Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii.
ABSTRACT Genome-wide computational analysis of alternative splicing (AS) in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs.
Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy.
The extent of AS in Chlamydomonas that we observed is much smaller than observed in land plants, but is much higher than in simple unicellular heterotrophic eukaryotes. The percentage of different alternative splicing events is similar to flowering plants. Prevalence of constitutive and alternative splicing in Chlamydomonas, together with its simplicity, many available public resources, and well developed genetic and molecular tools for this organism make it an excellent model system to elucidate the mechanisms involved in regulated splicing in photosynthetic eukaryotes.
- SourceAvailable from: Chun Liang[Show abstract] [Hide abstract]
ABSTRACT: Messenger RNA (mRNA) 3'-end formation is an essential post-transcriptional processing step for most eukaryotic genes. Different from plants and animals where AAUAAA and its variants are routinely found as the main poly(A) signal, Chlamydomonas reinhardii uses UGUAA as the major poly(A) signal. The advance of sequencing technology provides enormous amount of sequencing data for us to explore the variations of poly(A) signals, alternative polyadenylation (APA), and its relationship with splicing in this algal species. Through genome-wide analysis of poly(A) sites in C. reinhardtii, we identified a large number of poly(A) sites: 21,041 from Sanger ESTs, 88,184 from 454 and 195,266 from Illumina sequence reads. In comparison with previous collections, more new poly(A) sites are found in coding sequences (CDS), intron and intergenic regions by deep-sequencing. Interestingly, G-rich signals are particularly abundant in intron and intergenic regions. Prevalence of different poly(A) signals between CDS and 3'-UTR implies potentially different polyadenylation mechanisms. Our data suggest that the APA occurs in about 68% of C. reinhardtii genes. Using Gene Ontolgy (GO) analysis, most of the APA genes involve in RNA regulation and metabolic process, protein synthesis, hydrolase and ligase activities. Moreover, intronic poly(A) sites are more abundant in constitutively spliced introns than retained introns, suggesting an interplay between polyadenylation and splicing. Our results support that APA, as in higher eukaryotes, may play significant roles in increasing transcriptome diversity and gene expression regulation in this algal species. Our datasets also provide useful information for accurate annotation of transcript ends in C. reinhardtii.G3-Genes Genomes Genetics 03/2014; · 1.79 Impact Factor
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ABSTRACT: The species Brassica rapa (2n=20, AA) is an important vegetable and oilseed crop, and serves as an excellent model for genomic and evolutionary research in Brassica species. With the availability of whole genome sequence of B. rapa, it is essential to further determine the activity of all functional elements of the B. rapa genome and explore the transcriptome on a genome-wide scale. Here, RNA-seq data was employed to provide a genome-wide transcriptional landscape and characterization of the annotated and novel transcripts and alternative splicing events across tissues. RNA-seq reads were generated using the Illumina platform from six different tissues (root, stem, leaf, flower, silique and callus) of the B. rapa accession Chiifu-401-42, the same line used for whole genome sequencing. First, these data detected the widespread transcription of the B. rapa genome, leading to the identification of numerous novel transcripts and definition of 5'/3' UTRs of known genes. Second, 78.8% of the total annotated genes were detected as expressed and 45.8% were constitutively expressed across all tissues. We further defined several groups of genes: housekeeping genes, tissue-specific expressed genes and co-expressed genes across tissues, which will serve as a valuable repository for future crop functional genomics research. Third, alternative splicing (AS) is estimated to occur in more than 36% of intron-containing B. rapa genes, and 65% of them were commonly detected in more than two tissues. Interestingly, genes with high rate of AS were over-represented in GO categories relating to transcriptional regulation and signal transduction, suggesting potential importance of AS for playing regulatory role in these genes. Further, we observed that intron retention (IR) is predominant in the AS events and seems to preferentially occurred in genes with short introns. The high-resolution RNA-seq analysis provides a global transcriptional landscape as a complement to the B. rapa genome sequence, which will advance our understanding of the dynamics and complexity of the B. rapa transcriptome. The atlas of gene expression in different tissues will be useful for accelerating research on functional genomics and genome evolution in Brassica species.BMC Genomics 10/2013; 14(1):689. · 4.40 Impact Factor
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ABSTRACT: Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation.The Plant Cell 10/2013; · 9.25 Impact Factor
RESEARCH ARTICLE Open Access
Genome-wide analysis of alternative splicing in
Adam Labadorf1, Alicia Link2, Mark F Rogers1, Julie Thomas2, Anireddy SN Reddy2*, Asa Ben-Hur1*
Background: Genome-wide computational analysis of alternative splicing (AS) in several flowering plants has
revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is
part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago.
Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain
insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic
and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas
reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs.
Results: Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional
units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is
the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in
constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively
spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal
than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with
simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a
truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-
PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All
cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy.
Conclusions: The extent of AS in Chlamydomonas that we observed is much smaller than observed in land plants,
but is much higher than in simple unicellular heterotrophic eukaryotes. The percentage of different alternative
splicing events is similar to flowering plants. Prevalence of constitutive and alternative splicing in Chlamydomonas,
together with its simplicity, many available public resources, and well developed genetic and molecular tools for
this organism make it an excellent model system to elucidate the mechanisms involved in regulated splicing in
The coding regions (exons) of most eukaryotic genes are
interrupted by non-coding sequences (introns). The
intronic sequences from primary transcripts (also called
precursor-mRNAs or pre-mRNAs) are removed and the
exons are spliced to generate functional mRNAs . In
many organisms, pre-mRNAs are alternatively spliced to
generate multiple mRNAs from a single gene . It is
becoming clear that alternative splicing generates dis-
tinct proteins with altered functions from a limited set
of genes [2-5]. The effects of alternative splicing on pro-
teins include production of protein isoforms with loss or
gain of function, altered subcellular localization, protein
stability and/or posttranslational modifications [1,3].
Furthermore, alternative splicing plays a role in regula-
tion of gene expression through processes such as regu-
lated unproductive splicing and translation (RUST) and
mRNA recruitment [6,7]. Alternative splicing is also
implicated in evolution of organisms . The availability
of the complete genome sequences of many multicellu-
lar eukaryotic organisms and large sets of full-length
* Correspondence: email@example.com; firstname.lastname@example.org
1Computer Science Department, Colorado State University, Fort Collins, CO,
2Department of Biology and Program in Molecular Plant Biology, Colorado
State University, Fort Collins, CO, USA
Labadorf et al. BMC Genomics 2010, 11:114
© 2010 Labadorf et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
cDNAs and ESTs has permitted comprehensive analysis
of alternative splicing. More recently, global analysis of
alternative splicing has also been performed using spli-
cing sensitive mircroarrays and new generation sequen-
cing technologies [5,8,9]. These analyses have shown
that alternative splicing is highly prevalent in multicellu-
lar eukaryotes. In humans, 95% of multiexon genes
undergo alternative splicing resulting in the generation
of two or more transcripts from a single gene . Ana-
lysis of alternative splicing in flowering plants by align-
ing the available cDNAs/ESTs to genome sequences has
shown that pre-mRNAs from ~ 30% of genes are alter-
natively spliced [10,11]. Alternative splicing in some spe-
cific gene families such as genes encoding serine/
arginine-rich proteins is extensive, resulting in a five-
fold increase in transcriptome complexity [12,13]. In
addition, stresses have been shown to dramatically alter
the splicing pattern of many plant genes [3,12-16]. In
mammalian systems, exon skipping is most prevalent,
whereas in flowering plants up to 55% of alternative
splicing events are intron retention [3,10,11,17]. It is
suggested that the variations in frequencies of different
types of alternative splicing events between plant and
non-plant systems reflect the differences in gene archi-
tecture and pre-mRNA splicing between these organ-
isms [3,11]. In contrast to multicellular organisms, very
little is known about the prevalence and types of alter-
native splicing in simple unicellular photosynthetic
eukaryotes from which land plants have evolved. Recent
completion of the Chlamydomonas genome and the
availability of a fairly large number of ESTs [18-20] per-
mit global analysis of post-transcriptional events includ-
ing alternative splicing in a unicellular photosynthetic
Chlamydomonas shares many features with cells of
more complex eukaryotic plants and animals. Chlamy-
domonas, like land plants, is an autotroph and contains
a chloroplast. Furthermore, like animals, it can grow as
a heterotroph and is mobile. Chlamydomonas diverged
from land plants about one billion years ago . About
93% of the 120 Mb genome of Chlamydomonas rein-
hardtii has been sequenced [18,19]. Gene models in the
latest version (v4) of the Chlamydomonas genome
sequence predict 16,709 protein-coding genes and about
half of these gene predictions have cDNA/EST support.
Analysis of the Chlamydomonas genome revealed that it
contains many genes that are specific to both plant and
animal lineages, reflecting its unique position in evolu-
tion . Because of the many advantages Chlamydomo-
nas offers, it is considered to be the “green yeast” for
studying various eukaryotic cellular processes . For
over five decades, Chlamydomonas has been used as a
model system to study many aspects of photosynthesis,
structure and function of flagella, and a variety of other
biological processes. More recently, it is being used to
investigate mechanisms that regulate biofuels production
. Analysis of alternative splicing in Chlamydomonas
allows comparison of alternative splicing between simple
unicellular photosynthetic eukaryotes and highly evolved
Furthermore, this will also aid in understanding how
alternative splicing has evolved during the evolution of
land plants. Hence, we have performed a comprehensive
analysis of alternative splicing in Chlamydomonas rein-
hardtii. We have used the BLAT tool  and a modi-
fied version of the Sircah software for the detection and
visualization of alternative splicing . Detailed results,
including alignments and splice graphs for each cluster
exhibiting alternative splicing are available on our
“Chlamydomonas AS” site http://combi.cs.colostate.edu/
as/chlamy/. Our results show that alternative splicing is
prevalent in Chlamydomonas, although the extent of it
is less than in land plants. The relative frequency of dif-
ferent splicing events in Chlamydomonas is very similar
to higher plants.
Results and Discussion
Properties of introns
Unlike other unicellular eukaryotes (e.g. yeast), the vast
majority of genes (~ 88%) in Chlamydomonas have
introns (see Table S1 in the Additional file 1 for a com-
parison of the properties of the Chlamydomonas gen-
ome with humans and Arabidopsis). The percentage of
intron-containing genes in Chlamydomonas is higher
than in plants and humans.
Previous comparative studies on gene structure in
flowering plants and animals have revealed a number of
significant differences in their gene architecture [25,26].
For example, land plant genes are shorter than animal
genes with fewer exons and shorter introns .
Furthermore, plant introns are rich in T or T/A,
which is necessary for the recognition of splice sites and
efficient splicing of pre-mRNAs [25,26]. Chlamydomo-
nas shares both plant and animal features in its gene
architecture. The average number of introns in Chlamy-
domonas is similar to humans. However, the median
size of exons (132 bases) and introns (232 nucleotides)
is similar to flowering plants. The GC content of Chla-
mydomonas is 64%, which is significantly higher than
the GC content of multi-cellular organisms. Introns in
protein coding genes of metazoans have four signals
that are necessary for accurate splicing of pre-mRNAs.
These include two consensus sequences at the 5’ and 3’
splice sites with conserved GT and AG dinucleotides,
respectively, a polypyrimidine tract at the 3’ end of the
intron, and a branch point located about 17-40 nucleo-
tides upstream of the 3’ splice site . However, in
land-plants the branch point is not obvious; the 3’ end
Labadorf et al. BMC Genomics 2010, 11:114
Page 2 of 10
of plant introns are rich in T nucleotides . Although
the 5’ and 3’ splice sites in Chlamydomonas introns are
similar to land plants and humans (Figure S5 in Addi-
tional file 1) the 3’ end of introns in Chlamydomonas is
enriched in C in place of a polyprimidine tract (Figure
S6 in Additional file 1).
Extent and types of alternative splicing
We developed a pipeline for detection and visualization
of alternative splicing in Chlamydomonas based on
EST-to-genome alignments using BLAT , and a
modified version of the Sircah alternative splicing detec-
tion software . Details are found in the Methods sec-
tion and Additional file 1. For EST data we used a
recently constructed EST dataset containing 252,484
ESTs processed using cDNA termini to anchor tran-
scripts to their correct positions in the genome .
ESTs were aligned to the genome and grouped into
clusters that overlap in their genomic coordinates and
occur on the same strand. Our alignment and alterna-
tive splicing detection pipeline resulted in 498 clusters
that show 611 alternative splicing events. The alternative
splicing events in each cluster are summarized with
splice graphs . Example splice graphs are shown in
Figure 1. A companion website provides visualization of
the EST alignments and splice graphs for each cluster
showing alternative splicing, as well as access to the
alignments themselves and additional information (see
http://combi.cs.colostate.edu/as/chlamy/). Of the clusters
that show alternative splicing, 484 were associated with
predicted genes in version 4.0 of the Chlamydomonas
We classified all observed alternative splicing events
into the following five groups: Intron Retention (IR),
Alternative 5’ splice site (Alt5’), Alternative 3’ splice site
(Alt3’), events where both the 5’ and the 3’ end of an
intron are alternatively spliced (AltB), and Exon Skip-
ping (ES). The relative frequency of the various types of
alternative splicing events is very similar to those
observed in other plant species, with intron retention
making up almost half of the events. Detailed statistics
are provided in Table 1.
Splice site strength
We compared the splice site strength of the 5’ and 3’
splice sites in all types of alternative splicing events to
those of constitutively spliced genes using the protocol
described in . Consistent with observations in other
organisms , splice sites that participate in alternative
splicing are weaker than constitutive splice sites, and all
the differences are statisitically significant (see Table 2).
The most significant difference is found at the 3’ splice
site of Alt3’ events. In each alternative splicing event, we
identified the prevalent splice form as the one supported
by the largest number of ESTs. We observed that in the
case of Alt5’ and Alt3’ events the splice sites for the
non-prevalent splice forms are weaker than those of the
prevalent splice form; the latter are weaker than those
observed in constitutive splicing (see Table 3 and Figure
2). These differences are also highly statistically
Length and GC content of retained introns and skipped
Alternatively spliced introns and exons in multicellular
organisms were shown to have different length and
nucleotide composition than their constitutively spliced
counterparts [11,30]. We compared the length of
retained introns and skipped exons with those that did
not exhibit alternative splicing. This analysis revealed
that retained introns are shorter than those that did not
exhibit alternative splicing. The median size of retained
introns is 127 bp compared to a median size of 232 bp
in constitutively spliced introns. The difference is more
pronounced in Chlamydomonas than in Arabidopsis,
where median sizes are 93 bp compared to 100 bp .
Skipped exons are shorter than exons that are not
known to exhibit alternative splicing with a median size
of 84 bp compared to a median size of 132 bp in consti-
tutively spliced exons.
In land plants, introns have high AT content, whereas
exons are GC rich. Subsequently, a high percentage of
A/T or T was reported to be important for efficient spli-
cing of introns in flowering plants. Proteins that bind to
U-rich stretches in pre-mRNA have been reported in
plants [26,31]. In Chlamydomonas, we found that
retained introns have a GC content of 57% as compared
to 62% for constitutive introns. Furthermore, short, in-
frame introns have an even lower GC content (56%).
Similarly, skipped exons have a lower GC content as
compared to constitutive exons (63% versus 66%). All
these differences are highly statistically significant (t-
tests yielded p values of 4.1475e-35, 2.5601e-06, and
0.0031, respectively). In Arabidopsis, the opposite trend
is observed; retained introns have a higher GC content
Impact of alternative splicing on predicted proteins
Alternative splicing often results in the occurrence of a
premature termination codon (PTC) [1,6]. Transcripts
with PTCs are potential targets for degradation through
non-sense mediated mRNA decay (NMD) [32,33]. Sev-
eral recent studies suggest that the alternative splicing
of pre-mRNAs is coupled to mRNA degradation
through regulated unproductive splicing and translation,
(RUST) [6,7,34]. To analyze predicted proteins, we
focused on clusters that have full-length cDNAs and a
single alternative splicing event so that we can predict
Labadorf et al. BMC Genomics 2010, 11:114
Page 3 of 10
the effect of alternative splicing on the resulting protein.
Out of the 498 clusters showing AS, 483 correspond to
annotated genes. Of these, 416 have published start
codons, and 77 had a single AS event, and include a
stop codon within a full-length EST. In 76 out of these
77 clusters, whenever alternative splicing occurred in
the coding region, the non-prevalent splice form led to
a shorter protein because of a PTC, resulting in a pro-
tein that is around 50% shorter (see Table 4). In Arabi-
dopsis 78% of alternative splicing events occur in the
coding region and about 50% of these have a PTC .
It has been shown in plants that transcripts with a PTC
undergo NMD, and some of the components involved
in NMD have been reported in plants [34-37]. Interest-
ingly, the predicted proteome of Chlamydomonas con-
tains components of NMD such as UPF3 and exon-
junction complex proteins, suggesting that the NMD
might play a role in the regulation of gene expression.
Alternative splicing motifs
Since introns have four loosely conserved signals, it is
thought that other sequences may be involved in regu-
lated splicing. In metazoans and land plants, protein fac-
tors such as SR proteins and hnRNPs have been shown
to regulate splicing by binding to such splicing regula-
tory elements either in exons or introns and to enhance
or prevent the usage of a splice site [38,39]. We per-
formed a motif analysis of retained introns in compari-
son to constitutive introns using the DME program 
(see details in the Additional file 1). While we didn’t
find statistically significant motifs in retained introns,
constitutively spliced introns consistently produced such
Figure 1 Example splice graph. Shown are two splice graphs along with the relevant EST evidence for the gene fgenesh2_pg.
C_scaffold_39000087, which exhibits intron retention and Alt3’ (top), and for the gene estExt_fgenesh2_kg.C_380020, which exhibits intron
retention, exon skipping, and Alt3’ (bottom). Figures were generated by Sircah as part of our pipeline.
Labadorf et al. BMC Genomics 2010, 11:114
Page 4 of 10
motifs. All the motifs were tandem repeats of di-nucleo-
tides or tri-nucleotides. The consensus sequence for the
top scoring motif was TGCTGCTG. A complete list of
motifs with their associated p-values is presented in
Table S4 in Additional file 1. Simple repetitive elements
have been shown to bind splicing regulatory proteins
such as SRs and hnRNPs and contribute to regulated
splicing . In Chlamydomonas there are several SR
and hnRNP proteins that share significant sequence
similarity with splicing regulators in multicellular
Experimental verification of alternative splicing
For experimental verification, we chose two of the clus-
ters corresponding to ornithine decarboxylase 1 (ODC1,
gene ID: OVA2_SAN_estEXT_fgenesh2_kg.C_340012)
and asparagine synthase (ASyn, gene ID: estExt_fgen-
esh2_kg.C_280076), and performed reverse transcription
PCR (RT-PCR). Amplification of DNAse-treated RNA
with primers corresponding to these genes did not yield
any products (Figure 3A), suggesting no DNA
contamination in our RNA. Our RT-PCR analysis with
primers corresponding to the first and last coding exons
showed six splice variants with ODC1 (Figure 3B) and
two splice variants with ASyn (Figure 3C). Alignments
of previously-available ESTs predicted two alternative
splicing events in ODC1 and three in Asyn. To identify
the type of splicing events in each of these splice var-
iants, we have cloned and sequenced all amplified pro-
ducts. The types of alternative splicing events and their
influence on the predicted proteins are presented in Fig-
ures 3D and 3E. Complete nucleotide and predicted
amino acid sequence of all splice variants for both genes
are provided in Supplement 2. Our RT-PCR results
show that ODC1 produces more isoforms than pre-
dicted from EST alignments, suggesting that the avail-
able ESTs/cDNAs because of their limited number, do
not predict all alternative splicing events in a gene.
Sequence analysis has revealed that five of the six
forms are due to alternative splicing of the 4th intron,
which is the largest in this gene. In a study of alternative
splicing of SR genes, which undergo extensive alterna-
tive splicing in Arabidopsis, it was found that in almost
all SR genes the longest intron was involved in generat-
ing multiple transcripts by alternative splicing. The
alternative splicing events observed in ODC1 include
intron retention, Alt5’ and Alt3’ events. Only one of the
six isoforms produced the full-length protein of 542
amino acids, which contains all of the seven conserved
signature motifs of ODC1; the remaining five splice var-
iants are predicted to produce three different truncated
proteins with 151 to 172 amino acids due to in-frame
translation termination codons. None of these three pro-
teins contain conserved regions found in ODC1, hence
are not likely to be functional. In humans, splice var-
iants that have a premature termination codon at more
than 50 nucleotides upstream of the last 3’ exon-exon
junction are known to be degraded by NMD . Five
of the six splice variants of ODC1 meet this criterion,
hence are likely to be the targets of the NMD surveil-
lance system. Interestingly, all five splice variants with a
PTC are abundant and in some cases are present in
higher levels when compared to the functional transcript
(Fig. 3C, compare the lower band to the rest of the
bands), suggesting some type of regulatory role for these
other transcripts. Three of the four splice variants of the
ASyn gene also encode truncated proteins (Figure 3E,
Additional File 2). Two of these isoforms (isoform 3 and
4) are also likely targets of NMD. Of the three predicted
splice variants for the ASyn gene we verified one of
them (isoform 1), and detected a novel splice variant
(isoform 2). ASyn transfers the amide group of gluta-
mine to aspartate to form asparagine and plays a role in
nitrogen metabolism . Although land plants have
Table 1 The prevalence of different types of alternative
IR 305 (50.0%)
Alt5’ 71 (11.6%)
Alt3’ 158 (25.8%)
AltB 4 (0.7%)
This table shows the number and frequency of each type of alternative
splicing. Percentage of the total number of events is shown in parentheses.
The statistics for Arabidopsis and rice are from .
Table 2 Splice site strength in alternative and
Alt 5’ 7.304 6.373 (7.175e-20)
Alt 3’ 8.594 8.434 (0.00176)
7.790 7.492 (6.258e-44) 6.925 (8.734e-11)
6.701 6.156 (1.465e-09) 7.7356.921 (4.402e-12)
Average splice site scores and p-values for alternative splicing events and
constitutive splicing are shown here. All scores are computed with respect to
the splice site motif of the constitutive splice form, following the protocol
used in . In all cases, the scores for the alternatively spliced form are lower
than for constitutive splicing. The p-values are based on a comparison of the
scores for each type of alternative splicing event with the scores for
constitutive splicing, and are computed using the Wilcoxon signed-ranks test.
Except for the case of exon skipping, the 5’ and 3’ sites refer to the splice
sites of an excised intron. In exon skipping the 5’ and 3’ sites are the splice
sites flanking the skipped exon.
Labadorf et al. BMC Genomics 2010, 11:114
Page 5 of 10
one or more ASyn genes it is not known if the pre-
mRNAs from these undergo alternative splicing .
Ornithine decarboxylase is a key rate-limiting enzyme
in the biosynthesis of polyamines, which are required
for cell growth and cell division in Chlamydomonas and
other organisms . It catalyzes the formation of
putrescine from ornithine. ODC is present in algae and
animals. However, higher plants such as Arabidopsis do
not have ODC and synthesize polyamines via a different
pathway . ODC pre-mRNA in animals also under-
goes alternative splicing. However, the 5’ untranslated
region is alternatively spliced in animals and this event
controls ribosomal entry on the ODC mRNA . The
physiological significance of ODC1 isoforms in Chlamy-
domonas remains to be studied. It is possible that the
PTC forms may be involved in regulating the level of
the functional isoform through regulated unproductive
translation and splicing. In addition to these two genes
(ODC1 and ASyn) splice variant predictions for a few
other genes were verified by others [47-52]. A recent
analysis of thylakoid membrane proteins provides sup-
port for alternative splicing resulting in the generation
of two proteins from a single gene .
During the last six years, the estimates of the extent of
alternative splicing in flowering plants has increased
from 5% to 30%  due to an increase in available EST
and full-length cDNA sequences. It is likely that the
percentage of Chlamydomonas genes known to undergo
alternative splicing will increase as more ESTs/cDNAs
become available. Deep sequencing of the Chlamydomo-
nas trascriptome under various conditions using next
generation sequencing technologies should provide
information on the real extent of alternative splicing.
However, it is also likely that the prevalence of alterna-
tive splicing is roughly four-fold less than in flowering
plants. We found that 3% of Chlamydomonas genes are
alternatively spliced; in Arabidopsis, with a similar num-
ber of ESTs/cDNAs used in this analysis, about 12% of
genes were predicted to be alternatively spliced .
There are only a few cases of alternative splicing
reported in Chlamydomonas [47-52]. In several of these
the protein coded by splice variants was found to be dif-
ferent, suggesting that proteins generated by alternative
splicing may have different functions. In support of this,
alternative splicing in some of these genes has been
Table 3 Splice site strength for prevalent and non-prevalent splice forms.
AS event non-prevalent vs prevalent
non-prevalent avg. score
prevalent vs constitutive
prevalent avg. score
prevalent avg. score
constitutive avg. score
Splice site scores and p-values for the 5’ and 3’ sites of prevalent and non-prevalent splice forms. The table shows data that support two hypotheses: (i) non-
prevalent splice sites are weaker than splice sites associated with the prevalent splice form; (ii) prevalent splice sites are weaker than splice sites associated with
constitutive splicing. The “avg. score” columns provide the average score of splice site occurrences. For the comparison of non-prevalent with prevalent splice
forms, the scores are computed with respect to a motif model of prevalent instances; for the comparison of prevalent and constitutive splicing the scores are
computed with respect to a model of the constitutive splice sites.
Figure 2 Comparison of splice site motifs for prevalent and non-prevalent splice forms. WebLogo  images of 5’ and 3’ splice site
motifs for the prevalent and non-prevalent Alt5’ and Alt3’ splice forms. In the case of Alt5’ there is a difference between the prevalent and non-
prevalent forms only in the 5’ splice site, and similarly for Alt3’, there is a difference only in the 3’ splice site.
Labadorf et al. BMC Genomics 2010, 11:114
Page 6 of 10
shown to have a physiological role. Hence, the observed
alternative splicing events reported here are likely to be
important in regulating gene expression and protein
function. Furthermore, alternative splicing may also con-
tribute to the regulation of functional transcript levels
Our analysis indicates that alternative splicing is pre-
valent in Chlamydomonas reinhardtii. However, the
extent of alternative splicing is much lower than what is
observed in land plants. The frequency of different alter-
native splicing events is similar to flowering plants, with
about half of all splicing events representing intron
retention. Our finding that a large number of genes in
Chlamydomonas undergo alternative splicing, together
with the simplicity of the system and the availability of
powerful experimental tools (molecular and genetic)
suggest that this organism can serve as an attractive
experimental system to understand the mechanisms
involved in regulated splicing.
Detection of alternative splicing
To detect potential alternative splicing events, we obtained
252,484 high-fidelity Chlamydomonas EST sequences that
Liang et al. corrected using cDNA termini to anchor tran-
scripts to their correct positions in the genome . The
ESTs were aligned to the Chlamydomonas genome using
the BLAT program . We performed several filtering
steps to obtain high quality alignments. The resulting
alignments were clustered into putative transcriptional
units and processed using a modified version of the Sircah
tool to detect alternative splicing events. Details of our
alignment processing and alternative splicing detection are
found in Additional file 1.
In our experiments we used two strains of Chlamydo-
monas reinhardtii (the wall-less strain (cc503) and wt
(cc1690)). Both were obtained from the Chlamydomonas
Center culture collection at Duke University. These
strains were then grown in TAP medium . The cul-
tures were maintained at 22°C on a shaking platform in
a growth chamber set on a 12:12 light/dark cycle .
Cells were subcultured during log phase at a starting
density (determined by a hemocytometer) of 105cells/
mL . For RNA isolation, a 2 ml aliquot was collected
during log phase in 2 ml tubes and centrifuged at 0.2 g
for 2 minutes. Supernatant was removed and the proce-
dure repeated until cells from 4 to 6 mls were harvested
in the same tube. The resulting pellet was then frozen
immediately in liquid N2and stored at -20°C.
RNA Isolation and cDNA synthesis
Total RNA was isolated using an RNeasy Plant Mini Kit
(Qiagen, http://www.qiagen.com/. Prior to RNA isola-
tion, the cell pellet was thawed on ice and frozen in
liquid N2. This procedure was repeated 2-3 times in
order to lyse the cells and the total RNA was isolated
according to the protocol provided by the kit manufac-
turer. RNA amount was quantified spectrophotometri-
cally at 260 nm. The RNA sample was treated with
DNase I according to the manufacturer’s instructions
(Invitrogen). The quality of RNA was verified by run-
ning an aliquot on a 1% agarose gel. DNase-treated
RNA (1.5 μg) was used to synthesize first-strand cDNA
with an oligo (dT) primer using SuperScriptII
PCR of ODC1 and ASyn transcripts
One-twentieth of the first-strand cDNA was used for
PCR amplification in a reaction volume of 20 μl. The
primers were designed using the Primer3 Input http://
frodo.wi.mit.edu/ software. Touchdown PCR (TD-PCR)
was performed using a temperature range of 50 - 60°C
based upon the primer Tm. An extended hot-start
method was utilized in which the PCR sample was
Table 4 The effect of splicing on predicted proteins.
AS in Coding Sequence
ORF Shortened By
bp% AS in UTR # events
We considered a subset of the clusters with a full-length cDNA, a single alternative splicing event, and a published start codon in the JGI version 4.0 genome
annotation. For these clusters we show the number of events in a UTR and the number of events in the coding sequence, where UTRs were detected by the
location of where AS occurred with respect to the published start codon and the first stop codon in the reading frame. For events in the coding sequence, we
show the average reduction in the length of the predicted ORF that results when comparing the prevalent splice form with the non-prevalent splice form. In all
cases but one, the non-prevalent splice form is shorter as a result of a premature termination codon. For IR, the prevalent form is always the one where the
intron is spliced, and the non-prevalent form retains the intron. For ES, the prevalent form always contained the exon while the non-prevalent form skipped it.
Labadorf et al. BMC Genomics 2010, 11:114
Page 7 of 10
allowed to incubate at 95°C for 1.5 hrs prior to PCR
cycling. The following TD-PCR conditions were used:
initial denaturation performed at 95°C for 3 minutes,
followed by 10 cycles where denaturation was at 95°C
for 30 seconds, and an annealing temperature of 60°C
for 45 seconds. The annealing temperature was set to
decrease 0.5°C every cycle until the 10 cycles were com-
plete. Elongation was at 72°C for 3.5 minutes. The next
20 cycles had a denaturing temperature of 95°C for 30
seconds, an annealing temperature of 50°C for 45 sec-
onds, and an elongation temperature of 72°C for 3.5
minutes. The final extension was at 72°C for 5 minutes.
Amplified PCR productswereresolved by
electrophoresis in 1% agarose gels. All PCR reactions
were performed using Takara EX Taq™ polymerase.
Bands were extracted using a razor blade and stored at
-20°C until gel extraction was performed.
TOPO Cloning and Sequencing
Gel extraction was performed prior to TOPO cloning
using the GeneJET™ Gel Extraction kit (Fermentas).
After DNA was extracted, the sample was dried using
the Speed-Vac and dissolved in 4 μl of water. The DNA
was cloned using the TOPO TA Cloning Kit (Invitro-
gen). Plasmid from white colonies was isolated using the
QIAprep Spin Miniprep kit (Qiagen). Inserts in plasmids
were verified using PCR as well as digestion with EcoRI.
Clones with an insert were then sequenced at Colorado
State Macromolecular Center. Analysis of sequences was
performed using the Spidey program .
Additional file 1: Supplementary Material. The supplementary material
contains additional tables and figures, and a more in-depth description
of the alternative splicing detection and visualization pipeline.
Click here for file
Additional file 2: ODC1 and ASyn sequence information. The file
contains complete nucleotide and predicted amino acid sequences of all
ODC1 and ASyn splice variants.
Click here for file
This study was funded by NSF award 0743097. We thank Dr. Irene Day for
her comments on the manuscript and Jessye Maddox for her help in
culturing Chlamydomonas reinhardtii.
1Computer Science Department, Colorado State University, Fort Collins, CO,
USA.2Department of Biology and Program in Molecular Plant Biology,
Colorado State University, Fort Collins, CO, USA.
This study was conceived by AR, and designed by AR and AB. Bioinformatics
analysis was carried out by AB, AL, and MR, and supervised by AB. Wetlab
experiments were performed by ALink with the help of JT. AR and AB
drafted the manuscript, with contributions from AL, MR, ALink, and JT. All
authors read and approved the final manuscript.
Received: 16 June 2009
Accepted: 17 February 2010 Published: 17 February 2010
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Cite this article as: Labadorf et al.: Genome-wide analysis of alternative
splicing in Chlamydomonas reinhardtii. BMC Genomics 2010 11:114.
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