(solution) Can anyone explain me better Table 2 and Figure 8? I cant

(solution) Can anyone explain me better Table 2 and Figure 8? I cant

Can anyone explain me better Table 2 and Figure 8? I cant interpret/understand what the figures really mean, or whats the message

| INVESTIGATION The Interplay of Temperature and Genotype on
Patterns of Alternative Splicing in
Drosophila melanogaster
Ana Marija Jaks? ic´ *,? and Christian Schlötterer*,1 *Institut für Populationsgenetik, Vetmeduni Vienna, 1210 Vienna, Austria, and ?Vienna Graduate School of Population Genetics,
Vetmeduni Vienna, 1210 Vienna, Austria ABSTRACT Alternative splicing is the highly regulated process of variation in the removal of introns from premessenger-RNA
transcripts. The consequences of alternative splicing on the phenotype are well documented, but the impact of the environment on
alternative splicing is not yet clear. We studied variation in alternative splicing among four different temperatures, 13, 18, 23, and 29°,
in two Drosophila melanogaster genotypes. We show plasticity of alternative splicing with up to 10% of the expressed genes being
differentially spliced between the most extreme temperatures for a given genotype. Comparing the two genotypes at different
temperatures, we found ,1% of the genes being differentially spliced at 18°. At extreme temperatures, however, we detected substantial differences in alternative splicing?with almost 10% of the genes having differential splicing between the genotypes: a magnitude similar to between species differences. Genes with differential alternative splicing between genotypes frequently exhibit dominant
inheritance. Remarkably, the pattern of surplus of differences in alternative splicing at extreme temperatures resembled the pattern seen
for gene expression intensity. Since different sets of genes were involved for the two phenotypes, we propose that purifying selection
results in the reduction of differences at benign temperatures. Relaxed purifying selection at temperature extremes, on the other hand,
may cause the divergence in gene expression and alternative splicing between the two strains in rarely encountered environments.
KEYWORDS alternative splicing; temperature; plasticity; dominance S PLICING, the removal of introns from precursor messenger RNAs (mRNAs) together with the subsequent ligation
of exons, is an integral part of gene expression regulation.
Alternative splicing is the combination of different exons
from the same precursor mRNA and provides the basis for
the impressive diversity of gene products originating from a
substantially smaller set of genes (Pan et al. 2008; Nilsen
and Graveley 2010; Brown et al. 2014). There are several
types of alternative splicing; such as the exclusion of exons,
sometimes mutually exclusive; or the retention of intronic
sequence in the mature transcript. Furthermore, the alternative selection of 59 or 39 splice sites, a special form of exon Copyright © 2016 by the Genetics Society of America
doi: 10.1534/genetics.116.192310
Manuscript received June 3, 2016; accepted for publication July 8, 2016; published
Early Online July 19, 2016.
Available freely online through the author-supported open access option.
Supplemental material is available online at www.genetics.org/lookup/suppl/doi:10.
1534/genetics.116.192310/-/DC1.
1
Corresponding author: Institut für Populationsgenetik, Vetmeduni Vienna,
Veterinärplatz 1, 1210 Vienna, Austria. E-mail: [email protected] skipping (Koren et al. 2007), has been shown to make an
important contribution to transcript diversi?cation.
Splicing, in particular alternative splicing, is a highly regulated process that depends on cis-regulatory sequences (splicing
enhancers and suppressors) and trans-regulatory splicing factors, such as heterogeneous nuclear ribonucleoproteins and
SR and SR-related proteins (Nilsen and Graveley 2010).
The repertoire of isoforms, different mature mRNAs originating from a single gene, differs widely among tissues, developmental stages, and environmental conditions (Barberan-Soler
and Zahler 2008; Gan et al. 2010; Barbosa-Morais et al. 2012;
Bartok et al. 2013; Leviatan et al. 2013; Long et al. 2013; Reyes
et al. 2013; Telonis-Scott et al. 2013; Brown et al. 2014; Chang
et al. 2014; Vitulo et al. 2014). It could, therefore, be considered as a prototype for phenotypic plasticity on the molecular
level (Mastrangelo et al. 2012; Chen et al. 2015b).
Phenotypic plasticity describes the ability of a given genotype to display a range of phenotypes as a response to
environmental heterogeneity. On the organismal level, phenotypic plasticity has been of key interest to evolutionary
biologists as it provides the opportunity to respond quickly Genetics, Vol. 204, 315?325 September 2016 315 to environmental changes. On the cellular level, phenotypic
plasticity is the impressive manifestation of cellular differentiation of multicellular organisms; a property favored by
natural selection.
While the selective advantage of both the presence or
absence of phenotypic plasticity is conceptually appealing,
their relative importance is not yet clear. Traditionally, plasticity has been studied using high-order phenotypes, such as
morphology and life history traits, which integrate the effects
of many genes. Nevertheless, the advances in molecular biology have opened the possibility to expand these studies to
lower-level phenotypes such as gene expression and alternative splicing. Over the past years, an impressive amount of data
has been collected demonstrating plasticity of gene expression
and alternative splicing in different tissues and developmental
stages (Jin et al. 2001; Wang et al. 2008; Graveley et al. 2011;
Zhou et al. 2012; Smith et al. 2013; Brown et al. 2014; Etges
et al. 2015). Much less is known about the in?uence of environmental conditions on this plasticity (Levine et al. 2011;
Yampolsky et al. 2012; Telonis-Scott et al. 2013; Brown et al.
2014; Chang et al. 2014; Sikkink et al. 2014; Vitulo et al. 2014;
Yampolsky et al. 2014; Chen et al. 2015a; Zhao et al. 2015),
and the conservation of these patterns across genetically diverged organisms (Barberan-Soler and Zahler 2008; Etges
et al. 2015; Chen et al. 2015a; Zhao et al. 2015).
Temperature is one of the key environmental parameters,
in particular for ectotherms such as Drosophila. A broad range
of morphological, behavioral, and physiological responses to
temperature has been described, but few studies attempted
to compare the patterns of gene expression plasticity across
temperatures. Most of these studies compared the pattern of
gene expression at two temperatures (Sikkink et al. 2014;
Zhao et al. 2015) and found a large number of genes significantly affected by temperature. Recently, Chen et al. (2015a)
attempted a more re?ned characterization of the temperature
effect on gene expression by describing the reaction norm of
gene expression across a broad temperature range (13?29°).
Remarkably, they found that the reaction norm did not only
cluster genes according to function, but also explained some of
the underlying regulatory architecture (Chen et al. 2015b).
Extending the plasticity analysis to diverged genotypes
often found signi?cant differences in the reaction norm between genotypes. In studies that compared gene expression
between differentially evolved genotypes, differences in gene
expression plasticity were good indicators for direct or indirect
selection targets (Telonis-Scott et al. 2009; Yampolsky et al.
2012). An interesting pattern was found when Chen et al.
(2015a) contrasted the pattern of gene expression between
two genotypes at different temperatures. At 18° the authors
observed almost no differences in gene expression intensity
between two inbred Drosophila laboratory strains, but at
more extreme temperatures the expression divergence increased. This pattern was interpreted as evidence for canalized gene expression at 18°, which becomes lost when ?ies
are exposed to more extreme environments (decanalization)
(Chen et al. 2015a). 316 A. M. Jaks? ic´ and C. Schlötterer Figure 1 Temperature-dependent differential splicing between temperatures and between genotypes. Differentially spliced genes for pairwise
temperature comparisons (blue) are shown for each of the genotypes,
above the diagonal for Oregon-R and below the diagonal for Samarkand.
Differential splicing between genotypes at a given temperature is shown
on the diagonal (green). The heat map of pairwise temperature comparisons of differential splicing shows strong plasticity in both strains when
the most extreme temperatures (13 and 29°) are compared. Alternative
splicing between the more benign temperatures, 18 and 23°, however,
exhibited only a weak plastic response to temperature in both genotypes.
The data are based on reads mapping to the 39 side of the transcript (see
File S1 for more details). Despite its well-characterized in?uence on the phenotype,
alternative splicing plasticity has been studied only in the
context of exposure to acute stress conditions (Mastrangelo
et al. 2012; Long et al. 2013; Telonis-Scott et al. 2013; Vitulo
et al. 2014). Very little is known, however, about how longterm exposure to typically encountered environments modulates alternative splicing.
Here, we have used the data from the gene expression
study by Chen et al. (2015a), studied the in?uence of temperature on the pattern of alternative splicing in Drosophila
melanogaster, and compared this response between two genotypes. We contrasted the patterns of alternative splicing
to those of gene expression intensities from a study by Chen
et al. (2015a). Like for gene expression intensities, we found
that temperature has a strong effect on alternative splicing,
resulting in up to 568 (10.4%) genes being differentially
spliced between the two most extreme temperatures for
a given genotype. Even more surprising was the consistency of the pattern of increasing differences between
the genotypes on both levels of the phenotype at extreme
temperatures: at 18° only very few genes were differentially spliced between the two genotypes, whereas at extreme temperatures we detected the largest number of
genes with differential splicing. Despite the similarity of
this pattern, the involved genes did not overlap more than
expected by chance. Figure 2 Distribution of splice types for genes with
differential splicing between 13 and 29° for each of
the two genotypes. The most prevalent splice type is
exon skipping (red), followed by alternative 39 splice
site usage (yellow), 59 splice site usage (green), and
intron retention (blue). Overall, our statistically inferred
splicing differences are also re?ected by Sashimi plots
based on reads covering exon junctions (Figure 3). Materials and Methods
Females (f) from Oregon-R (O) and Samarkand (S) laboratory
strains were crossed with males (m) from both strains (Of 3
Om, Of 3 Sm, Sf 3 Sm, Sf 3 Om) in three replicates. After
2 days of egg laying at 23°, the eggs were transferred to one
of the four assaying temperatures (13, 18, 23, and 29°). Virgin females were used for extraction and sequencing of
mRNA. Further details on ?y rearing can be found in Chen
et al. (2015a) and Supplemental Material, File S1. Library
preparation and sequencing are described in Chen et al.
(2015a). Raw sequence reads (National Center for Biotechnology Information accession number SRP041398 and
SRP041395) were trimmed based on sequencing quality using PoPoolation2 (Ko?er et al. 2011) and mapped to the D.
melanogaster reference genome (Flybase assembly 5) using
the genomic short-read nucleotide alignment program (GSNAP)
(Wu and Nacu 2010). All mapped RNA sequencing (RNA-seq)
reads were randomly downsampled to the same coverage
and counted with a DEXSeq counter. Differential exon usage
analysis was conducted using the DEXSeq R package (Anders
et al. 2012). Due to 39 gene-transcript coverage bias in some
samples, we restricted some analyses by using only the reads
mapping to the 39 side of the transcript (Figure A, File S1).
Inheritance assignment followed the procedures described
in McManus et al. (2010) and Chen et al. (2015a) and is
described in detail in File S1. Splice types were assigned
based on the D. melanogaster annotation (see File S1). Gene
ontology (GO) analysis was performed using Gowinda (Ko?er
and Schlötterer 2012) and accounted for different splicing
opportunities (i.e., intron numbers) among GO categories. Gene set overlaps were assessed using receiver operating
characteristic (ROC)-like curves, which indicate if the overlap between two sets of ranked data are higher or lower
than expected by chance (curve above and below the diagonal). Further details about the methods used are described in File S1.
Data availability All raw sequence data used in this study is deposited in the
National Center for Biotechnology Information Sequence
Read Archive with accession numbers SRP041398 (Oregon-R
and Samarkand) and SRP041395 (F1). All un?ltered read
counts, custom scripts, and protocols will be available at
DataDryad.org. Results
We used 100-bp paired-end RNA-seq reads from two D.
melanogaster genotypes, Samarkand (S) and Oregon-R (O),
which were exposed to four different developmental temperatures ranging from 13 to 29° (Chen et al. 2015a). Each
genotype?temperature combination was analyzed in three
replicates. We measured alternative splicing by means of
exon usage (Anders et al. 2012), using only those multiexon genes with an average of at least 50 reads across all
samples in the analysis (Table C, File S1).
Temperature-mediated plasticity of splicing Pairwise comparisons of alternative splicing revealed a substantial effect of temperature, with up to 10.4% (568 out of
5463) of the multi-exon genes showing differential splicing Alternative Splicing Plasticity 317 Figure 3 Genotype- and temperature-dependent alternative splicing. Sashimi plots for exon skipping of the third exon (marked in yellow) of the gene
CG42351. Dark blue, Oregon-R 13°; dark red, Oregon-R 29°; light blue, Samarkand 13°; pink, Samarkand 29°. between two temperatures for a given genotype. The highest
plasticity of splicing was seen between the two extreme
temperatures, but as few as seven genes differed in splicing
between 18 and 23° in Oregon-R. Overall both D. melanogaster strains showed the same pattern of differential splicing with exons being more commonly retained at 13° and
spliced out at 29° in both strains (Figure G, File S1). Oregon-R
was more plastic than Samarkand (Figure 1).
The splicing differences between the two most extreme
temperatures (13 and 29°) within genotypes were mostly
caused by exon skipping (O = 67%, S = 65%) followed by
both alternative 39 (O = 13%, S = 17%) and 59 (O = 14%,
S = 11%) splice site selection, and with least changes caused
by intron retention (O = 4%, S = 5%; Figure 2).
Temperature-dependent differences in alternative
splicing between genotypes Despite the overall similarity of the two strains in splicing
patterns across temperatures, we systematically tested for
differential splicing between the two genotypes (Oregon-R
and Samarkand) at each of the four developmental temperatures (Figure 3 and Figure 4). The highest similarity in splicing between the two strains for a given temperature was
observed at 18°, with 1.21% of all tested multi-exon genes
(97 out of 8021) showing signi?cantly different splicing patterns. However, at the other three temperatures, 13, 23 and
29°, splicing differed between the two genotypes for 1.95%
(173 out of 8858), 7.99% (646 out of 8090) and 12.81%
(1049 out of 8186) genes, respectively; (Figure 4) suggesting
that difference in alternative splicing between strains is
strongly dependent on the assaying temperature. This pattern was previously observed for gene expression intensities 318 A. M. Jaks? ic´ and C. Schlötterer (Figure 4 inset). The difference in alternative splicing between the two genotypes at 13° becomes clearer after adjusting for variance in the 39 gene-body read coverage across
replicates (see File S1).
With temperature stress resulting in increasing differences in the splicing pattern between the two strains, we
were interested to understand this better. Since different
reaction norms of alternative splicing between the two
strains may have caused the differences between genotypes
at a certain temperature, we related these patterns to the
intrastrain plasticity between different temperatures. Plotting the fold change in exon expression between genotypes
within a temperature for each exon (corrected for overall
gene expression) and fold changes of exons with splicing
plasticity (differences between temperatures, within a
strain) against each other, clearly indicated that the two
are not congruent. Hence, we conclude that differences between strains are not a consequence of different reaction
norms for alternative splicing of the two genotypes (Figure
5). Further support for this lack of congruence comes from
ROC for exon expression intensities as well as a difference in
GO term enrichment for genes with signi?cant genetic differences and plasticity (Figure H, File S1).
Out of all splicing events that differed between the strains
for a given temperature, exon skipping was the most frequent
one (76%), followed by 39 alternative splicing site usage (14?
30%), and 59 alternative splice site usage (3?5%). The least
frequent event was intron retention (1%). This pattern was
very similar across the entire temperature range, with a trend
toward more exon skipping at higher temperatures (Figure
6). Importantly, a similar distribution of alternative splicing
events has been described previously (McManus et al. 2014). Figure 4 Genotype-speci?c alternative splicing. At 18° only a few
genes differ in splicing between
Oregon-R and Samarkand, while
at more extreme temperatures the
splicing patterns become increasingly
different. This pattern resembles the
one seen for gene expression intensity (blue inset; redrawn from Chen
et al. 2015a). Candidate genes for differential exon skipping Previously, Chen et al. (2015a) showed enrichment in the GO
categories ?spliceosome? and ?mRNA splicing, via spliceosome?,
indicating that the expression differences in the core splicing
machinery could result in the differences in alternative splicing
between the genotypes. Dominant inheritance of alternative
splicing between the genotypes also suggested that alternative
splicing regulation is guided mostly by trans-acting factors. To
test this hypothesis further, we took advantage of trans-acting
factors with genome-wide in?uence on alternative splicing.
The exon junction complex serves a central role in splicing
(Tange et al. 2004). Knockdown of two members of the exon
junction complex, mago nashi and eIF4AIII, increases the rate of
exon skipping (Tange et al. 2004; Ashton-Beaucage et al. 2010;
Wang et al. 2014). The three core exon junction complex genes
that can be found in the nucleus and can, therefore, have the
ability to interact with the splicing process, show a consistent
expression pattern across temperatures. At 13° they are more expressed in Oregon-R, while at 29° Samarkand has the higher expression level (Table 1). If the exon junction complex is involved
in the splicing differences between the two strains, we expect to
?nd more exon skipping in Oregon-R at 13°, while Samarkand
would have more exon skipping at higher temperatures. In support of this hypothesis, in Samarkand ?ies we ?nd on average downregulation of differentially spliced exons at 13°, and
the opposite pattern at higher temperatures (Table 1). While
these results strongly suggest a substantial in?uence of the exon
junction complex on the alternative splicing, the observation of
different genes being alternatively spliced across temperatures
indicates that other splicing factors may also shape the plasticity
of alternative splicing. Brooks et al. (2015) recently reported
56 splicing factors and their target genes. We used this set of
splicing factors to further test our hypothesis. In our data, 49 of
the factors reported by Brooks et al. were expressed (on average
at least 20 mapped reads across all samples). A total of 31 (63%)
of the splicing factors showed a similar pattern as the exon junction complex genes: they were upregulated in one strain at 13°
and upregulated in the other strain at 29°. Genes with differential
splicing between the two genotypes were enriched with genes
regulated for 19 splicing factors (Table D, File S1). Five splicing
factors, snRNP-U1-70K (FBgn0016978), RpS3 (FBgn0002622),
SC35 (FBgn0265298), RnpS1 (FBgn0037707), and Hrb27C
(FBgn0004838) showed the same concordance of expression
level and exon skipping as core exon junction complex genes
(Table D, File S1). The protein components of the spliceosome,
snRNP-U1-70K and SC35, are strong candidates for regulating Alternative Splicing Plasticity 319 Figure 5 Genetically vs. environmentally induced differences in alternative splicing. Log2-fold changes between Oregon-R and Samarkand at (A and B)
13° and (C and D) 29° are plotted against log2-fold changes between 13 and 29° in (A and C) Oregon-R and in (B and D) Samarkand. Exons that are
signi?cantly differentially spliced between genotypes (GD, green) mostly do not overlap with the exons that are plastic (ED) in Oregon-R (red) or
Samarkand (blue). Density plots on the top and on the right show the distribution of plotted points with corresponding colors. ED, environmental
differences; GD, genetic differences. differential splicing between the two genotypes. The auxiliary
protein component of the exon junction complex RnpS1 provides
further support for the importance of the exon junction complex
for the alternative splicing patterns seen in this study.
Similar patterns of temperature-dependent differences
among strains for gene expression and
alternative splicing Interestingly, the striking temperature dependence of differential splicing between Oregon-R and Samarkand is mirrored
for gene expression intensity (Figure 4) (Chen et al. 2015a). 320 A. M. Jaks? ic´ and C. Schlötterer While at 18° the differences in both splicing and gene expression intensity between genotypes are very small, at extreme
temperatures the differences increase. Given these parallel
patterns, we were interested in whether the same genes were
affected and compared the expression intensity differences of
the entire gene against the expression differences in each exon
(Figure 7; Figure I, File S1). Independent of the developmental temperature, genes with signi?cant differences in gene expression intensity have only limited overlap with genes with
differential splicing (Figure 7). These results suggest that
despite the overall similarity in temperature dependence of Table 1 Expression of nuclear core exon junction complex
negatively correlates with exon skipping at extreme temperatures
13°
FDR
mago nashi
tsunagi
eIF4AIII
Mean log2FC expression of exons
differentially spliced between
Oregon-R and Samarkand log2FC 0.077
0.915
0.769
0.136
0.035
0.914
20.26 29°
FDR
0.006
0.001
0.024 log2FC
20.923
21.024
20.659
0.3 FDR, false discovery rate; FC, fold change. Figure 6 Contribution of splice types to differential splicing contrasting
Oregon-R and Samarkand at each of the four temperatures. Exon skipping is the most frequent splice type among genes with differential splicing between genotypes and with increasing temperatures this pattern
becomes even more pronounced. differential splicing and gene expression intensities, both
processes are regulated by different mechanisms. This conclusion is further substantiated in a comparison of GO categories
that are enriched for genes with signi?cant differential splicing
or gene expression intensities at 23 and 29°. Despite both
categories harboring a signi?cant enrichment for some genes,
there is very little similarity in the enrichment patterns (GO
categories) between differential splicing and gene expression (Figure I, File S1). A similar pattern has been observed
by Brooks et al. (2015) who found that expression levels of
splicing factors that regulate alternative splicing of thousands of genes do not in?uence their expression intensities
(Brooks et al. 2015).
Dominance prevails for differential splicing The mode of inheritance of alternative splicing can be studied by contrasting two parental genotypes to offspring of
a cross between them. Between 92 and 99% of the genes did
not differ signi?cantly from the splicing pattern of both parents.
Splicing of most (83?96%) remaining genes matched one of
parents (i.e., were dominant; Table 2). Unexpectedly, this
dominance was not evenly distributed between the two parental genotypes and differed strikingly among temperatures
(Figure 8). This pattern was most extreme at 13 and 29°.
While at 13° the splicing pattern of Samarkand was dominant
for the majority of genes (58%) and made up for 70% of all
genes with dominant splicing inheritance, at 29° splicing of
most genes in F1 individuals matched Oregon-R (92%) and
corresponded to 96% of all dominant genes. At 18°, no
such imbalance of dominance was found (44% Oregon-R dominant vs. 56% Samarkand dominant). To…