Evidence of Limited Structural Organization in Globin
Intron Sequences of Messenger RNA
Wayne Dawson 1

Kenji Yamamoto 2

dawson@ims.u-tokyo.ac.jp

backen@lute.is.s.u-tokyo.ac.jp

1 Human Genome Center, Institute of Medical Science, University of Tokyo

4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan
University of Tokyo, 6-28-3 Mejirodai, Bunkyo-ku, Tokyo 112, Japan
Abstract
The structural signi cance of introns in and globin genes were evaluated in terms of thefolding (or stacking) free energy of the nascent RNA sequences as obtained from secondary structure calculations. From calculations of the folding free energy of exons and introns, it was foundthat introns tend to have a more folded (globular) structure compared to the exon counterparts segments. The results suggest that signi cant biological information is present in all intronic sequences
including spliceosome type introns.
2 Lab. Clinical Microbiology and Immunology, Bun'in Hospital, Medical School1

Introduction

Since the discovery of introns, considerable progress has been made at understanding the generalmechanisms that drive the intron splicing apparatus in eukaryotic messenger RNA (mRNA). Notwithstanding, in many respects, introns seem to carry no functional role in the pre-RNA (heterogeneous
nuclear RNA (hnRNA)) sequence other than to interrupt the protein coding sequence of mRNA.
Yet despite nearly a billion years of evolution [10, 33], this functionless intron has remained in the
eukaryotic genes. Moreover, the number of introns has often increased in the pre-RNA sequence particularly for the higher forms of life [33]. To the contrary then, there is reason to think that the intron
important to the genetic apparatus.
In some cases, the 3D structure of the intron in nascent mRNA exhibits something akin to an
enzymatic role in which the intron splices itself out of the gene [15, 25]. However, we also have introns
that depend on a complex splicing apparatus to successfully extricate themselves from the pre-RNA
sequence [22, 26]. Moreover, in some cases, much of the intron can be deleted or exchanged withanother intron sequence, yet (unless there are cryptic sites involved) there is change in the splicepoints [35]. Hence, not only do we lack understanding about the purpose of the intron, we still don't
really know how much information (if any) is contained on a given intron sequence.
The majority of the introns depend on the same consensus sequence \GU 1 1 1 AG" in which \GU"is located at the 5 end of the intron and \AG" is located at the 3 end [31]. This dinucleotide patternis the basis for the so-called \GU 1 1 1 AG rule". Generally, the exons do not appear to in uence any
steps in this splicing process neither chemically nor enzymatically although more recent ndings aresuggesting some in uence [12]. Presently, four major types of introns are known.
The spliceosome type introns are the most common in which a host of small nuclear RNA (snRNA)sequences [21, 22, 23, 26] (and usually a number of auxiliary enzymes called SR proteins) [22] arerequired to cleave the introns from the hnRNA sequences. The precise cleaving mechanism of hnRNAis still under investigation but generally appears to involve a complex interplay between the snRNA,
the SR proteins, and several short consensus sequences on the intron where the spliceosome apparatus(snRNA) hybridizes and eventually splices the intron out. We refer to this large splicing apparatus as
the small nuclear ribonucleoprotein complex (snRNP).
is

no

0

0

49

Another type of intron has similar properties to the snRNA mentioned above, but the splicing
sequence follows an \AT 1 1 1 AC rule". The proportion of these introns is much smaller than theformer, however, they compose roughly 1/10000 of the population of snRNA and they often require
their own special snRNP (U12) to carry out splicing [31].
The other types of introns so far isolated are called self splicing introns: type I and type II. GroupI exons are found in the lower eukaryotic cells, ciliates like, and slime molds
like[3]. Group II introns are found in organelles and bacteria. For these
intron sequences, the secondary structure and tertiary structure determine the catalytic behavior of
the introns [14, 15]. These type of structures are sometimes referred to as ribozymes.
The most complete way to examine the current problem of intron interactions within the hnRNAsequences would be a quantum chemistry calculation at thelevel. However, such calculations
on these complex biological system are completely inaccessible to the current genre of computers. The
next level is semiempirical calculations in a subset such as the 3D structure. However, at present,a robust calculation scheme of the general 3D structure remains rather elusive. MD simulation of
the relaxation of large biological structures (minimum of 1 - 1000 s for a 400 nucleotide sequence)are extremely expensive when one considers the number of atoms involved, the computation time foreach iteration, and the increment (1 fs) for each iteration [39]. The demands on computer memory
can also be daunting for a simple calculation even at the semiempirical level. Finally, these expensive
calculations require a well developed strategy to interpret the complex interactions found in molecular
dynamics simulations. It is the purpose of this work to help clear a path for this strategy.
Hence, to gain a foothold on future calculations of this far more complex 3D structural analysis,
we have begun by resorting to a much simpler strategy of estimating the free energy obtained fromthe secondary structure of these sequences. Secondary structure calculation have been found to assist
researchers in development the full 3D structure [40]. Generally, the 3D structure can be obtained byfolding the secondary structure into the more complex tertiary structure [13, 40].
Estimation of the free energy of these sequences is useful in establishing how compact or globular
these structures are. A very compact (highly folded) structure also has a large negative free energy
and a loosely bonded structure (marginally folded) should exhibit a small negative free energy. Free
energy calculations are very inexpensive to carry out in a secondary structure calculation.
We chose the globin family because it is common throughout the Chordata phylum and spans all
the vertebrates. The GenBank database contains about 40 representative complete(CDS), which is fairly large for a typical database of CDS. With such alarge data set,
the results of this investigation are expected to
the general properties of introns and exonsin the globin gene family. Furthermore, to the best of our knowledge, globin hnRNA only exhibits
constitutive splicing which eliminates any unusual splicing related e ects. Finally, no recognizable
Alu, L1, or MER1/2 sequences are found in any of the introns in these sequences; although Alu andL1 repeats are contained within sections of the entire globin gene [9]. All of the genes in the globin
family appear to have three exons and two introns. The exon sequences have a number of common
conserved elements, but the introns are often vastly di erent even between related families of species.
In this work, we report that, in the globin family, there isevidence for internal structure inthese snRNP based introns [6, 38]. We also suggest that this research may also serve to complimentsearch techniques for 5 /exon/intron/3 boundaries in unknown sequences.
Tetrahymena thermophile

Physarum

polycephalum

ab initio

coding sequencescomparatively

approximate

some

0

2

0

Methods

Unless otherwise noted, all calculations were carried out using the SGI Origin 2000 computer at the
Human Genome Center, Institute of Medical Science University of Tokyo. The software used in thiswork included a program written by the authors [6, 38], and the Wisconsin GCG Package which
includes M. Zuker's mfold and foldrna programs [40].
50Gather data:(1) Obtain hnRNA CDSa of gene.
(II) Evaluate the distribution:(2) divide CDS into introns, exons, 5 ,(1) nda max(1G) = 1Gj , min(1G) = 1Gi ,and 3 subsequences.
(2) evaluateb 2 = abs(1Gj 0 1Gi )(3) For each subsequence: calculate 0!
0!
the free energy (1G) for N -shued( )21 N 9(1 i 1 j )k[limN !1 (1 i ) ! (1 )limN !1 (1 j ) ! (1 )]sequencesb.
For a \spontaneous" (favorable) reaction the free energy must
(I)

0

0

a

i; j

G

and

G ;

G

max

G

min

max

G

min

G ;

G

b

a
b

yield a negative value.

CDS = complete coding sequence sample set of shued sequences.
N

Analyze the m-bin histogram:(1)use
the m-bin histogram indices as a(III) Construct a histogram:sequence independent normalizationa.
(1) divide 2 into m sections.
(2) Find the index of maximum density(2) calculate G(l) = max(1G) 0 1Gl,
for the shued sequences.
l 2 N ).
0!
,! (where
(
i+1)2
i
2
(3) Find the index of original unshued(3) locate G(l) k m G(l) < msequence.
(where i < m, and l 2 N )(4) Compare the indices.
(4) construct an m-bin histogram based on
assignment in (3).
For all histograms compared in this way,(IV)abe the same value.

m

Figure 1: Flow chart of the approach used to evaluate the exon and intron structure in hnRNA.
In this work, the word \structure" will be used to refer to both the(as in thegeometrical arrangement of atoms or molecules in a sequence of hnRNA) and(as
in the chemical bonding and functional groups of a particular arrangement of atoms or molecules).
When referring to secondary structure, or to bulk structural features (such as hair pins) we will specify
these higher levels of abstraction as. The word \function" will be used to refer tothe biological function of a large protein or nucleotide sequence.
physical structurechemical structure

secondary structure

2.1

Computational methods

A ow chart of the approach used in this work to examine the exon and intron structure is diagrammedin Fig. 1.
First, we obtained complete CDS of the hnRNA. In the current work we found 10 -globin species
with 24 complete CDS, and 12 -globin species with 20 complete CDS.
Next, we sliced the hnRNA into its intron, exon, 5 and 3 sections. A set of N -shued sequences
for each section was created and the free energy (FE) was evaluated from the respective secondary
structure.
From the results of the calculated FE values in Fig. 1(I), and the distribution of FE obtained inFig. 1(II), a histogram of m indices is constructed (Fig. 1(III)).
Finally, maintaining the same binning between histograms, we then compared the FE of the original
sequence with the mean free energy of the shued sequences and assigned the appropriate index. Thismethodology is carried out for all introns and exons in a given hnRNA.
A typical histogram of the FE is shown in Fig. 2. The horizontal axis represents the bin number(index) which also carries the function of normalization when the number of bins is kept constantfor all histograms (Fig. 1(IV) and 2). In Fig. 2, the numbered columns along the horizontal axis
0

51

0

represent the normalized FE (or a m-bin normalization of the free energy) in which the bin index
numbers extend from the 1st-bin to the mth-bin. In the current convention, the 1st -bin represents thelargest ( : :, the most negative) free energy, and 11th-bin represents the smallest free energy ( : :, theleast negative) obtained in the shued distribution. The mean FE is located between the 6th -bin and7th -bin. The FE of the original introns and exons are then plotted with respect to the distribution
found in Fig. 2 and are shown in the exon-intron plot of Fig. 3.
i e

2.2

i e

Analysis

The very rst problem one encounters in examining the energy distribution of all possible shuedsequences generated from a given coding sequences of length n, is that evaluating possible conformations is intractable. For example, suppose we have a sequence of 20 nucleotides (nt) and equal
percentages of AUGC contained in the sequence. The number of possible sequences will be equal toa multinomial distribution
n!=(na !nu !ng !nc!)(1)
where n is the sequence length, n = na + nu + ng + nc , na is the number of A in the sequence, nu thenumber of U, etc. For a sequence of length 20 and na = nu = ng = nc = 5, we obtain 20!=(5!)4 =1:17 2 1010 . The number of secondary structure conformations would be at least half this number,due to symmetry considerations and degeneracy e ects in the secondary structure conformations, butwould be of similar order. Moreover, we would still need to evaluate all sequences to determine thisdegeneracy and symmetry precisely. A typical exon or intron of mRNA contains sequences in excess of
100 nucleotides, which means that we would need to evaluate more than 1057 sequences to determine
all possible conformations and completely describe the distribution.
Since such a complete evaluation is obviously outside the power of even the most advanced computers of this age, we have reasoned that shuing a given sequence of length n into a set of N possible
shued sequences will provide anof this distribution. As reasoned in Fig. 1(II), as thenumber of random sequences N increases, the observed FE distribution will gradually approach thetrue FE distribution [7]. In the calculations done in this work, we have used values of N such thatN 400.
To estimate the error in this small number of calculations, we evaluated the uctuation of the mean
FE value for a given sequence (length: 100 nt) [4]. Each mean FE was determined by shuing the
given sequence N times. From the distribution of 200 separate mean FE calculations, we found thatthe ratio of the standard deviation () to the range of the FE (2) comes out roughly to =2 < 0:01
(see Fig. 1). This means that the separation between bins in Fig. 2 is equal to roughly 10 (where therange is divided into 11 bins: , m = 11). Other authors have found that 100 sequences is sucient
to make these estimations at a con dence level of 95% [20]. Hence, a threshold of 400 shued sequences
should be sucient to obtain the mean value of the distribution based on our calculation, and others[8, 18, 20].
We also examined the tendency of these sequences as a function of nt length. For sequences with
xed AUGC percentages, the property is linear as a function of nt length. For sequences with di erent
AUGC concentration, the slopes are di erent but the linear dependence is the same [4].
The distribution seen in Fig. 2 is typical and appears to be fairly independent of the nt-length orpercentages of AUGC components, although there
di erence in the degree of dispersion(variance). In the sequences evaluated so far, the variance appears to be stable. If there is any
signi cant uctuation in the variance, it is most likely to appear at the limits of these distributions. For
sequences with moderately equivalent percentages of AUGC (between 20 and 40%), the FE distribution
obtained from secondary structure calculation appears to be a stable distribution [4]. Hence, we expectthat this nite sampling method re ects a sucient representation of the actual distribution of thesesequences. In particular, this should be the case for the sequences analyzed here, since the distributionof AUGC in these globin genes ts within the 20  40 percent range. Moreover, all these distributions
allapproximationi.e.

could be some

52

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Figure 2: Distribution of free energy in shued sequences of a globin exon.
strongly resemble the familiar normal distribution. There is some indication that the distribution islog normal, but the shift is very close to a typical Gaussian distribution with a small contributionof skew symmetry ( 00:5). Hence, we have adopted Gaussian statistics in our analysis. However,
because of the small component of skew in the FE distributions, we will refer to the plot in Fig. 2 asa Gaussian-like density distribution (GLDD).
The next issue that one encounters in evaluating the free energy of a secondary structure calculation, is that secondary structure calculations remain a very imprecise technique for estimating theminimum free energy of a nucleotide sequence. At present, the estimated certainty is claimed to beabout 70% accurate [13]. It should be noted, however, that the introduction of secondary structure
analysis has greatly assisted researchers in other techniques such as searching for conserved sequences
to deduce the structural relationship of hnRNA and mRNA. Hence, with some judicious considerations, we can expect to extract someunderstanding. In particular, if numerous FE calculationsof families of intron sequences consistently yield a similar result, this trend is reasonable to accept as
genuine, but a lone calculation that has an exceptionally di erent value is probably questionable. As
a result of these underlying issues, we have focused mainly on the general trend or behavior of these
intron and exon sequences.
Thus, we have made the following assumptions in this work. First, we have used a normal distribution to evaluate the mean FE of the GLDD. Second, we have assumed that the optimal secondary
structure of the shued sequences can make a suciently qualitative representation of the true stacking energies to make these estimates useful. Finally, we have assumed that the tertiary structure will
not signi cantly change this result and yield a far di erent mean FE.
general3

Results

The results in this report should only be considered suggestive; however, using only the secondary
structure as a discriminating tool, we have found what appears to be a signi cant di erence in thefree energy of secondary structures of introns and exons found in the globin family.
53

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Figure 3: Distribution of free energy in intron (dark gray) and exon (light gray) sequences of globin
gene from various warm blooded species. The sequence data were obtained from GenBank.
The distribution of exons and introns in the / -globin sequences are plotted in Fig. 3. The
FE is roughly 6 in both Fig. 2 and 3. Due to the small negative skew ( 00:5) of the FE distribution,
the actual mean is slightly larger than 6 (mean: 6.6). However, since the error in the FE estimation
using secondary structure stacking energies is likely to be a much greater source of error, we will quote
6 as the mean FE value. Sequences with a FE lower than 6 possess a more stable structure (morefolded or globular). Sequences with a FE greater than 6 have a less stable structure and consequently,
are less folded (or globular) than the mean sequences.
The wild type exons are distributed from bin 1 to bin 9 in Fig. 3(light gray) with a broad maximum
at roughly bin 5 or 6 (near the mean FE). The dispersion of the exon FE values tends to resemble
a normal distribution which broadly spans most of the FE range. The exon distribution shows
bias toward a larger negative FE, but less that the introns.
The distribution of intron FE values Fig. 3(dark gray) does not appear to take on a normal
distribution about the mean FE. One group of introns appear to cluster around bin 5 (1 to 2 bins
more negative than the mean FE) and another set of introns appears to have a very negative FE(centering at bin 1 in Fig. 3). On the other hand,of the introns appear to have a FE thatis signi cantly greater than the mean FE of the shued sequences. Hence, we can likely assume thatthe majority of the introns in and -globin sequences are globular in nature, and that as a whole,this is theof intron sequences.
The exon sequences show considerable conservation even when all the sequences are viewed together
for the and globin genes. On the other hand, there is very little conservation between distantintrons (Table 1). It should be expected that there will be strong conservation of the exon sequences
since they are used in the protein coding sequences. Since the preserved homology for the introns isalmost non-existent, this means that the biasing e ect seen in the introns is probably real, and not an
artifact of sequence conservation.
In the present analysis, we did not attempt to classify the nature of the resulting secondary
structure. However,it was found that the initial fragments of the intron sequences havea shape in which the GU and AG ends are partially free but roughly a 5  10 nt sequence is present
mean

some

very

tendency

in some cases

54

few

gene family

ex(1)

ex(2)

ex(3)

in(1)

in(2)

-globin

0.75

0.75

0.75

0.07

0.10

-globin

0.50

0.60

0.65

0.07

0.12

Table 1: Average similarity score for exon and intron sequences of / -globin used in the calculations.
which joins the 5 and 3 ends in a similar fashion. However, there are a number of other patterns
that commonly occur. This may be part of the origin of the longer (but not well de ned) \consensus
sequence" that surrounds the doner and acceptor sites. This consensus sequence has been found bothexperimently [2] and by conditional probability analysis [30]. This may also indicate some part of theactual structure of the intron. However, the tertiary structure (of which little is known) is also likely
to be signi cant for short intron sequences. These issues of structure and function will be considered
in a future monograph.
040Discussion

We preface this section by reiterating that, since the results are based on secondary structure calculations of the stacking free energies, only the general trend of the wild type intron and exon distributions
will be considered. Whereas the results reported here are not nearly as impressive as recent results
found for the cytochrome c family of genes [6], similar trends are observed in the globin family as well.
4.1

General considerations

We rst address some general matters in relation to interpreting the calculated FE given here.
One point to consider is that it is not clear whether the secondary structure of hnRNA should
be expected to segregate into intron and exon regions as we have \forced" the sequences to do inthese calculations. However, others have tried xed windows to scan a gene and have observed similar
trends as reported here [8, 18]. There is certainly no precedentusing a sliding window andselecting the regions of interest. Rather this may even be important (: section 4.4).
As far as we know, the globin genes studied in this work are not subject to alternative splicing[12, 32]. It would appear that introns spanning regions of alternative spliced genes ( ,geneof[18]) exhibits theresults found here ( , intron 5 is less folded). Our
calculations show roughly intron 5 (in ref. [18]) has a FE located at roughly bin 7 or 8. However,alternative splicing mechanisms are still poorly understood and are likely to require various regulatory
proteins to govern the selection process [12, 32]. We don't anticipate that the behavior should be thesame as genes that obey a constitutive splicing mechanism.
Further work is continuing on the functionality issues. We need to examine whether these results
will come out the same when a select sub-set of the intron sequence is shued instead of the entire
intron. More work is needed in studying the homology for clues of the intron structure. This might
help to narrow down where the structural features occur.
againstvide infra

e.g.

D. Melanogaster

4.2

4.2.1

opposite

Mcl1

i.e.

Intron Distribution

Bimodality in the intron sequences

The intron distribution in Fig. 3 suggests that there could be two types of introns in globin. There isno correlation between intron number, but there istendency for the shorter introns (n< 150nt)
to center around bin 5 whereas the longer introns tend to center around bin 1. The origin of this\bimodality" is unclear.
some

55

At present we think this e ect could be the result of uncertainties due to unaccounted for solvent
e ects. These short nt segments are likely to critically depend on the stabilization energies introducedby the local concentration of various metallic ions such as Mg2+ and Na1+. These are known to play
an important role in the structure and possibly the function of type I introns [28, 5]. Although there isno data for snRNP related genes, ion stabilization is expected for most nucleic acid sequences based on
their chemistry. Solvent e ects are likely to play a signi cant role in stabilizing (or destabilizing)
nucleotide sequences because of the greater degree of exposure of the central core. Longer sequences
appear to be highly stabilized by comparison due to their globular character. Hydrophobic/hydrophilic
e ects are more likely to play a central role in long intron sequences [8].
Ultimately, to interpret the results in Fig. 3, we will need to compute the free energy generated
by the 3D structure with the inclusion of solvent interactions and charged ions. However, since thepurpose of this current work is to show that there is a detectable e ect resulting from a mere secondary
structure calculation, we can safely say that there seems to beevidence of structure in the intron
sequences. We are currently looking into tertiary structure issues in relation to these shorter sequences.
One remaining issue is what level of con dence to place on any FE calculations based on a secondary
structure calculation. Whereas the secondary structure is likely to generate more \scatter" than theactual distribution (or at least a \di erent kind of scatter"), a deviation of 40 (between bin 1 andbin 5) seems unlikely even from such an extremely crude calculation. This would suggest that thebimodal character of the intron distribution is expressing some real property of introns.
There is also a matter of concern whether there could be some other artifact causing the higher
degree of organization. One such possibility would be a strong homology conservation in the intron
sequences since the exon sequences are strongly conserved. However, whereas the exons retain roughly
60% of their homology within their respective or families, the introns only retain roughly 10% .
Therefore, whatever the origin of the intron band, it is unlikely that the bimodality is the result of some
artifact in the conserved homology between species. Furthermore, there is no relation between the
intron number and the magnitude of the FE. Another possibility would be various repeating sequences
like s. The s are generally more globular than the random sequence. However, whereas thesesequences have been found on some of the genes analyzed here, none of them were located in any partof the hnRNA sequence [9]. Other e ects such as accounting for dinucleotide bias does not appear tochange this result substantially, but is still under investigation.
short

some

Alu

4.2.2

Alu

Origin of the Lower Free Energy of the Intron

In Fig. 3, it is apparent that the majority of the data from the secondary structure calculation suggest
limited degree of structural organization in the introns. This contradicts the general notion that
introns only rely on a trivial set of conserved sequences (the GU 1 1 1 AG rule and the \A" branch
point; , see [35]). These conclusions were based on the fact that parts of an intron sequence could
be spliced o with no apparent change in the cleavage of the introns as long as there were no inserted
sequences which could also contain intron character. Such a lack of selectivity would strongly suggestthat every property between intron selection to the transesteri cation could be handled by the snRNP,
and that the intron sequence provides no signi cant information other than its consensus sequence.
However, since the 3D structure is basically unknown for most of the hnRNA, it is still possible that
there was no catalytic function associated with the deleted sections of the intron sequence in theseexperiments.
It is generally accepted that the dinucleotide pair GU 1 1 1 AG and the branch point is not sucient
information to fully de ne a constitutive splicing region. A cursory examination of any hnRNAsequence will show numerous dinucleotide sequences of \GU" and \AG" in any exon or intron region.
Currently this plethora of possibilities in the hnRNA sequences are resolved by laborious examinationof the mRNA and hnRNA sequences by well trained experts and selection is rarely determined strictlyby the consensus sequence alone [2]. However, since the intron is spliced out of the hnRNA with
some

e.g.

56

almost perfect precision, this would suggest that the interaction mechanism is more complex thanmere sequence information.
We suggest that structural organization is necessary for the intron in order for the snRNP complex
tothe intron. More precisely, there is likely to be both chemical and structural aspects
involved in the binding of SR proteins and snRNA to the intron-exon junctions. Such a large andcomplex apparatus ( , recognition system) is likely to be \designed" to attack very speci c chemical
conformational con gurations and functional groups. These chemical and con gurational (steric)factors have been found already in type I introns [1, 11, 25, 28, 34] and type II introns [14, 15]. Hence,
it is clear that specialized introns structured andform complex enzymatic-like structural
architectures.
From a di erent angle, each part of the snRNP functions much like a subunit. However, becauseof the bulk size of this complex, most of the subunits must be in close proximity to work e ectively
since the primary rate limiting steps are probably both di usion limited and rotationally restricted by
the large hydrogen bonded network of water molecules. Likewise, it would not be thermodynamicallyecient for these large subunits to seek out these consensus sequences, then to seek out all the othersnRNA subunits (meanwhile dragging large sections of the pre-RNA substrate with them throughhistones, DNA, ), and only then to nally carry out catalysis. Rather, the intron should alreadybe in the \proper con guration" ( , the biologically active structure) whereupon the snRNP complex
simply \recognizes" this \con guration", then carries out the two step transesteri cation process. Sucha sequence of events is more thermodynamically feasible, and greatly reduces the rate limiting factors
in the physical interaction. Furthermore, since thermodynamically ecient organisms are more likely
to possess a selective advantage, reducing or optimizing the number of di usion limiting steps is likely
to have a relatively rapid xation time in the population of pre-Cambrian proto-eukaryotic organisms[8, 20].
Moreover, the compact structure of hnRNA appears to in uence the splicing order carried out by
the SR proteins and snRNA yielding a unique electrophoresis pattern. Such splicing order strongly
suggest steric e ects and that splicing begins at the surface of a hnRNA molecule [19]. Moreover,the splicing does not appear to occur in a linear progression starting from the 5 end of hnRNA andending with the 3 end. Rather splicing occurs according to some subset of possible orders [19]. All
this suggests that somein the mRNA structure is required to initiate splicing.
Finally, there is already clear evidence of order in type I and type II introns, suggesting that at
least
subsections of all intron sequences
convey some kind of signi cant information, whichthen controls, moderates or regulates the activity or function of the splicing apparatus. Although the
majority of the catalytic potential of the spliceosome intron has obviously been lost, if all these introns
share some common evolutionary history, then it should be reasonable to expect that some minimal
catalytic structure isin snRNP introns. Catalytic functions generally exhibit structural
constraints as well as chemical functional group requirements. Thus, it should be reasonable to expectthat con gurational information must be preserved in various parts of the intron in order for the
snRNP complex to successfully splice the intron. (See Appendix A for more information about intron
types.)
recognize

i.e.

are

can

etc.

i.e.

0

0

shape

some

might

necessary

4.3

Exon Distribution

The exons also exhibit some visible evidence of biasing in the overall exon FE distribution in Fig. 3.
Although the biasing is less pronounced than the corresponding intron sequences, it is signi cant. We
suggest that this is the consequence of conserved sequences that discourage the formation of excessive
CpG islands. It is well known from mutation studies of mRNA of rabbit -globin that certain typesof sequences are conserved because the CpG islands are likely to result in hot spots for mutation[29]. Perhaps, as a result of this, the tertiary structure of the globin exon gene also shows someorganization in its sequencing as a means of protecting against undesirable mutations in the protein
57sequence, and/or as a result or serendipitous positioning of conserved sequences associated with this
phylogenetic conservation.
The biasing in the exon distribution in Fig. 3 may also be related to the extent to which thesequences are conserved in the globin family genes. In Table 1, it can be seen that the both andglobin exons have a very high degree of sequence conservation (over 60%) despite the broad diversity of
species analyzed in this work. This biasing is likely to be observed in the FE distribution. Moreover, it
may be bene cial for the nal mRNA to form a globular structure because the mRNA could be easily
be exposed to the possibility of lethal mutations if all sections of its sequence were left unhybridized.
4.4

Relation to Bioinformatics

Bioinformatics has contributed substantially to our understanding of gene splicing. Recent work in
conditional probability techniques combined with heuristic methods has helped to extend the simple
consensus sequence (GU1 1 1AG) [30]. Other work using k-gram analysis, in which a sequence of nt isanalyzed as though it were a coded instruction set and the average size of a \word" in that sequenceis analyzed. Intuitively, the exons produce a strong 3-gram signal, but introns appear to vary andgenerate 2, 4, 5, and 7-gram signals [16]. The k-gram studies suggest that the information content in
the intron sequences consists of dinucleotide, tetranucleotides etc., but the information content in thecoding regions consists of multiples of trinucleotide sequences [16]. If one assumes that the snRNP
complex strictly utilizes a \text" analyzing mechanism to process the sequence information in the
intron, then both these approaches are likely to yield success. Indeed, at least some signi cant partof the properties of introns is suggestive of this \instructions manual" type of concept.
However, it also appears that a simple text analyzing approach is not sucient to completely
explain how the introns are recognized by the snRNP complex. Many false positives are obtainedin this form of analysis. Therefore, based on thenature of the results reported here, it
seems reasonable that the current approach could complement research in nding
intron:exonboundaries. We suspect that the results we have obtained contain an important clue to the splice point
detection mechanism, and could contribute to better search methods. We are currently looking forways to apply this secondary structure aspect to searching unknown sequences.
suggestive

unknown

5

Conclusions

The current analysis indicates that, contrary to established notions of intron behavior, there isevidence for greater structural order and ribozyme-like function in the spliceosome introns. Thissuggest that all introns carry some degree of catalytic behavior, where spliceosome introns are theweakest of the known intron types. These results should help establish the critical mechanisms toconsider when attempting to establish how introns are spliced out of the nascent RNA and the orderin which they are excised. In addition, this may be another clue as to the function that introns play inthe eukaryotic cell. Some limited structure is also observed for exons in the globin family which is likely
a consequence of conserved sequences that protect evolutionary hot spots in these genes. Lastly, thesestudies of intron and exon structure may provide a complementary approach to detecting intron-exonboundaries in nascent RNA.
some

Acknowledgments

Research funding was supported through a grant from the Japan Society for the Promotion of Science.
Support was provided by University of Tokyo Human Genome Center sta . Thanks are also extended
to Prof. Yonezawa, Prof. Takagi, Dr. Morishita, and A. Nakaya, for their helpful discussions. Special
thanks to Dr. T. Tsunoda for commenting on the original manuscript.
58

Appendix A. Type I, type II and spliceosome introns

Let k be the index of a given exon (or intron) such that ex(k) (or in(k)) represent the kth exon (orintron) in a hnRNA sequence. If we use a left to right sequence convention, then the doner site is
ex(k):in(k) and the acceptor site is in(k):ex(k + 1).
Type I introns have been studied extensively and appear to strongly depend on the 3D-structure.
Roughly ten domains are generally found and typically ve triple helical junctions are also observed[24, 36]. The 3D-structure and chemical kinetics of the type I intron have been studied extensively anda number of the structures have been worked out [24, 36]. In this case, the majority of the structure anddomains could be resolved by examining the consensus sequences alone [1, 3, 5, 11, 24, 25, 28, 34, 36].
However, in the case of type I introns, the length of the consensus sequences are considerable comparedto the non-structural segments of these introns. Only a small part of these sequences can be completely
deleted without a probable loss of functionality. For a detailed description of their structure andfunction see [3, 11, 24, 25].
The type II introns somewhat resemble precursors of the spliceosomal RNA apparatus [14, 15]. A
typical feature of this hnRNA is the presence of 6 domains in the secondary structure. Experimentson plasmid sequences of domain 5 bear a strong resemblance to the function of U2/U6 in the snRNA[37]. However, the basic structure of type I and type II introns are very di erent and are not likely tohave originated from the same evolutionary pathway [37]. The full tertiary structural details of type
II introns are still unclear; however, some aspects of the consensus sequence are found in both type II
introns and the hnRNA which utilize the snRNA (particularly the GU 1 1 1 AG splice points and the\A" branch point).
In the spliceosome process the snRNA U1 attaches to the 5 end (ex(k):in(k)) of the intron sequence,U2 connects to a branch point on the intron and then connects to U1. The branch point structure
and location are partially characterized by a consensus sequence of form YNYYRAY; where Y is apyrimidine, R is a purine and N can be any base. In this sequence, \A" is the only conserved element.
U4 appears to act as an inhibitor for U6 in the nal splicing operations. U5 binds to the 3 end of theintron (in(k):ex(k +1)) in the last step of the transesteri cation process where the 3 end of the intron
is excised and the 5 end of ex(k + 1) is mated with the 3 end of ex(k) to form ex(k):ex(k + 1). This
step is accomplished after U5 appears to have triggered the release of U4 such that U6 can attach toU2 and also release U1 from the 5 position. As U1 is released, U6 replaces U1 at the 5 position of
the intron. For further details, see [22].
0

0

0

0

0

0

0

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