantly enriched Gene Ontology terms associated with
a list of genes. Bioinformatics 20: 3710–3715.
Brar, G. A., M Yassour, N Friedman, A Regev, N. T. Ingolia et al., 2012. High-
resolution view of the yeast meiotic program revealed by ribosome pro-
filing. Science 335: 552–557.
Burns, N., B. Grimwade, P. B. Ross-Macdonald, E. Y. Choi, K. Finberg et al.,
1994 Large-scale analysis of gene expression, protein localization, and
gene disruption in Saccharomyces cerevisiae. Genes Dev. 8: 1087–1105.
Carlson, M., and D. Botstein, 1982Two differentially regulated mRNAs
with different 59 ends encode secreted with intracellular forms of yeast
invertase. Cell 28: 145–154.
Cheah, M. T., A. Wachter, N. Sudarsan, and R. R. Breaker, 2007
alternative RNA splicing and gene expression by eukaryotic riboswitches.
Nature 447: 497–500.
Cherry, J. M., C. Adler, C. Ball, S. A. Chervitz, S. S. Dwight et al., 1998
Saccharomyces Genome Database. Nucleic Acids Res. 26: 73–79.
Chu, D., D. J. Barnes, and T. von der Haar, 2011
ribosome competition in coupling the expression of different mRNAs in
Saccharomyces cerevisiae. Nucleic Acids Res. 39: 6705–6714.
Cliften, P, P. Sudarsanam, A. Desikan, L. Fulton, B. Fulton et al.,
2003 Finding functional features in Saccharomyces genomes by phylo-
genetic footprinting. Science 301: 71–76.
Coelho, P. S. R., A. C. Bryan, A. Kumar, G. S. Shadel, and M. Snyder,
2002A novel mitochondrial protein, Tar1p, is encoded on the antisense
strand of the nuclear 25S rDNA. Genes Dev. 16: 656–664.
Cvijovi? c, M., D. Dalevi, E. Bilsland, G. J. L. Kemp, and P. Sunnerhagen,
2007 Identification of putative regulatory upstream ORFs in the yeast
Differential expression analysis for se-
The role of tRNA and
genome using heuristics and evolutionary conservation. BMC Bioinfor-
matics 8: 295.
David, L., W. Huber, M. Granovskaia, J. Toedling, C. J. Palm et al., 2006
high-resolution map of transcription in the yeast genome. Nat. Biotech-
nol. 103: 5320–5325.
Frischmeyer, P. A., A. van Hoof, K. O’Donnell, A. L. Guerrerio, R. Parker
et al., 2002An mRNA surveillance mechanism that eliminates tran-
scripts lacking termination codons. Science 295: 2258–2261.
Gaba, A., Z. Wang, T. Krishnamoorthy, A. G. Hinnebusch, and M. S. Sachs,
2001 Physical evidence for distinct mechanisms of translational control
by upstream open reading frames. EMBO J. 20: 6453–6463.
Garneau, N. L., J. Wilusz, and C. J. Wilusz, 2007
of mRNA decay. Nat. Rev. Mol. Cell Biol. 8: 113–126.
Goffeau, A., B. Barrell, H. Bussey, and R. Davis, 1996
Science 274: 546–567.
Guittaut, M., S. Charpentier, T. Normand, M. Dubois, J. Raimond et al.,
2001Identification of an internal gene to the human Galectin-3 gene
with two different overlapping reading frames that do not encode
Galectin-3. J. Biol. Chem. 276: 2652–2657.
Hood, H. M., D. E. Neafsey, J. Galagan, and S. Sachs Matthew,
2009Evolutionary roles of upstream open reading frames in mediating
gene regulation in fungi. Annu. Rev. Microbiol. 63: 385–409.
Kim Guisbert, K. S., Y. Zhang, J. Flatow, S. Hurtado, J. P. Staley et al.,
2012 Meiosis-induced alterations in transcript architecture and non-
coding RNA expression in S. cerevisiae. RNA 18: 1142–1153.
Kochetov, V., 2006Alternative translation start sites and their significance
for eukaryotic proteomes. Mol. Biol. 40: 705–712.
Kumar, A., P. M. Harrison, K. H. Cheung, N. Lan, N. Echols et al.,
2002Serial analysis of gene expression. Nat. Biotechnol. 20: 58–63.
Landry, J.-R., D. L. Mager, and B. T. Wilhelm, 2003
role of alternative promoters in mammalian genomes. Trends Genet. 19:
Langmead, B., C. Trapnell, M. Pop, and S. L. Salzberg, 2009
memory-efficient alignment of short DNA sequences to the human ge-
nome. Genome Biol. 10: R25.
Law, G. L., K. S. Bickel, V. L. MacKay, and D. R. Morris, 2005
undertranslated transcriptome reveals widespread translational silencing
by alternative 59 transcript leaders. Genome Biol. 6: R111.
Lawless, C., R. D. Pearson, J. N. Selley, J. B. Smirnova, C. M. Grant et al.,
2009Upstream sequence elements direct post-transcriptional regulation
of gene expression under stress conditions in yeast. BMC Genomics 10: 7.
Lee, M. V., S. E. Topper, S. L. Hubler, J. Hose, C. D. Wenger et al., 2011
dynamic model of proteome changes reveals new roles for transcript
alteration in yeast. Mol. Syst. Biol. 7: 514.
Levin, J. Z., M. Yassour, X. Adiconis, C. Nusbaum, D. A. Thompson et al.,
2010 Comprehensive comparative analysis of strand-specific RNA se-
quencing methods. Nat. Methods 7: 709–715.
Mangone, M., A. P. Manoharan, D. Thierry-Mieg, J. Thierry-Mieg, T. Han
et al., 2010 The landscape of C. elegans 39UTRs. Science 329: 432–435.
McNabb, D., and R. Reed, 2005 Dual luciferase assay system for rapid
assessment of gene expression in Saccharomyces cerevisiae. Eukaryot. Cell
Mignone, F, C. Gissi, S. Liuni, and G. Pesole, 2002
mRNAs. Genome Biol 3: REVIEWS0004.1–0004.10.
Miura, F., N. Kawaguchi, J. Sese, A. Toyoda, M. Hattori et al., 2006
scale full-length cDNA analysis to explore the budding yeast transcrip-
tome. Proc. Natl. Acad. Sci. USA 103: 17846–17851.
Mueller, P. P., and A. G. Hinnebusch, 1986
dons mediate translational control of GCN4. Cell 45: 201–207.
Nagalakshmi, U., Z. Wang, K. Waern, C. Shou, D. Raha et al., 2008. The
transcriptional landscape of the yeast genome defined by RNA sequenc-
ing. Science 320: 1344–1349.
Nagalakshmi, U., W. Karl, and S. Michael, 2010
comprehensive transcriptome analysis. Curr. Protoc. Mol. Biol. 89:
Neafsey, D. E., and J. E. Galagan, 2007
upstream open reading frames. Mol. Biol. Evol. 24: 1744–1751.
The highways and byways
Life with 6000 genes.
Complex controls: the
Untranslated regions of
Multiple upstream AUG co-
RNA-Seq: a method for
Dual modes of natural selection on
Volume 3February 2013|Transcript Diversity in Yeast|
Ozsolak, F, P. Kapranov, S. Foissac, S. W. Kim, E. Fishilevich et al.,
2010Comprehensive polyadenylation site maps in yeast and human
reveal pervasive alternative polyadenylation. Cell 143: 1018–1029.
Parkhomchuk, D., T. Borodina, V. Amstislavskiy, M. Banaru, L. Hallen et al.,
2009 Transcriptome analysis by strand-specific sequencing of comple-
mentary DNA. Nucleic Acids Res. 37: e123.
Perepelitsa-Belancio, V., and P. Deininger, 2003
mature polyadenylation attenuates human mobile element activity. Nat.
Genet. 35: 363–366.
Perocchi, F., Z. Xu, S. Clauder-Münster, and M. Steinmetz Lars,
2007 Antisense artifacts in transcriptome microarray experiments are
resolved by actinomycin D. Nucleic Acids Res. 35: e128.
Petersen, T. N., S. Brunak, G. von Heijne, and H. Nielsen, 2011
discriminating signal peptides from transmembrane regions. Nat. Meth-
ods 8: 785–786.
Riordan, D. P., D. Herschlag, and P. O. Brown, 2011
recognition elements in the Saccharomyces cerevisiae transcriptome.
Nucleic Acids Res. 39: 1501–1509.
Ross-Macdonald, P., P. S. Coelho, T. Roemer, S. Agarwal, A. Kumar et al.,
1999 Large-scale analysis of the yeast genome by transposon tagging
and gene disruption. Nature 402: 413–418.
Selpi, S., C. H. Bryant, G. J. Kemp, J. Sarv, E. Kristiansson et al.,
2009 Predicting functional upstream open reading frames in Saccha-
romyces cerevisiae. BMC Bioinformatics 10: 451.
Shendure, J., and H. Ji, 2008 Next-generation DNA sequencing. Nat. Bio-
technol. 26: 1135–1145.
RNA truncation by pre-
Identification of RNA
Trapnell, C., L. Pachter, and S. L. Salzberg, 2009
junctions with RNA-Seq. Bioinformatics 25: 1105–1111.
van der Velden, A. W., and A. A. Thomas, 1999
translated region of an mRNA in translation regulation during develop-
ment. Int J Biochem Cell Biol 31: 87–106.
Vilela, C., and J. McCarthy, 2003Regulation of fungal gene expression via
short open reading frames in the mRNA 59untranslated region. Mol.
Microbiol. 49: 859–867.
Vilela, C., B. Linz, C. Rodrigues-Pousada, and J. McCarthy, 1998
transcription factor genes YAP1 and YAP2 are subject to differential
control at the levels of both translation and mRNA stability. Nucleic
Acids Res. 26: 1150–1159.
Waern, K., U. Nagalakshmi, and M. Snyder, 2011
Methods Mol. Biol. 759: 125–132.
Wang, Z., M. Gerstein, and M. Snyder, 2009
for transcriptomics. Nat. Rev. Genet. 10: 57–63.
Xu, Z., W. Wei, J. Gagneur, F. Perocchi, S. Clauder-Münster et al.,
2009Bidirectional promoters generate pervasive transcription in yeast.
Nature 457: 1033–1037.
Yoon, O. K., and R. B. Brem, 2010 Noncanonical transcript forms in yeast
and their regulation during environmental stress. RNA 16: 1256–1267.
Zhang, Z., and F. Dietrich, 2005 Identification and characterization of up-
stream open reading frames (uORF) in the 59 untranslated regions (UTR)
of genes in Saccharomyces cerevisiae. Curr. Op. Genet. 48: 77–87.
TopHat: discovering splice
The role of the 59 un-
RNA-Seq: a revolutionary tool
Communicating editor: B. J. Andrews
|K. Waern and M. Snyder