Relating Whole-Genome Expression Data with Protein-

Protein Interactions

Ronald Jansen1, *, Dov Greenbaum2, * & Mark Gerstein1, 3, †

Departments of Molecular Biophysics & Biochemistry1,

Genetics2 and Computer Science3
266 Whitney Avenue, Yale University
PO Box 208114, New Haven, CT 06520
(203) 432-6105, FAX (360) 838-7861

[email protected]
[email protected]
[email protected]

* These authors contributed equally to this work.

† Corresponding author

Jansen et al. -1-

Abstract
We investigate the relationship of protein-protein interactions with mRNA expression
levels, by integrating a variety of data sources for yeast. We focus on known protein
complexes (from the MIPS catalog) that have clearly defined interactions between their
subunits. We find that subunits of the same protein complex show significant co-
expression, both in terms of similarities of absolute mRNA levels and expression profiles
-- e.g. we can often see subunits of a complex having correlated patterns of expression
over a time-course. We classify the yeast protein complexes as either permanent or
transient, with permanent ones being maintained through most cellular conditions. We
find that, generally, permanent complexes, such as the ribosome and proteasome, have a
particularly strong relationship with expression, while transient ones do not. However, we
note that several transient complexes, such as the RNA polymerase II holoenzyme and
the replication complex, can be subdivided into smaller permanent ones, which do have a
strong relationship to gene expression. We also investigated the interactions in
aggregated, genome-wide datasets, such as the comprehensive yeast two-hybrid
experiments, and found them to have an only weak relationship with gene expression,
similar to that of transient complexes. (Further details on
genecensus.org/expression/interactions and
bioinfo.mbb.yale.edu/expression/interactions.)

Jansen et al. -2-

Introduction
Analysis of gene expression data is currently one of the most exciting areas in genomics.
Computationally, it involves clustering and grouping individual expression measurements
and interrelating them to other sources of information, such as phenotypes, functional
classifications, or cellular responses (Golub et al. 1999; Brown et al. 2000; Califano et al.
2000; Gaasterland and Bekiranov 2000; Raychaudhuri et al. 2001; Subrahmanyam et al.
2001). In particular, functional assignment of uncharacterized genes can take place
through transferring the annotation from a characterized gene (gathered from databases
such as MIPS or GO (Ashburner et al. 2000; Mewes et al. 2000)) to an uncharacterized
gene when their expression profiles are strongly related by a similarity criterion (such as
the correlation coefficient). While this procedure is usually not sufficient to
unambiguously determine the function of an uncharacterized gene, it can be the starting
point (e.g. in target selection) for further genetic experiments, functional characterization,
or high-throughput proteomic analysis (Luscombe et al. 1998; Westhead et al. 1999;
Christendat et al. 2000a; Christendat et al. 2000b; Eisenberg et al. 2000; Emili and
Cagney 2000; Gerstein and Jansen 2000).

An important component of functional annotation is characterizing protein interactions as

these often circumscribe (or effectively define) protein function. Moreover, protein
interactions can often be described more precisely than protein functions. Thus, rather
than directly dealing with the general relationship between protein function and
expression, we look here at a sub-problem: the relationship between mRNA expression
and protein-protein interactions, especially those in protein complexes. A priori it seems
reasonable that there should be a well-defined relationship between the expression levels
of the subunits in a complex: since the functionality of many complexes hinges on the
presence of all the subunits, a haphazard and independent expression of any one subunit
would be energetically costly. For instance, the components of the ribosome are
regulated in a complex way but there is usually agreement that they should be present in
equimolar amounts, although this has not yet been measured directly (Nomura et al.
1999; Li et al. 1999; Woolford et al. 1991; Planta et al. 1997).

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We investigate this relationship for many of the known protein complexes in a
comprehensive, global fashion by interrelating many of the yeast datasets for protein
interactions and expression. The diversity and number of yeast experiments provide
high-quality data under varied conditions. Additionally, we investigate the relationship
between other types of protein-protein interactions (e.g. aggregated physical and genetic
interactions) and mRNA expression. Our work follows up on many recent analyses of
protein-protein interactions (Fellenberg et al. 2000; Hishigaki et al. 2001; Teichmann et
al. 2001; Walhout and Vidal 2001).

In general, our goal was to integrate and cross-correlate already existing data from
different sources and find general trends in it. This is an exploratory study prior to any
type of prediction. In a sense, this study can be understood as an exploration of the
knowledge already implicit in the current data but not yet obvious because, previously, it
has not yet been integrated and put together in this way.

Results
In our survey of existing data, we have used two different approaches to analyze the two
different types of expression data available: the computation of normalized differences
for absolute expression levels and a more standard analysis of the correlation of profiles
of relative expression levels (expression ratios). We explain these two approaches in
more detail in the following two sections.

Calculation of Normalized Differences between Absolute Expression Levels

In order to compare absolute mRNA expression levels between subunits of a protein
complex, we define the normalized difference Dij as follows:
Ei  E j
Dij  [1]
Ei  E j

where Ei and Ej are the mRNA expression levels of subunits i and j. This quantity defines
the difference as a fraction of the sum of the expression levels, thus allowing for a

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comparison of gene pairs of both high and low expression. Values for the normalized
difference range from 0 to 1.

For a group of N proteins in a complex we generally compute the normalized difference

not only for the pairs that are in direct physical contact, but for all (N2 - N)/2 theoretically
possible pairs, thus arriving at a distribution of normalized differences of these pairs for
each complex. We can then investigate this distribution of normalized differences and
compare it with those among randomly chosen proteins. In our following discussion we
often refer to the median of the (N2 - N)/2 protein pairs as a key summarizing statistic.

In general, we assume stoichiometric ratios of 1:1 between subunits, although equation

[1] could be adjusted to account for other ratios. But even then, as shown in the Methods
section below, we would not expect this quantity to always be close to zero due to the
relationship between mRNA and protein and also the noise in the expression data.

It should also be noted that there are obviously many limitations in treating GeneChip
and SAGE data as absolute measurements of mRNA expression (Schadt et al. 2000).

In order to judge the statistical significance of normalized differences for particular

groups of proteins we compare them to the control distribution of randomly chosen
protein pairs (see figure 1). An interesting theoretical aspect in this context is that if Ei
and Ej are random variables with an exponential distribution (which is a close
approximation to the actual distribution of expression of levels in the reference
expression set), then Dij is distributed uniformly between 0 and 1 (Pitman 1993). This
explains why we can observe a nearly uniform distribution of normalized differences for
randomly selected pairs of proteins (see figure 1).

Correlation of Expression Profiles for Relative Expression Levels

Analysis of expression profiles may be more useful than that of absolute levels for
characterizing interacting proteins that exist in unequal but stoichiometrically related
amounts (e.g., 3:1) as it refers to the relative shape of expression profiles. It can be

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carried out on data from cDNA microarrays (such as the Rosetta data) because only
relative rather than absolute expression levels are necessary. Specifically, we look at the
distribution of Pearson correlation coefficients for pairs of genes as the measure of
similarity. (Other measures of similarity are possible as well (D'haeseleer 1997; Wen et
al. 1998; Heyer et al. 1999; Qian et al. 2001).)

As the input for our procedure we use the expression vectors or profiles of all the
subunits of a complex and then compute their pair-wise correlations. Like for the
normalized difference, we compute the correlation coefficients for all protein pairs in a
complex, thus gaining a distribution of correlation coefficients. If the complex consists
of N subunits, this yields (N2 - N)/2 different combinations of protein pairs and thus
correlation coefficients. To summarize these distributions, we calculate the “average
correlation” (by which we mean the average of all pair-wise correlations within a
complex). As a suitable control to assess statistical significance, we use the distributions
of correlation coefficients for random groups of proteins and their averages (see
methods). We would expect correlations of close to 1 for subunits in a tight complex.
However, as we show in the Methods section this will not be exactly the case due to the
relationship between mRNA and protein abundances.

Results
We first outline some results obtained for specific protein complexes, then we proceed to
a more general overview of complexes.

Specific Complexes

Ribosome
It has long been known that the mRNA expression levels of the ribosomal proteins are
strongly correlated with one another (Johannes et al. 1999). Figure 1 shows the observed
distribution of normalized differences for protein pairs in the large subunit of the
cytoplasmic ribosome. The median of this distribution is 0.23, much lower than the
median of 0.5 for randomly selected protein pairs. While there is a wide range of
normalized differences (which may partially result from the fact that many proteins in the

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ribosome are known not to be expressed in a 1:1 ratio (Kruiswijk et al. 1978)), the
ribosomal distribution is clearly skewed towards zero. Distributions of the correlation
coefficients for protein pairs within the large ribosomal subunit are shown in figure 2.
For both the cell cycle and the Rosetta data the correlations tend to be much higher than
the random control.

Similar observations can be made for the proteins in the small cytoplasmic ribosome.
Key statistics are summarized in figure 3 in comparison to those for other protein
complexes. Furthermore, the two separate ribosome particles are strongly co-regulated.
In fact, the large and the small ribosomal particles cannot be differentiated by our
measures of expression similarity.

Proteasome
A second example of a complex whose individual subunits are strongly co-regulated is
the proteasome, which is involved in protein degradation and responsible for the rapid
breakdown of ubiquitinated proteins. Like the ribosome, the 26S proteasome can be
divided into two sub-particles: the 20S and the 19S (or 19S/22S regulatory particle). The
20S particle is present as a dimer in the center of the complex structure and contains the
catalytic core, whereas two 19S particles are attached to both ends of the 20S particle
dimer (Coux et al. 1996; Wilkinson et al. 1999).

The distribution of the normalized differences for all possible protein pairs in the 20S
proteasome is shown in figure 1. Like the ribosome, it is clearly skewed towards zero,
compared to the control, with a median of 0.29. Figure 2 shows the distribution of
correlation coefficients, which is strongly shifted to the right of the control, though to a
lesser extent than that for the ribosome. An investigation of the crystal structure of 20S
particle (Whitby et al. 2000) did not reveal any relationship with the gene expression
differences (e.g. proteins with slightly more random correlations tending to be more on
the surface of the particle).

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Similar results can be observed for the 19S particle of the proteasome (figure 3A). Also,
in terms of both measures of co-expression (normalized differences and correlation of
expression profiles) the 19S and the 20S particles of the proteasome form a single unit
that is difficult to separate by gene expression analysis. Part of the reason for this may be
that the common classification into 19S and 20S particles is based on the purification
procedure for the proteasome (Hochstrasser 2001) and thus does not necessarily reflect
functional or biochemical properties in a direct way.

One subunit, Doa4p, exhibits a very low average correlation (-0.02). Biochemical studies
have previously shown that not all proteasomes have Doa4p bound and that the Doa4p-
proteasome interaction is more likely to be transitory (Papa and Hochstrasser 1993; Papa
et al. 1999).

RNA Polymerase II Holoenzyme

We have seen above that the ribosome and proteasome can be regarded as strongly
associated and co-regulated multi-particle complexes. However, in some cases a
complex contains more loosely associated components. An example is the RNA
polymerase II holoenzyme, which contains the core RNA polymerase II together with the
more loosely associated SRB complex (Kornberg's mediator) and other smaller
components (such as the SWIF/SNF complex and the TAFIIs).

It is known that, unlike the RNA polymerase II core enzyme, the SRB complex and the
other holoenzyme components are only needed for the transcription of a fraction of genes
(Holstege et al. 1998). In other words, the holoenzyme is an example of a complex of
transitory nature with a permanent core. This permanent-and-transitory structure is
clearly evident in the gene expression analysis. For the core enzyme, the average
correlation in both the cell cycle and Rosetta data sets are significantly higher than for the
random control (Figure 3). However, for the SRB complex and a variety of other, smaller
components (e.g. the TAFIIs) the average correlations are virtually indistinguishable from
the random control.

Replication Complex

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Another example of a transient complex is the replication complex, which binds to DNA
and is needed for the initiation of replication. The replication complex can be subdivided
into a number of sub-components: the MCM proteins, the origin recognition complex and
the DNA polymerases  and (Aparicio et al. 1997).

As a whole, the replication complex exhibits a low average correlation not significantly
different from that of the random control (figures 3 and 4). However, figure 4 shows how
the entire complex breaks into subcomponents in terms of correlations in the cell-cycle
experiment. The individual correlations for each of the subcomponents are much higher
than that of the complex as a whole. This indicates that the replication complex is
composed of independent units in terms of expression regulation. Using the permanent-
transient terminology, each subcomponent behaves similarly to an independent
permanent complex, whereas the replication complex as a whole can be characterized as
transient. The permanent sub-components can be seen to come together to form a
transient functional entity. (Note, this effect is more evident in the cell cycle experiment
than the Rosetta data, as it should only be observable in a synchronized population of
cells, not those averaged across the cell cycle.)

Complexes in General: Permanent vs. Transient

In discussing the specific examples above, we have found the permanent or transient
nature of the association to be an important feature. This distinction is, in fact, valuable
in a more general context. As shown in figure 3, we have a priori formalized a division
between "permanent" complexes, which are maintained throughout the cell cycle and
most cellular conditions, and "transient" ones, which we define here as a group of
proteins that do not consistently maintain their interactions. That is, the existence of a
transient complex is temporal and specific to a part of the cell cycle or a subset of cellular
states. We are aware that the division into the two absolute categories "permanent" and
"transient" is perhaps somewhat oversimplifying as there can be varying degrees and
combinations of these attributes (see Discussion).

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In figure 3, we show a general classification of the large MIPS complexes into permanent
and transient classes, together with key statistics (details of the classification method are
given in the caption). We list all complexes with more than 10 subunits (which together
account for ~80% of all the protein-protein interactions in the MIPS complexes), with
smaller complexes listed on our website. Figure 3B shows a graphical representation of
the complex list, synthesizing the correlations for both the Rosetta and cell-cycle
experiments with the normalized differences. It clearly shows that there is a greater
tendency for permanent complexes to have higher average correlations than for transient
ones.

Comparing the average correlations in Figure 3A against random controls allows us to

derive P-values for the statistical significance of the correlation. As shown in the figure,
these are less then 10-4 for most of the permanent complexes. On the other hand, they are
considerably higher, and thus less significant, for transient complexes. The separation
between permanent and transient complexes is also evident in terms of the normalized
difference statistics, although not as strongly.

Aggregated Protein-Protein Interaction Sets

From our analysis above it seems reasonable to conclude that there is indeed a strong
relationship between mRNA expression and the protein-protein interactions in
“permanent” complexes. This raises the question whether similar observations can be
made for other types of protein-protein interactions. We briefly summarize here the
degree to which the interactions in the aggregated interaction datasets, such as the yeast
two-hybrid, are related to expression.

Figure 1 shows the distribution of normalized differences and figure 2 the distributions of
correlation coefficients between interacting proteins in the aggregated data sets. The
distributions of normalized differences are relatively similar to those of the transient
protein complexes. The physical interactions show the smallest median normalized
difference while the yeast two-hybrid interactions have a median normalized difference
closest to the random control (~0.5). Figure 2 shows that the correlation distributions for

Jansen et al. - 10 -
the aggregated data sets are fairly similar among themselves and only slightly shifted
towards the right of the distribution curve for random protein pairs. This, again, is very
similar to the behavior of transient protein complexes.

Thus, overall, it seems fair to conclude that the aggregated protein-protein interactions
are related to mRNA expression in a similar fashion as the transient protein complexes.

Discussion and Conclusion

We have investigated the relationship of protein-protein interactions and mRNA
expression levels, integrating and surveying a variety of data sources for yeast. We have
focused our investigation on the protein interactions wi\thin specific complexes. While
we have demonstrated a strong relationship between expression data and most permanent
protein complexes, this relationship is much weaker for transient protein complexes as
well as for the aggregated sets of protein-protein interactions (i.e. physical, genetic and
yeast-two hybrid interactions).

Issues with Permanent-Transient Classification

Our complex classification scheme -- separating most complexes into either permanent or
transient -- while useful cannot account for all complexes in the MIPS database. Some
complexes may not clearly fit into the permanent-transient classification. We list a few of
these as "other" in figure 3. Moreover, the complexes list is a compilation of current
biochemical knowledge and therefore reflects its inherent limitations (sometimes not all
subunits are known or some proteins are mistakenly assigned to a complex).

Of course, even for the complexes that we do classify, the terms "transient" and
"permanent" are somewhat of an over-simplification. In particular, our detailed
discussions of the RNA polymerase II holoenzyme and the replication complex above are
precisely two examples where our simplified terminology fails to completely explain the
situation since these complexes are somewhere between fully "transient" and
"permanent".

Jansen et al. - 11 -
One can think about the distinction between permanent and transient in terms of the
mathematical model introduced in the Methods section. Whenever a complex is formed,
its subunits tend to be expressed at equimolar protein concentrations: Pi  Pj and

dP dt i  dPj dt dPi dt  dPj dt (where Pi and Pj are the protein concentrations of two

subunits i and j). If the complex is "permanent", then these conditions should be
approximately or vaguely met. If the complex is "transient", then these conditions can be
relaxed in those situations where the complex is not formed. There are some complexes,
that are always formed ("permanent") whereas the "transient" complexes are only formed
under particular conditions. There can be different degrees of being transient: for
instance, complexes that are formed under 80% of conditions or those that are formed
under 20% of conditions. The transient complex formed under 80% of conditions
behaves almost like "permanent" (i.e., 100% of conditions), whereas the transient
complex formed only 20% of the time would be expected to show less significant
normalized differences and correlations.

If one goes as far as to accepts the premise that the subunits in a complex should be
present at equimolar amounts, then it is perhaps circular reasoning to say that they should
also be co-expressed.

Complexes versus the Aggregated Interactions: the Need for Structures

We found it difficult to discern expression-based relationships in the aggregated data sets.
This may be due to the generalized and heterogeneous nature of the aggregated data sets,
(e.g. inconsistent physiological conditions, false positives and false negatives).
Moreover, both the aggregated sets and the transient complexes suffer partially from the
limited amount of mRNA expression data as their interactions may occur under particular
physiological conditions that may not be sampled by mRNA expression data. Our
results, thus, illustrate the difficulty in drawing general conclusions for the pair-wise
interaction sets and highlight the important role clearly resolved crystal structures of
complexes, detailing protein interactions between subunits, have in studying protein-
protein interactions.

Jansen et al. - 12 -
Noise in the expression and interaction data
In general, the interactions in the aggregated datasets exhibited surprisingly little
deviation from randomness in terms of the co-expression of interaction pairs. This was
most strongly observed for the yeast two-hybrid data. It is true that, overall, this
deviation from randomness is statistically significant. All the same, the gene expression
data and the aggregated protein interaction data do not reinforce each other strongly and
it seems that the prediction of these type of interactions from expression data would be of
little benefit.

Perhaps the most optimistic view of this situation is that the strong degree of
independence of the two types of data makes both of them suitable for use in machine-
learning approaches to characterize genes of unknown function: if they were strongly
correlated, then one type of data could perhaps well replace the other since it represents
very similar information. A negative view would be that the reason for the surprisingly
weak relationship between the aggregated interactions and mRNA expression are to be
found in the problems with the either the expression or the interaction data.

We feel confident that our results are robust to the noise in the expression data for the
following reasons. With respect to the correlation analysis of expression profiles roughly
the same results (in terms of statistical significances) can be obtained for two independent
data sets (the cell-cycle timecourse and the Rosetta knockout series). The normalized
difference analysis is perhaps more sensitive to problems with the data, in particular,
considering that the measurement of absolute expression levels with gene chips is
problematic to start with. However, we have looked at an integrated dataset from various
chip experiments and the SAGE data, thus averaging out errors to some degree (see
Methods). In addition, for both the correlation and the normalized difference analysis, we
have concentrated on the statistical significance of distributions rather than relying on the
error-prone data for individual protein pairs, thus observing more robust, aggregate trends
for whole complexes and groups of proteins.

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Part of the aggregated data, in particular the yeast two-hybrid data, represent a relatively
new approach to studying protein-protein interactions and it is interesting to note that it,
obviously, includes some interactions implied by the complexes. However, the degree of
intersection with possible complexes interactions ranges from 35% for the physical
interactions to only approximately 6% for the yeast two-hybrid data (as a fraction of the
number of interactions in the aggregated datasets). This is surprisingly low, given that
the yeast two-hybrid data is from experiments that covered the complete genome (Uetz et
al. 2000; Ito et al. 2001). Independently, Ito et al. (2001) have reported that only a small
fraction of the previous yeast two-hybrid data (Uetz et al. 2000) overlapped with their
own yeast two-hybrid results. (Although Ito and colleagues assumed that their core data
was similar in quality as the Uetz data, the fraction of interactions present in both datasets
was only 16.8% for the Ito core and 20.4% for the Uetz data).

mRNA vs. Protein Expression

The co-regulation of subunits in a protein complex should be primarily observable in
terms of protein abundance and only indirectly in terms of mRNA expression. Several
recent studies have attempted to investigate the relationship between mRNA and protein
expression levels in yeast cells and found them to be correlated to various degrees
(Anderson and Seilhamer 1997; Futcher et al. 1999; Gygi et al. 1999; Greenbaum et al.
2001; Lian et al. 2001). Other factors beyond transcription, such asGenerally, post-
transcriptional regulation, are is more difficult to investigate given the sparse data
resources currently available for protein abundance levels. It is possible that in some
situations co-regulation occurs mostly on the protein level, almost independent of cellular
mRNA levels. Particularly, those permanent complexes that do not have high levels of
correlation in our analysis may be indicative of translational or post-translational control
and could be a starting point for further experimental investigation. See also the Methods
section for further discussion.

(Additional information can be found at genecensus.org/expression/interactions and

bioinfo.mbb.yale.edu/expression/interactions.)

Jansen et al. - 14 -
Methods

Interactions Data Sources

The primary focus of this paper are the interactions occurring within specific complexes.
These were obtained these from the MIPS complexes catalog (Fellenberg et al. 2000),
which represents a carefully annotated, comprehensive dataset of protein complexes
culled from the scientific literature. In addition, we looked at other types of protein-
protein interactions from large "aggregated" datasets collecting many heterogeneous pair-
wise interactions. We collected these from the MIPS catalogs of physical and genetic
interactions(Fellenberg et al. 2000), databases of interacting proteins (DIP and BIND)
(Bader and Hogue 2000; Xenarios 2000), and a comprehensive collection of yeast 2-
hybrid experiments (Y2H) (Cagney et al. 2000; Ito et al. 2000; Schwikowski et al. 2000;
Uetz et al. 2000; Uetz and Hughes 2000; Ito et al. 2001). These interactions are
subdivided into groups based on their method of discovery. They include physical
interactions (e.g., collected through co-immunoprecipitation and co-purification), genetic
interactions (e.g., determined through genetic means such as synthetic lethality or
suppression experiments), and yeast two-hybrid pairs.

Expression Data Sources

We included two different types of expression measurements in our analysis: absolute
expression levels in vegetative yeast cells as determined by SAGE or gene chip
experiments, and profiles of ratio-type expression data from microarray experiments. For
the first type, we use a comprehensive reference set, which we merged and scaled
together from a variety of Affymetrix GeneChip and SAGE datasets (Velculescu VE
1997; Holstege FC 1998; Roth et al. 1998; Jelinsky and Samson 1999) into a single
representative data source (scaling details on our website; Greenbaum et al. 2001). For
the expression profiles, we focused on two different datasets: a cell cycle experiment
(Cho et al. 1998) and the Rosetta yeast compendium (Hughes et al. 2000). The two
datasets provide a fairly good sampling of the possible cellular states of yeast and
represent different experimental methodologies. The cell-cycle data contains expression
profiles obtained from synchronized cells over the course of two cell cycles, whereas the

Jansen et al. - 15 -
Rosetta data contains genome-wide expression ratios for 300 stationary cell states, which
are derived from 280 gene deletions and the 20 drug interaction experiments.

Efficient Calculation of the Average Correlations

For two expression ratio profiles Xi and Xj (transformed to average 0 and standard
deviation 1), the Pearson correlation coefficient ij is given by the dot product:
1
ij  Xi  X j ,
M 1
where M is the number of elements in the profiles Xi and Xj. The profile X can be
computed as a ‘Z-score’ from the measured expression ratio profile x, through the

xk  x
relation X k  , where x denotes the average and σx the standard deviation of
x
values in x, and Xk and xk are the kth components of their respective profiles.

Given a group of N genes we can compute the correlation coefficient matrix R, where
each element ij of the matrix denotes the Pearson correlation coefficient between genes i
and j. We can then compute the average correlation coefficient  by averaging the
matrix elements (excluding the main diagonal). This statistic gives an idea of the overall
similarity of the expression profiles in a group of genes. Although there are O(N2)
elements in R, the computation time for  can be kept proportional to O(N) by using the
linearity of the correlation to calculate  as follows:
1 N  1  1 
 
  R ij  N  2
  XT  XT  N  ,
N  N  i, j  N  N  M 1
2

N
where X T   X n is the sum of all expression profiles in the group of N genes.
n1

Kinetic Model of the Relationship between Protein and mRNA

Concentration
For a protein complex that is perfectly co-regulated we can assume that its components
are present at equimolar amounts and change similarly over time. So for the protein
concentrations Pi and Pj of two different subunits i and j we would get: Pi  Pj and

Jansen et al. - 16 -
dPi dt  dPj dt . Using a simple model for the relationship between mRNA and protein

concentrations, we can see how even under these ideal conditions similarity measures
based on the mRNA concentrations would deviate from perfect results. For instance, A a
linear kinetic model for the protein concentration Pi and the mRNA concentration Ri of a
subunit i in a complex is given by:
dPi
 k Ri Ri  k Pi Pi
dt
where kRi is an mRNA translation rate constant and kPi is a protein degradation constant.

Why expression profile correlations have to be less than one

For two subunits in a complex with Pi  Pj  P and dPi dt  dPj dt dP dt i  dP dt j ,
we can deduce:
k Ri Ri (t )  k Rj R j (t )   k Pi  k Pj  P(t )

It is clear that only under the strong assumption that the two protein degradation
constants are equal (kPi = kPj)
Ri (t ) k Rj
  const
R j (t ) k Ri

from which would follow corr(Ri, Rj) = 1. Otherwise, corr(Ri,Rj) < 1.

Why normalized differences are greater than zero

Furthermore, assuming steady-state (that is, dPi dt  dPj dt  0 dPi dt  0 ), we can
deduce the following relationship for the relationship between the mRNA levels of two
complex subunits:
k Rj k Pi
Ri  Rj
k Pj k Ri

Thus, the two mRNA expression levels are only expected to be equal if the ratios of the
rate constants for translation and degradation are the same for both proteins. This is not
necessarily the case for the subunits of a complex and therefore normalized differences
should not be expected to be zero.

Jansen et al. - 17 -
It is clear that the arguments above are based on a variety of simplifying assumptions. In
reality, there are additional factors (such as the noise in the expression data, the stochastic
nature of gene expression) that add even more difficulty to the analysis of mRNA levels.

Jansen et al. - 18 -
Figure Captions

Figure 1
Distributions of normalized differences for various groups of proteins in boxplot
representation. The normalized difference Dij is a measure of the relative similarity of
two absolute gene expression levels Ei and Ej. The middle panel shows the distribution
for two protein complexes (the large ribosomal subunit and the 20S proteasome). Note
that we considered all theoretically possible protein pairs within the protein complex (as
indicated in the schematic drawing above the panel). The right panel shows the
distribution for the aggregated datasets of protein-protein interactions (Y2H is yeast two-
hybrid) (Bader and Hogue 2000; Cagney et al. 2000; Fellenberg et al. 2000; Ito et al.
2000; Schwikowski et al. 2000; Uetz et al. 2000; Uetz and Hughes 2000; Xenarios 2000;
Ito et al. 2001). Unlike in the complexes, where we consider interactions among a whole
group of proteins, the interactions in the aggregated datasets are specific to individual
protein pairs (see schematic drawing). The left panel shows two control distributions of
the normalized difference, on the left for pairs of nuclear and cytoplasmic proteins --
which presumably, because of spatial separation, do not interact -- and on the right for
any random protein pair ("all transcripts") in yeast. The distribution of nuclear versus
cytoplasmic proteins is strongly skewed towards one (the maximum value of the
normalized difference), which is partially explained by the fact that cytoplasmic proteins
tend to have higher expression levels than cytoplasmic ones (Drawid 2000; Drawid and
Gerstein 2000). The distribution of all transcripts is nearly uniform (with a median of
0.5) -- see Methods. The complexes distributions are clearly skewed towards zero with
medians between 0.2 and 0.3. The medians of the distributions of the aggregated datasets
are still somewhat smaller than the control median, most notably for the physical
interactions dataset; on the other hand, there is virtually no difference between the control
and the distribution of the yeast two-hybrid dataset.

The aggregated data, obviously, includes some interactions implied by the complexes,
with the degree of intersection ranging from 35% for the physical interactions to
approximately 4%6% for Y2H.

Jansen et al. - 19 -
Figure 2
Distributions of correlation coefficients between expression profiles. In part A we show
distributions of the average correlation  N of N genes for the cell cycle experiments.
The gray curve in the background represents the case N = 2 (i.e., simply the distribution
of pair-wise correlations). In the case of N > 2,  N is defined as the average of all
possible (N2-N)/2 pairwise correlations among the N genes. We show here, as examples,
the distributions for N = 3 and N = 5. The distributions obviously become narrower,
reflecting the fact that it becomes more unlikely to find large groups of strongly
correlated genes at random as N increases.

These distributions provide a suitable control for the observed correlations between pairs
of genes (N = 2) or for the average correlations among the subunits of a complex (N > 2).

We have developed a method to efficiently sample the distribution curves f(  N ) (see

AppendixMethods). Based on the distribution function of f(  N ) we can calculate a one-
sided P-value:
1
P(  N )   f (
N
N )d N

This P-value then represents the chance that a group of N randomly selected genes could
exhibit an average correlation greater than or equal to that of a complex with N proteins
(see figure 3).

Part B and C show the distribution of pair-wise correlations for both the cell cycle and
the Rosetta experiments in two protein complexes (the ribosome and the proteasome) as
well as for the aggregated datasets (genetic, physical and Y2H). The gray curves in the
background are the control distributions for N = 2 as explained above. The distributions
for the ribosome and the proteasome are strongly shifted to the right of the control; this
effect is much weaker for the datasets of aggregated interactions.

Jansen et al. - 20 -
Figure 3
Part A consolidates various key statistics shown in figures 1 and 2 for the ribosome and
proteasome as well as for a large number of protein complexes. We list all protein
complexes from the MIPS catalog having at least 10 ORFs. The complexes are divided
into three classes: permanent, transient or "other" (see below). Some complexes can be
divided into smaller sub-complexes (e.g., the ribosomes) as indicated. The table lists
(from left to right) the average expression level of the complex, the median normalized
difference (see figure 1A), the average correlation for the cell cycle and Rosetta
experiments (see figure 2), the negative logarithm of the P-value of the average
correlations in both experiments (see figure 2), and the size of the complex in terms of
the number of ORFs.

In general, the P-values for the average correlations are very low for most of the
permanent protein complexes (accordingly, -log10(P) is very high), indicating that these
averages are significantly greater than for random groups of proteins of the same size.
The same cannot be observed for the transient protein complexes, for which the
correlation averages are usually much smaller.

The section "other" at the bottom of part A contains complexes that are either difficult to
classify as permanent/transient or for which, due to very small turnover rates, down-
regulations of mRNA levels take a very long time to affect protein abundance. The H+-
transporting ATPase can be thought of as containing a mixture of permanent and transient
components at the same time(Kane 2001). The nuclear pore complex (NPC) and the
TRAPP complex are known to have low turnover rates (Bucci and Wente 1997; Winey et
al. 1997; Sacher et al. 1998; Barrowman et al. 2000). The NPC has relatively small
average correlations, but this still yields P-values of 10-3 (cell cycle) and <10-4 (Rosetta)
because the nuclear pore complex is a relatively large aggregation of proteins, and even
these weak average correlations are very unlikely to occur for random groups of proteins
of this size. The TRAPP protein complex, while existing throughout the cell cycle, has a
low turnover rate and as such its mRNA expression data would not be sufficient for our
analysis.

Jansen et al. - 21 -
The RNA polymerase holoenzyme is composed of both permanent and transient
components. Note that the MIPS complexes catalog does not include the SWI/SNF
chromatin-remodeling complex and a subset of basal transcription factors (Wilson et al.
1996) as part of the holoenzyme, thus we list them separately here.

The list does not include those categories from the MIPS complexes catalog that do not
really represent protein complexes per se but rather aggregations of disparate proteins
that are involved in similar types of complex interactions, such as the "actin-associated"
and "tubulin-associated" protein groups.

Part B shows a graphical representation of part of the protein complex statistics from part
A. The abscissa and ordinate represent the average correlations in the cell cycle and the
Rosetta data, while the bubble sizes are a function of the normalized differences (larger
bubbles represent larger normalized differences). In general, the permanent complexes
tend to be located in the upper right region of the plot, whereas transient complexes are
closer to the random control in the lower left.

Figure 4
Part A of the figure shows a representation of the replication complex and its components
on the same coordinates as the protein complexes in figure 3B. The transient replication
complex can be decomposed into smaller complexes: the origin recognition complex, the
MCM proteins, and the DNA polymerases  and . Whereas the whole replication
complex exhibits an average correlation close to zero (in both the cell cycle and the
Rosetta data), the four smaller complexes show greater correlations in the cell cycle
experiment. The four sub-complexes behave more like permanent complexes than the
replication complex as a whole.

Part B shows the correlation coefficient matrix for the subunits of the replication
complex derived from the cell cycle data. The upper triangle of the correlation matrix
shows the individual correlation coefficients for particular gene pairs (with darker colors

Jansen et al. - 22 -
indicating higher correlations). The lower triangle shows the average correlations for
subgroups of proteins (representing the MCM proteins, the two DNA polymerases, and
the origin of the replication complex) within the complex as a whole. The table on the
right side shows which genes belong to which subgroups in different colors. The genes
were ordered with unsupervised clustering (average linkage) without regard to their
classification according to the three subgroups. It can be seen that this order reflects the
separation according to the subgroups very well (only the proteins in the two DNA
polymerase cannot be separated into two groups). An exception is the CDC45 protein
that belongs to the MCM proteins but tends to cluster with the DNA polymerases.

Jansen et al. - 23 -
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