A Novel Role for Minimal Introns: Routing mRNAs to the Cytosol
Citation: Zhu J, He F, Wang D, Liu K, Huang D, et al. (
A Novel Role for Minimal Introns: Routing mRNAs to the Cytosol
Jiang Zhu 0
Fuhong He 0
Dapeng Wang 0
Kan Liu 0
Dawei Huang 0
Jingfa Xiao 0
Jiayan Wu 0
Songnian Hu 0
Jun Yu 0
Xiaoyu Zhang, University of Georgia, United States of America
0 CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences , Beijing , China , 2 Graduate University of Chinese Academy of Sciences , Beijing , China
Background: Introns and their splicing are tightly coupled with the subsequent mRNA maturation steps, especially nucleocytoplasmic export. A remarkable fraction of vertebrate introns have a minimal size of about 100 bp, while majority of introns expand to several kilobases even megabases in length. Principal Findings: We carried out analyses on the evolution and function of minimal introns (50-150 bp) in human and mouse genomes. We found that minimal introns are conserved in terms of both length and sequence. They are preferentially located toward 39 end of mRNA and non-randomly distributed among chromosomes. Both the evolutionary conservation and non-random distribution are indicative of biological relevance. We showed that genes with minimal introns have higher abundance, larger size, and tend to be universally expressed as compared to genes with only large introns and intron-less genes. Genes with minimal introns replicate earlier and preferentially reside in the vicinities of open chromatin, suggesting their unique nuclear position and potential relevance to the regulation of gene expression and transcript export. Conclusions: Based on these observations, we proposed a nuclear-export routing model, where minimal introns play a regulatory role in selectively exporting the highly abundant and large housekeeping genes that reside at the surface of chromatin territories, and thus preventing entanglement with other genes located at the interior locations.
Funding: This work was supported by the National Basic Research Program (973 Program) from the Ministry of Science and Technology of the People’s Republic
of China (2006CB910404 to J.Y.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Gene expression program, rather than a simple assembly line
to process mRNAs, is a complex network systematically
coordinating many cellular pathways including transcription
initiation-elongation-termination, RNA processing,
transcription-coupled DNA repair, nuclear export of mRNAs, translation
and RNA/protein degradation [
]. In concomitance with
transcription and pre-mRNA processing, a dynamic repertoire
of proteins are recruited to package mRNA forming the
messenger ribonucleoprotein particle (mRNP). The interactions
among the protein components of mRNP and other expression
machineries can enhance or reduce the rate/efficiency of the
coupled reactions, constituting a complex network of
co-/posttranscription regulation [
]. The architectural organization of
nucleus also provides another level of expression control.
Nuclear positions of chromosomes, gene loci and specific
genome regions, as well as the spatial interactions among them
play important roles in transcriptional regulation [
Therefore, expression regulation involves not only the binding
of site-specific transcription factors/cofactors but extensive
coupling and coordinating among relevant machineries and
processes. All of these events are spatially and temporally
integrated within the nucleus.
An intriguing example of the coupling among the expression
machineries is the observation that RNA splicing influences many
subsequent steps of mRNA metabolism such as nucleocytoplasmic
]. It was reported that the efficiency of mRNA export can
be enhanced 6- to 10-fold for spliced mRNAs relative to their
cDNA counterparts in mammalian cells [
]. The current working
model for the splicing-dependent nuclear export proposes that the
TREX (transcription/export) complex, containing key export
factors Aly and UAP56, colocalizes with the splicing machinery in
the nuclear speckles. It is recruited to mRNA as a component of
the exon junction complex (EJC) at ,20 bp upstream of the
exonexon junction during splicing. Aly binds to the mRNA export
receptor Tap:p15 heterodimer that interacts with the FG
nucleoporins in the pore channel to move the mRNP through
the nuclear pore [
]. The magnitude of the
splicinginduced enhancement appears to vary from gene to gene [
may depend on certain genomic parameters, such as the length
and position of introns in the unprocessed transcript. However, the
observation that TREX complex can be recruited to cDNA
transcripts, although less efficient, implies that splicing can
enhance, but is not obligatory, for mRNA export [
naturally intron-less transcripts, export factors were proposed to be
recruited by co-transcriptional mechanism or through some
specific sequence elements [
]. These results demonstrate that
whether a gene has introns and where the introns are have
significant influence on gene’s nuclear export.
We previously reported a conspicuous feature of vertebrate
introns that a remarkable fraction of introns have a lineage-specific
minimal size (,100 bp), which were termed as ‘‘minimal introns’’
]. Based on a sequence variation study on human populations
and the primate lineage, we proposed that these minimal introns
are not ‘‘junk’’ DNA, but may have potential roles in regulating
the export of spliced mRNAs from nucleus [
]. In this study, we
further analyzed minimal introns in human and mouse genomes.
We showed that minimal introns are evolutionarily conserved in
terms of both length and sequence as compared to large introns.
Minimal introns preferentially locate toward 39 end of mRNA and
are non-randomly distributed among chromosomes. Both the
evolutionary conservation and non-random distribution indicate
their biological relevance.
In order to understand their functions, we analyzed genes with
minimal introns and found some unique characteristics associated
with the presence of minimal introns. In general, genes with
minimal introns have higher abundance and larger size, and tend
to be universally expressed as compared to genes with only large
introns and intron-less genes. The presence of minimal intron is
also correlated with the replication timing and chromatin structure
of the gene locus, implying specific nuclear positions of these
genes. Based on these observations, we proposed a nuclear export
routing model where minimal introns play regulatory role to
selectively export some highly abundant and large housekeeping
genes that reside at the surface of chromatin territory, thus
preventing the entanglement with other genes located at the
interior locations. Although this model is largely descriptive and
hypothetical, it provides a necessary framework to design
experiments to test the exact role of minimal introns in
Minimal introns are evolutionarily conserved
A remarkable fraction of vertebrate introns have a minimal size
(,100 bp) peaking at the low end of the size distribution, while
majority of introns expand to several kilobases even megabases in
length (Figure 1A). Over large evolutionary timescale, intron loss
and gain have frequently occurred, limiting the identification of
orthologous intron [
]. In this study we chose human and
mouse for further analysis. They were diverged about 100 million
years ago, and intron position relative to coding sequence was
proposed to be nearly constant . We can use this
correspondence of intron position to identify orthologous introns and
monitor intron dynamics.
We analyzed 18,468 human and 18,889 mouse RefSeq loci,
containing 175,723 and 165,351 introns, respectively. We defined
introns with length of 50–150 bp as minimal introns and those
with length of .150 bp as large introns (Figure 1A). In total, 9.4%
of human and 10.5% of mouse introns are minimal introns, and
34.3% of human and 33.8% of mouse genes bear minimal introns.
From 13,382 human-mouse orthologs, we identified 74,615
reliable human-mouse orthologous intron pairs as those from
orthologous genes and having the same position relative to the two
coding sequences. These subsets of introns have very similar length
distribution as total introns in the two species (data not shown). We
observed that 97.1% of introns remain to be either minimal
introns or large introns, and exchanging between the two classes
occurs rarely (2.9%).
In order to understand the intron evolution between human
and mouse, we first analyzed the length differences of
humanmouse orthologous introns as a function of intron length. We
found that most of large introns fluctuate in length, with a median
length difference of 452 bp. In contrast, the lengths of minimal
introns are highly conserved, with a median length difference of
8 bp (Figure 1B). At present, the molecular mechanisms that
cause intron lengthening/shortening remain poorly understood.
Transposon insertion/deletion may be one of the primary
reasons, while the fixation of highly conserved sequences was
also reported to contribute to intron length dynamics [
Moreover, we observed enhanced sequence conservation in
minimal introns as compared to intronic sequences from large
introns and intergenic sequences (Figure 1C). The number of
substitutions per site in minimal introns (with a mean of 0.40) is
significantly lower than that in intronic sequences from large
introns and intergenic sequences (with means of 0.43 both;
Wilcoxon rank sum test, P,2.2610216). We concluded that
minimal introns are evolutionarily conserved in terms of both
length and sequence.
Minimal introns reside preferentially toward 39 end of
Introns in 59 UTR are often very large due to higher load of
regulatory elements, and introns rarely locate in 39 UTR
because, in most cases, this pattern triggers nonsense-mediated
mRNA decay (NMD) pathway [
]. Except for these
documented exceptions, we reasoned that if ancient introns
randomly lengthened during evolution and minimal introns
are reserved by chance, minimal introns would be evenly
distributed relative to the exon-intron structure. However, the
data denied this expectation. We indexed the ith intron in a
locus with N introns with a position value of i/N, and observed
that minimal introns preferentially reside toward the 39 end
within the sequential arrangement of exon-intron structure
(Figure 2A). It’s interesting that minimal introns are excluded to
be the last intron (with position value of 100%). We also
analyzed the intron position relative to the transcript. Because
39 most exon are generally very large (data not shown), introns
in general reside toward 59 end of the transcript. However, for
minimal intron, we also observed a preference toward 39 end of
the transcript (Figure 2B).
Minimal introns are non-randomly distributed among
We observed that minimal introns are non-randomly distributed
among chromosomes. Using the fraction of minimal intron within
a chromosome to index its enrichment, some chromosomes (19,
16, 17, 11) are significantly enriched by minimal introns whereas
others (21, 18, 4, 13, 5, 10, 15, 2, 7, 9, 1) are significantly deficient
It has been reported that chromosome features such as gene
density, GC content, replication time, compactness and nuclear
position are all correlated—open chromatins tend to locate at
the nuclear interior, replicate early, and have higher gene
density and GC content as opposite to close chromatins—
although the causal relationship among them remains to be
]. We observed that minimal intron
enrichment is also associated with these chromosome features.
Minimal intron enriched chromosomes tend to be gene dense
(Figure 3B; r = 0.8217, P = 8.561027), GC-rich (Figure 3C;
r = 0.7390, P = 3.761025), replicate earlier (Figure 3D;
r = 0.7021, P = 1.361024) and have more open chromatin
(Figure 3E; r = 0.6363, P = 8.361024).
Two striking example are chromosome 19 and 18 (Figure 3).
Although having similar DNA content (64 Mb and 76 Mb,
respectively), they differ significantly in their gene and GC
content. Chromosome 19 is gene-dense (20.3 genes/Mb) and
has high GC content (48.4%), whereas chromosome 18 is
genepoor (3.3 genes/Mb) and has low GC content (39.8%).
Chromosome 19 is the most minimal intron-enriched chromosome (with
18.7% of total introns, P = 3.76102196), but chromosome 18 is
among the most minimal intron deficient (with 4.0% of total
introns, P = 5.1610226). Chromosome 19 replicates early and has
open chromatin structure (with a mean S:G1 and open:input ratio
of 1.67 and 2.06, respectively) as compared to chromosome 18
(with a mean S:G1 and open:input ratio of 1.39 and 1.03,
respectively). In human lymphocyte nuclei, which exhibit an
spherical shape, chromosome 19 is consistently localized toward
the nuclear center without any detectable attachment to the
nuclear envelope, whereas chromosome 18 is positioned close to
the nuclear border [
], and this nuclear arrangement was
reported to be highly conserved [
Genes with minimal introns have unique characteristics
The evolutionary conservation, 39-positional preference and
non-random chromosomal distribution all indicate that minimal
introns may have special biological functions. We previously
showed that minimal introns are not randomly distributed among
]. Therefore, it is expected to observe their functional
relevance from the characteristics of genes with minimal introns.
We carried out gene ontology (GO) analysis on the functional
annotation of 6,327 (34.3%) genes with minimal introns (Table
S1), and found several significantly overrepresented functional
groups (Figure S1). Genes related to the cellular structure are
among the most enriched functional groups, such as nuclear
envelope and cytoskeleton. Most of these genes are related to
various housekeeping functions.
In order to look further into the features of minimal
intronscontaining genes, we separated genes into three classes: (1) genes
with minimal introns, (2) genes with only large introns and (3)
genes without introns. We observed that intron-containing genes
have significantly higher mRNA concentration and are
significantly larger than genes without introns, consistent with the
experimental observation that splicing can enhance mRNA
nuclear export [
]. Moreover, the mRNA level and length are
significantly greater in genes with minimal introns than in genes
with only large introns (Wilcoxon rank sum test, P,2.2610216;
Figure 4A and 4B). We previously showed that ubiquitously
expressed housekeeping genes are in general highly expressed and
have greater length in comparison with tissue-specific genes [
In consistence with this and above GO analysis, we observed
housekeeping genes are significantly enriched in intron-containing
genes and deficient in intron-less genes and the fraction of
housekeeping genes is positively correlated with the number of
minimal introns in intron-containing genes; the opposite trend was
observed for tissue-specific genes (Figure 4C).
Comparing replication timing and chromatin structure among
the three classes of genes, we found that the replication time of
genes with minimal introns (with a mean S:G1 ratio of 1.66) is
significantly earlier (Wilcoxon rank sum test, P,2.2610216) than
that of other spliced genes with only large introns (with a mean
S:G1 ratio of 1.58), which is again significantly earlier (Wilcoxon
rank sum test, P = 4.161026) than that of intron-less genes (with a
mean S:G1 ratio of 1.51; Figure 4D). The chromatin structure
surrounding genes with minimal introns (with a mean open:input
ratio of 1.73) is significantly more open (Wilcoxon rank sum test,
P,2.2610216) than that of other spliced genes with only large
introns (with a mean open:input ratio of 1.39), which is again
significantly more open (Wilcoxon rank sum test, P = 4.661025)
than that of intron-less genes (with a mean open:input ratio of
1.17; Figure 4E). Interestingly, the number of minimal introns
within a gene has a quantitative nature, i.e., the more minimal
introns a gene contains, the higher abundance, the larger size, the
earlier replication timing and the more open chromatin structure
In this study, we demonstrated that minimal introns are
evolutionarily conserved and non-randomly distributed within
genes and among chromosomes, indicating minimal introns have
biological function and are specifically conserved during evolution.
In order to unveil the function of minimal introns, we
comparatively analyzed three sets of genes: (1) genes with minimal
introns, (2) genes with only large introns and (3) genes without
introns, and found that the presence of minimal introns is
associated with gene’s length, expression level, replication timing
and chromatin structure. Based on these observatons, we proposed
a nucleocytoplasmic export routing model (Figure 5). Genes with
minimal intron reside at the surface of chromatin territories and
near nuclear speckles to facilitate a specific splicing process. They
generate large and highly abundant mRNPs that are directly
exported to the cytosol. Genes with only large introns and
intronless genes locate at interior locations and use distinctive pathways
to export. Cells use this routing strategy to selectively export the
three types of genes, preventing the entanglement of mRNPs and
maximizing the efficiency of nuclear export.
There are several pieces of evidence supporting our model.
Nuclear pore complex (NPC) contains 9-nm aqueous channels,
through which small water-soluble molecules are moved
backand-forth through passive diffusion; the channel of NPC expands
to about 25 nm when exporting macromolecules, such as protein
and mature mRNP, in an energy-dependent way [
nuclear transport is a highly orchestrated and rapid event; at least
10 molecules may traverse a NPC simultaneously . At these
busy and crowded ‘‘gates’’, specific regulation is necessary for
bulky and frequent objects. Striking examples are the insect
Balbiani Ring (BR) genes that contain four small introns and
generate mRNAs of more than 30 kb even after splicing. The
exceptionally large and highly abundant BR mRNAs form giant
mRNP particles that are folded into a compact ring-like structure
with a diameter of 50 nm in nucleus. When exporting through
the NPC, BR mRNP, one at a time, undergoes a series of
structural unfolding and moves through as a thin fibril with 59
end in the lead [
]. This regulated unfolding of giant BR
mRNPs before nuclear export may have evolved to be a specific
feature for highly abundant large genes. In this study, we showed
that genes with minimal introns tend to be universally expressed
housekeeping genes with higher cytoplasmic mRNA
concentration and of larger size as compared to genes with only large
introns and intron-less genes. These observations implied that
during evolution, minimal introns may have been conserved in
these highly abundant and large mRNAs to facilitate specific
regulation on nucleocytoplasmic export.
How minimal introns specifically regulate the export of these
highly abundant and giant mRNAs? In lower eukaryotes, introns
are generally small and could be directly recognized by the splicing
machinery—the intron definition model; but in higher eukaryotes,
such as vertebrates where most of introns have been expanded, the
splicing machinery would use exons as the unit of recognition to
facilitate the identification of exons among the intronic oceans—
the exon definition model [
]. The evolutionarily conserved
minimal introns may preserve the ancient intron-recognition
pathway, while the large introns in vertebrates have been evolved
to use the exon-recognition pathway. It is now known that
spliceosome positions exon junction complexes (EJC) at 20–24 nt
upstream of exon-exon junctions when introns are spliced out. It’s
reasonable to speculate that the two entirely different splicing
mechanisms may locate different EJC at the splicing junction. At
present, whether EJC is deposited at every exon-exon junction in
mRNA with multiple introns and the full composition of EJC at
every exon-exon junction remain to be elucidated ; but it is
believed that the composition of EJC imprints mRNA with
information required for many following steps of mRNA
metabolism such as nucleocytoplasmic export, and these
downstream processes are highly dependent on EJC position along the
]. Our observation that minimal introns are
preferentially located toward 39 end of mRNA is consistent with
this position-dependent effect. We proposed that intron-specific
alterations in EJC component, depending on intron position and
intron length, may be an important variable. EJC at exon-exon
junction surrounding minimal intron may have unique
composition, imprinting minimal intron-containing genes with distinctive
signals that guide a specific nucleocytoplasmic export pathway.
The current mammalian nuclear architecture model, i.e.,
chromosome territory - interchromatin compartment (CT-IC)
model, depicts that chromosomes in the nucleus are organized as
chromosome territories (CTs), which are sponge-like structure
built up from condensed high-order chromatin fibers. CTs occupy
spatially limited volume and are non-randomly positioned within
the nucleus [
]. Although the molecular mechanisms that
establish and maintain the nuclear architecture remain to be
elucidated, the radial position of chromosomes has been related to
some chromosome features such as gene density, chromosome
size, replication time and transcriptional activity [
Recently, it was proposed that the non-random position of CTs
is established in a self-organized way, i.e., the morphological
appearance and spatial organization of CTs are determined by the
sum of all functional properties of individual chromosome such as
distribution of replication and transcriptional activity [
showed that minimal intron enrichment of chromosome is also a
correlated feature. Therefore, the presence of minimal intron may
also be involved in shaping the nuclear architecture, although the
exact molecular mechanism needs further experiment to testify.
Interchromatin compartment (IC) is a contiguous network of
DNA-free space, starting at nuclear pores and expanding between
CTs and into their interior. IC can form lacunas with diameters of
up to several micrometers containing nuclear bodies such as
speckle, Cajal and PML bodies [
chromatins reside at the surface of CT and expand into the IC, whereas
the dense/compact chromatins are buried in CT interior.
Earlyreplicating chromatins tend to locate at nuclear interior, whereas
late-replicating chromatins locate at nuclear peripheral. We
showed that genes without introns, genes with only large introns
and genes with minimal introns have increasingly earlier
replication time and more open chromatin structure. According
to the present CT-IC model, the three classes of genes would
locate at different nuclear position: genes with minimal introns
would reside at CT surface and/or near the speckle domain, while
genes with only large introns are relatively interior and genes
without intron are deeply buried within CTs (Figure 5). Therefore,
genes with minimal introns would undergo a unique splicing
process and be directly exported to the nuclear pores through an
independent pathway, preventing the entanglement of mRNPs
produced in the interior of CTs.
So far, we still only have a very fragmented view of the
molecular mechanism associated minimal intron. As the EJC
composition can influence almost every stage of RNA metabolism,
including export, localization, translation and NMD pathway
], in this study, although we proposed that the most likely role
of minimal introns is to regulate nuclear export, we cannot rule out
other possible involvement of minimal intron at current stage.
Better knowledge of the detailed function of minimal introns
entails further experiments. For example, directly visualizing
nuclear spatial distribution of minimal intron by using interphase
fluorescent in situ hybridization (FISH) and establishing mutated
genes where minimal introns are artificially lengthened and/or
disrupted would provide very important evidence for their
function. Although the results presented in this study are largely
descriptive and correlative and the model are hypothetical, these
observations provided a necessary framework to design
experiments to test the exact role of minimal introns in
Materials and Methods
We aligned 25,127 human and 21,153 mouse RefSeq
transcripts (NCBI, March 13, 2008 update) onto their reference
genomes (UCSC, hg18 and mm9) using BLAT program [
clustered them into 18,468 human and 18,889 mouse loci based
on splicing-site-sharing for multi-exon transcripts and
exonoverlapping for single-exon transcripts. When a locus has multiple
alternatively spliced transcripts, the one with the greatest number
of exons and/or length was selected as the representative. Refseq
transcripts of G. gallus, X. tropicalis and D. rerio are processed in the
same way. Intron positions are derived from RefSeq alignments.
Human EST sequences and their genomic alignments were
retrieved from UCSC annotation database (March 11, 2007
update), and clustered into RefSeq loci as previously described
]. The number of ESTs associated with a RefSeq locus was
used to index expression level; only non-normalized EST libraries
with at least 100 ESTs were used for tag counting. The original
sampling counts were converted into TPM (transcripts per million)
for convenience. Expression breadth estimation across 18 human
tissues was retrieved from previous study [
]; genes expressed in
at least 16 of 18 tissues are defined as housekeeping genes and
those expressed in at most 3 tissues as tissue-specific genes.
We retrieved 13,382 human-mouse orthologs from NCBI
HomoloGene (Build 61), and aligned amino acid sequences by
using CLUSTALW2 [
]. Ka/Ks ratios were calculated using
CODEML program in PAML package [
]. Introns from
orthologous genes and with the same position relative to the two
coding sequences were defined as orthologous introns. 74,615
orthologous introns that have at least 3 flanking amino acids
exactly aligned on each side were considered as highly reliable and
were used for analysis in this study. For each human minimal
intron, we randomly selected a position in large introns and
intergenic regions and cut a stretch of sequence of equal length as
control. The alignments of intronic and intergenic sequences were
obtained according to the best chain alignment between human
and mouse genomes from UCSC annotation database (August 15,
2007 update). The number of substitutions per site was calculated
using BASEML program in PAML package.
Information on the replication time and chromatin structure of
human genome were obtained from previous studies [
where DNA from S/G1 phase and open/compact chromatin were
co-hybridized to a BAC clone array. Replicating time is measured
by S:G1 ratio, with the larger value representing the earlier
replication; chromatin status is measured by open:input ratio, with
the larger value representing the more open chromatin structure.
We retrieved 2,955 BAC clones from the array and mapped them
onto the human genome hg18 by using UCSC precomputed
coordinates (clonePos.txt, November 22, 2006 update). We
obtained 2,683 non-overlapping clones with, on average, length
of 150 Kb and spaced by 1 Mb. For each chromosome, the
parameters are averaged among clones and weighted by the clone
lengths. These clones interrogate 3768 loci which have similar
fractions of minimal introns and intron length distribution as the
total (data not shown). For each locus, the parameters of the
overlapped clones were assigned.
Table S1 List of 6,327 human genes with minimal introns.
Found at: doi:10.1371/journal.pone.0010144.s001 (1.30
Figure S1 Gene ontology annotation of human genes with
Found at: doi:10.1371/journal.pone.0010144.s002 (0.12 MB
Conceived and designed the experiments: JZ JY. Performed the
experiments: JZ FH JY. Analyzed the data: JZ FH JY. Contributed
reagents/materials/analysis tools: DW KL DH JX JW SH. Wrote the
paper: JZ JY.
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