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INTRODUCTION |
Although spliced introns and mRNAs are spliced from
pre-mRNA in equal molar amounts, far less is known about the fate
and stability of introns than of mRNAs (1). This may be because most introns have no known function. Apart from a few "specialty introns" that mediate antisense regulation (2), enhance transcription (3), encode small nucleolar RNAs that play a role in rRNA processing (4, 5), or encode proteins that mediate RNA splicing and transposition
(6), it is widely assumed that most introns have no specific functional
attributes. Instead, evidence suggests that most introns in modern
organisms arose serendipitously as a result of the following two
processes: the shuffling of small intronless primordial genes to
generate large genes with introns (the intron-early theory), and the
introduction of introns into intronless genes by transposition-type
events (the intron-late theory) (7).
What are the consequences of the fact that modern eukaryotic genomes
are saddled with large numbers of introns? First, evidence suggests
that introns play a major evolutionary role in shaping protein function
by virtue of their ability to promote exon shuffling (8). Second, it is
clear that introns are necessary for the efficient expression of most
mammalian genes (9-12). Presumably, the presence of introns in
pre-mRNA sends the newly transcribed RNA down the appropriate
pathway to permit high level accumulation of spliced mRNA.
What is less clear is the importance of introns after they
are spliced out of pre-mRNA. Intron sequences exceed the length of
exon sequences in most vertebrate genes (13, 14), and thus the major
component of spliced transcription units that are ultimately degraded
is introns. The rate of this degradation may influence the levels of
nucleotides available for further rounds of transcription. In addition,
intron turnover rate may influence RNA splicing. U2, U5, and U6 small
nuclear ribonuclear proteins and Ser-Arg-containing proteins remain
bound to released intron lariats (15), and therefore the metabolism of
introns may have a significant influence on the recycling of these
splicing factors. Vertebrate cells execute complex constitutive and
alternative splicing events that depend on precise concentrations of
splicing-regulatory factors. The rate of intron turnover may
significantly affect the availability of these splicing factors,
thereby regulating many cellular processes in multicellular organisms.
The importance of efficient intron turnover is supported by studies on
organisms deficient in debranchase, the enzyme that specifically
cleaves the 2'-5'-phosphodiester bond at the branch site of intron
lariats (16). Schizosaccharomyces pombe deficient in
debranchase because of a null mutation accumulate high levels of intron
lariats and exhibit a severe growth defect (17). That this growth
defect in fission yeast may stem from a toxic build up of undegraded
introns is suggested by a comparison with the budding yeast
Saccharomyces cerevisiae, which contains ~40 times fewer
introns than S. pombe and does not display obvious
phenotypic defects when rendered debranchase-deficient (18). Vertebrate genomes contain even more introns than S. pombe, and
therefore an interference with intron decay is also likely to have
serious consequences in vertebrates.
Given the probable importance of intron turnover, it is surprising that
very little is known about this topic. In part, this deficiency may
reflect the fact that very few spliced nuclear pre-mRNA introns
have been detected in vertebrate cells. Most of the spliced introns
that have been observed in vivo are derived from strongly
transcribed genes. For example, excised introns from the adenovirus-2
E2A pre-mRNA were identified in HeLa cells treated with
cycloheximide to increase the rate of transcription (19). A
-globin
intron was detected from rabbit liver, a source rich in
-globin
pre-mRNA (20). A spliced immunoglobulin-
intron was observed in
a stimulated plasmacytoma cell line that transcribes high levels of an
immunoglobulin-
gene (21).
A widely held belief is that spliced introns accumulate at low levels
because they are rapidly degraded (within seconds) at their site of
origin in the nucleus (14, 22). However, there is little direct
evidence to support this view. The only mammalian intron whose fate and
stability have been examined in detail is the
IVS1C
1 intron from a mouse T-cell receptor (TCR)1-
gene. This intron
is easily detectable by the relatively insensitive Northern blot
procedure, despite being generated from only a modestly transcribed
gene (23). The half-life of IVS1C
1 in HeLa
cells was determined to be 6 min (24), which is much longer than was
originally proposed for introns in general (14, 22). Most spliced
IVS1C
1 lariats were found to be in the
nuclear compartment, consistent with their origin and degradation in
the nucleus (24).
In the present communication, we report on the fate, localization, and
stability of spliced introns from the Pem homeodomain gene,
a mammalian gene that contains typical mammalian introns ranging in
length from ~0.2 to 2.4 kb (25, 26). Unlike the other genes from
which spliced introns have been studied, the Pem gene is not
expressed in a cell type-specific manner. Instead, Pem is
expressed by different cell types in many different fetal and adult
tissues, as well as in tumor cells from several different lineages
(25-32). Because the Pem gene is not cell type-specific and
its introns appear to be typical, we hypothesized that Pem would be a good candidate to provide information on the metabolism of
mammalian introns in general.
We used Northern blot analysis and RPA to detect spliced Pem
introns, because these approaches have several advantages, including their ability to provide a quantitative measure of RNA levels and their
ability to resolve (by size) spliced introns from intron-containing pre-mRNAs. Other methods used to identify, localize, or measure the
stability of intron-bearing RNAs (e.g. pulse-chase analysis, reverse transcription-polymerase chain reaction (PCR) analysis, and
in situ hybridization) are either not quantitative or do not distinguish between spliced introns and pre-mRNAs (33-36). We used several independent approaches to verify that we had identified bona fide spliced Pem introns. Our analysis of
the three introns in the Pem coding region revealed that
they had a range of half-lives that were even longer than that of the
only other previously analyzed vertebrate intron,
IVS1C
1. The spliced Pem introns
were found in several fractions of the nuclear compartment, consistent
with their origin in the nucleus. Surprisingly, the Pem
introns were also present in the cytoplasmic fraction of several different cell types. Together, these findings may have important implications for RNA metabolism in general.
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EXPERIMENTAL PROCEDURES |
Cells--
The cells used for our study were the kind gift of
the following investigators: the human HeLa cell line (Susan Berget,
Baylor School of Medicine), the rat PS-1 prostate mesenchymal cell line (David Rowley, Baylor School of Medicine), the rat FRTL-5 thyroid cell
line (Christine Spitzweg, Mayo Clinic), and the rat 208-F, Rat-1/S
(src-transformed), and Rat-1/R (ras-transformed)
fibroblast cell lines (Bruce Magun, Oregon Health Sciences University).
The rat McA-RH8994 liver cell line was obtained from the American Type
Culture Collection. The myelin basic protein-reactive rat T-cell line
was obtained from Arthur Vandenbark and Halina Offner (Oregon Health
Sciences University).
RNA Isolation and Subcellular Fractionation--
Nuclear,
cytoplasmic, and total cellular RNAs were isolated as described
previously (30, 37). For each cell line, the length of time in the
lysis buffer (0.6% Nonidet P-40, 0.15 M NaCl, 10 mM Tris (pH 8), 0.1 mM EDTA) was optimized
(between 3 and 10 min) such that >90% of cells exhibited disrupted
cell membranes as judged by staining with toluidine blue O. Under the
conditions chosen, we found that none of the cell lines displayed
nuclear lysis, as judged by microscopic visualization of the released nuclei and by the fact that there was no evidence of viscous nuclear pellets (disrupted nuclei leak DNA, which causes aggregation of nuclei
and increased viscosity). Further assurance of purity was demonstrated
by methylene blue staining of nylon membranes onto which RNA from the
nuclear and cytoplasmic fractions had been transferred (38). We found
that cytoplasmic RNA had only mature 18 S and 28 S rRNA, whereas
nuclear RNA also had 32 S and 45 S precursor rRNAs, in accordance
with its expected properties (38). In cases in which a nuclear wash was
performed, the nuclei were resuspended in 0.5% sodium deoxycholate in
lysis buffer and immediately centrifuged.
Subcellular fractionation of HeLa cells was performed as described
(39). In brief, trypsinized cells were fractionated into cytoplasmic
(after Nonidet P-40 lysis), nuclear membrane (after Nonidet P-40 and
sodium deoxycholate wash), chromatin-associated (after DNase I
digestion), high salt-soluble (after incubation with a high
concentration of NaCl), and nuclear matrix (remaining pellet)
fractions. The RNA from these five fractions was purified by using
method 1 described in Ref. 37.
Northern Blot and RNase Protection Analysis (RPA)--
For
Northern blot analysis, RNA samples were electrophoresed in agarose or
polyacrylamide gels, blotted, and hybridized as described (24). Before
prehybridization, all blots were stained with methylene blue to
demonstrate RNA integrity, show equivalent loading and transfer, and
judge the purity of nuclear and cytoplasmic RNA (38). Standard RNA
molecular weight markers (RNA Molecular Weight marker I (Roche
Molecular Biochemicals)) or the 0.16-1.77- and 0.24-9.5-kb RNA
ladders (Life Technologies, Inc.) were used to determine the size of
RNA transcripts. The DNA probes used for hybridizing the Northern blots
were generated by PCR using the DNA oligonucleotides listed in Table
I. Table II
provides a list of the DNA probes. The names of the probes correspond
to the exons (E) or introns (I) in the probes. Most DNA probes were labeled using [32P]dATP and a Roche Molecular
Biochemicals random-primed DNA labeling kit. To increase the specific
activity of the IVS1 probe, both [32P]dATP and
[32P]dCTP were used. Band intensities of Northern blots
were determined by phosphorimage analysis or densitometry.
We determined the half-lives of the Pem introns by the same
procedures we described previously (24). In brief, when the cells
reached 70-80% confluence, they were incubated with 1 µg/ml tet
(Sigma) for the times indicated in the figures. Total cellular, nuclear, or cytoplasmic RNAs were isolated at various time intervals after the addition of tet. The kinetic loss of RNA was monitored by
Northern blot analysis. Levels of Pem RNAs for each
time point were normalized against the corresponding levels of
cyclophilin mRNA. The RNA half-lives were assessed by least squares
linear regression analysis as described previously (24).
RPA was performed essentially as we have done before (25, 30) using
riboprobes synthesized with T3 RNA polymerase and labeled with
[32P]UTP. The Pem IVS2/exon probe, which contains 234 nt
of IVS2 and 106 nt of the upstream exon, was made by PCR using the
oligonucleotides N and O, the latter includes a T3 polymerase-binding
and initiation site. In an analogous fashion, the IVS3/exon probe,
which contains 333 nt of IVS3 and 46 nt of the upstream exon, was
generated using the primers P and Q. The size of protected bands was
determined by comparison with a [32P]UTP-labeled RNA
Century ladder composed 100-, 200-, 300-, 400-, and 500-nt RNA
transcripts (Ambion, Inc., Austin, TX). Band intensities of RPAs were
measured using an Instant Imager (Packard Instrument Company, Meriden,
CT), which directly measures radioactivity.
Primer Extension Analysis and Debranching of Lariat
Introns--
Primer extension experiments were performed as described
previously (24). Oligonucleotide J (Table I), which is complementary to
the 3' end of the IVS2, was annealed to total cellular RNA from HeLa
cells stably transfected with pTARP. The sequencing ladder was
generated by the standard dideoxy-mediated chain termination DNA
sequencing method (40). Oligonucleotide M (Table I), which is
complementary to the 5' end of the exon immediately downstream of IVS2,
was used to anneal with the pTARP plasmid.
Debranching of intron lariats was performed by incubating total
cellular RNA (isolated from HeLa cells stably expressing rat Pem) at 32 °C for 50 min in a 50-µl reaction volume
containing 10 µl of S100 extract, 1 µl of 0.5 M EDTA,
and then brought up to a 50-µl reaction volume with 1× buffer D (20 mM Hepes (pH 7.9), 20% glycerol, 0.1 M KCl,
0.5 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride; dithiothreitol and phenylmethylsulfonyl
fluoride were added fresh just before use). The S100 extract was
isolated from HeLa cells as described (24). The particular debranching
reaction conditions chosen were selected after extensive pilot
experiments optimizing the debranching activities of the S100 extract,
while limiting the amount of nonspecific degradation, following the guidelines described by Ruskin and Green (16).
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RESULTS |
Detection of Spliced Introns, Pre-mRNAs, and mRNAs from the
Pem Homeobox Gene--
We chose the rat Pem gene to study
intron metabolism, as it is an average sized gene (~5 kb) that
contains typically sized introns (Ref. 25; GenBankTM
accession number U52034). To study the half-life of the Pem introns, we placed the Pem gene downstream of the
tet-regulated promoter (Fig. 1). This
construct, ptPem (Pem-89), was stably transfected into HeLa cells,
along with pTAN (EV-124), which encodes a tet-repressible
transactivator protein necessary for expression from the tet promoter
(24). Stably transfected HeLa cell clones that expressed high levels of
transcripts from ptPem were selected for further study. The cell clone
used for the data shown in this study (clone 5) and its culturing
requirements were described previously by Misteli et al.
(41). We also observed the same results with another HeLa cell clone
(clone 4) (data not shown).

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Fig. 1.
Pem homeobox gene transcripts
derived from a tet-regulated promoter. A,
Pem pre-mRNA transcribed from the ptPem construct. The
Pem introns IVS1, IVS2, and IVS3 are indicated (note that
additional introns in the 5'-untranslated region of Pem
pre-mRNA derived from the endogenous Pem distal promoter
are not in the ptPem construct). Pem coding sequences are in
the 3' portion of exon 1, all of exons 2 and 3, and the 5' portion of
exon 4. The location of the DNA probes and oligonucleotides used in the
present study are indicated. B, Pem pre-mRNA
and mRNA, showing the corresponding DNA probes and oligonucleotides
used for analysis.
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Northern blot analysis was performed with several different
Pem probes to determine the nature of the transcripts
expressed from the tet promoter-driven Pem gene. Both
nuclear and cytoplasmic RNA were analyzed to help with transcript
identification. A 0.6-kb transcript was identified as spliced intron 2 (IVS2), based on its size and the fact that it hybridized with both 5'
and 3' IVS2 probes (Fig. 2A
and data not shown) but not exon probes (Fig. 2, C and
D). Spliced IVS2 was not only present in the nuclear fraction (N), but also in the cytoplasmic fraction (C), a finding that
will be addressed below.

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Fig. 2.
Identification and characterization of
spliced Pem IVS2 and other Pem
transcripts in HeLa cells stably transfected with ptPem.
A-E, Northern blot analysis of nuclear (N) and
cytoplasmic (C) RNA (10 µ) electrophoresed in a 1.2%
agarose gel from the ptPem-transfected HeLa cell clone incubated with
tet (+) or media alone ( ) for 18 h. The blot was hybridized
sequentially with the probes indicated (see Table I and Fig. 1 for
description of the Pem probes; note that a 5' IVS2 probe is
used in A). The Pem transcripts recognized by the
probes are spliced IVS2, m1 (mature Pem mRNA
polyadenylated in IVS2), m2 (mature Pem mRNA
polyadenylated in the last Pem exon), and m3 (probably a 3' cleavage
intermediate). The housekeeping gene cyclophilin (E) served
as a control to show RNA levels from an endogenous gene not under the
control of the tet promoter. This blot and those used for all other
figures were stained with methylene blue to demonstrate equivalent
loading of all lanes and to show the purity of the nuclear and
cytoplasmic RNA (data not shown; see "Experimental
Procedures").
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We also detected three other major transcripts, which we call m1, m2,
and m3. m2 is mature Pem mRNA, based on its cytoplasmic localization and its hybridization with exon probes (Fig. 2,
B and D) but not intron probes (Fig.
2A). Its ~1.5-kb size indicates that m2 is polyadenylated
at the consensus site at the end of the last Pem exon (25).
In contrast, the shorter m1 transcript is a mature mRNA whose 3'
terminus is probably generated by use of the polyadenylation signal
consensus sequence (AAUAAA) present in the 5' portion of IVS2 (25). The
evidence for this assignment includes the following: its ~0.9-kb size
is consistent with a transcript that polyadenylates in the beginning of
IVS2; it hybridized with a 5' IVS2 probe (Fig. 2A) and an
exon 2-IVS2 probe (Fig. 2B) but not with a 3' IVS2 probe
(data not shown); and it was predominantly in the cytoplasm, as
expected for a mature mRNA. m3 is probably a 3'-cleavage splicing
intermediate because it is a nuclear transcript that hybridized
strongly with the exon 1-4 probe (Fig. 2C) and the exon
3/exon 4 probe (Fig. 2D) but not well with an exon 2 probe
(Fig. 2B).
We also detected several larger molecular weight transcripts that are
probably unspliced and partially spliced mRNAs. The evidence for
this assignment is that they are all present exclusively in the nuclear
fraction (Fig. 2, A and B), and they hybridized with both intron probes (Fig. 2, A and B, and
Fig. 3, A and B) and exon probes (Fig. 4,
D and E; longer exposures of the autoradiographs corresponding to Fig. 2 also revealed their presence; data not shown).
We found that spliced IVS2, m1-3, and the precursor RNAs were all
derived from the transfected tet-Pem gene, because
incubation with the specific transcriptional inhibitor tet strongly
reduced the levels of all of these transcripts (Fig. 2,
A-D). In contrast, the level of mRNA from the
endogenous housekeeping gene cyclophilin was not affected by tet (Fig.
2E). We conclude that the ptPem construct generates
precursor mRNAs, mature mRNAs, and spliced introns that are
under stringent tet-regulated control in HeLa cells, thus permitting us
to perform further molecular analyses.
Subcellular Fractionation Analysis--
The observation that
spliced IVS2 was detected in the cytoplasmic fraction (Fig.
2A) was surprising given that introns are known to be
spliced out of pre-mRNA in the nucleus and are believed (without
direct evidence) to be degraded rapidly within this same compartment
(14, 22). To determine whether the presence of Pem IVS2 in
the cytoplasmic fraction is a peculiarity of this intron or is instead
a property shared by other introns, we also examined the localization
of other Pem introns. Because Pem IVS1 is only
167 nt long (Table III), we had
difficulty detecting it with a probe generated by the typical
random-primed labeling approach. However, when we used a "double
labeling" approach to generate an IVS1 probe with higher specific
activity (see "Experimental Procedures"), we were able to detect
spliced IVS1 by Northern blot hybridization (Fig.
3A). Our analysis showed that
spliced IVS1 was present at higher levels in the cytoplasmic fraction than in the nuclear fraction, supporting the notion that a significant fraction of Pem introns is exported to the cytoplasm.
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Table III
Pem intron characteristics
The following abbreviations are used: M is A or C; R is A or G; N is A,
C, T, or G; yx is a stretch of mainly pyrimidines (C or T) for
at least 10 nt.
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Fig. 3.
Identification and characterization of the
spliced Pem introns IVS1 and IVS3. Northern blot
analysis of nuclear (N) and cytoplasmic (C) RNA
(10 µg) electrophoresed in a 1.2% agarose gel from the
ptPem-transfected HeLa cell clone incubated with tet (+) or media alone
( ) for 18 h. The blot was hybridized sequentially with the Pem
probes indicated in A and B. The c-myc
gene (C) served as a control to show RNA levels from an
endogenous gene not under the control of the tet promoter.
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We also considered the possibility that rather than undergoing
cytoplasmic export in vivo, the Pem introns
instead leaked into the cytoplasmic fraction during our cytoplasmic RNA
isolation procedure in vitro. In this case, we predicted
that there might be general nuclear leakage into the cytoplasm, and
therefore Pem pre-mRNAs would also be in the cytoplasmic
fraction. Contrary to this prediction, we found that Pem pre-mRNAs
(as detected with either IVS1 or IVS2 probes) remained exclusively in
the nuclear compartment (Figs. 2A and 3A). We
also considered the possibility that IVS1 and IVS2 preferentially leak
out of the nucleus during RNA preparation in vitro because
they are smaller than Pem pre-mRNAs. To test this
notion, we examined the localization of Pem IVS3, as it is
much larger (2.4 kb) than the other Pem introns (25). We
found that IVS3 was present in the cytoplasmic fraction at a level
approximately equal to that in the nuclear fraction, when an equal
amount of RNA from each fraction was loaded (Fig. 3B). Because ~85% of the RNA in HeLa cells is in the cytoplasmic fraction (based on our typical RNA yields), this indicated that more IVS3 was in
the cytoplasmic fraction than in the nuclear fraction on a per
cell basis. As a control, we hybridized the same blot with a
c-myc probe and found that, as expected, c-myc
mRNA was at much higher levels in the cytoplasmic fraction than in
the nuclear fraction (Fig. 3C). Given that c-myc
mRNA levels were ~5-fold higher in the cytoplasm than in the
nucleus and that ~85% of HeLa RNA is cytoplasmic, this meant that
~30-fold more c-myc mRNA was in the cytoplasm than in
the nucleus. Calculated in this way, IVS1 and IVS2 exhibited a
cytoplasmic-to-nuclear ratio between 3 and 10, whereas IVS3 had a
cytoplasmic-to-nuclear ratio between 2 and 5 (some variation was seen
from experiment to experiment, presumably because of different degrees
of enrichment).
To examine further the intracellular localization of the Pem
introns, we performed more extensive subcellular fractionation analysis. The procedure that we used separates cells into five operationally defined fractions: cytoplasmic (F1), nuclear
membrane-associated (F2), chromatin-associated (F3), high salt soluble
nuclear (F4), and nuclear matrix (F5) (39).
Analysis with IVS2 and IVS3 probes revealed that both spliced IVS1 and
IVS2 were present in most of the nuclear fractions. The
chromatin-associated (F3), high salt-soluble (F4), and nuclear matrix
(F5) fractions all contained spliced IVS2 and IVS3 (Fig. 4, A and B). As
expected, these same nuclear fractions also contained high levels of
unspliced and partially spliced Pem pre-mRNAs. The only
nuclear fraction that had few or no spliced introns was the nuclear
membrane-associated fraction (F2) (Fig. 4, A and
B). This nuclear membrane fraction also lacked precursor
mRNA, consistent with the fact that precursor transcripts are known
to be localized primarily in the nuclear interior where they undergo
splicing (42-44). This F2 fraction contained intact RNA, as
demonstrated by the signals for Pem m1 and m2 mRNA (Fig. 4,
D and E) as well as cyclophilin mRNA (Fig.
4C).

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Fig. 4.
Subcellular localization of spliced
Pem introns, pre-mRNA, and mRNA.
A-E, Northern blot analysis of total cellular RNA
(T) and RNA from five operationally defined fractions as
follows: cytoplasmic (F1), nuclear-membrane associated
(F2), chromatin-associated (F3), high
salt-soluble nuclear (F4), and nuclear matrix (F5) obtained
from the ptPem-transfected HeLa cell clone (10 µg of RNA loaded in
all cases). The blot was hybridized sequentially with the indicated
probes. F, Northern blot analysis of subcellular fraction
RNA from HeLa cells stably transfected with pT M, a plasmid
containing the TCR- minigene driven by the tet-regulated promoter
(24). The fractionation was performed in parallel with the
fractionation of ptPem-transfected cells. The identity of the TCR-
transcripts is described in Ref. 24.
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Spliced IVS2 and IVS3 were also in the cytoplasmic fraction (F1) (Fig.
4, A and B). This F1 fraction had the
characteristics expected of the cytoplasm: (i) higher levels of mature
mRNAs, including mature Pem mRNA (m1 and m2) (Figs.
4, D and E) and mature cyclophilin mRNA (Fig.
4C), than did the nuclear
fraction, and (ii) no Pem pre-mRNAs, as determined using
either IVS2 or IVS3 probes (Fig. 4, A and B).
More spliced IVS2 was in the cytoplasmic fraction than in any of the
nuclear fractions, when taking into consideration the fact that the
cytoplasmic fraction contained ~85% of the total HeLa RNA. Spliced
IVS3 displayed a similar distribution pattern as that of spliced IVS2,
except that its accumulation in the cytoplasmic fraction was less
marked.

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Fig. 5.
Localization of the spliced Pem
introns IVS2 and IVS3 to both the nuclear and cytoplasmic
fractions of rat cell lines expressing the endogenous Pem
gene. A, Northern blot analysis of total cellular
RNA (10 µg) electrophoresed in a 1.2% agarose gel from the rat cell
lines indicated and the stably transfected ptPem-HeLa cell clone. The
blots in the left and right panels were both
hybridized sequentially with the Pem E1-E4 (cDNA) and the
cyclophilin housekeeping gene probes. endog = 1.0-kb
transcript expressed from the endogenous Pem gene.
B, RPA of 5 µg of nuclear (N), 20 µg of
nuclear wash (NW), and 20 µg of cytoplasmic (C)
RNA from the cell lines shown. The negative controls are a rat T-cell
line (T) and untransfected HeLa cells (not shown), neither
of which protected the IVS2, IVS3, and mRNA bands when 20 µg of
total cellular RNA was hybridized. The upper two panels are
different regions (and exposures) of the same lanes hybridized with the
IVS2/exon probe. The size of the spliced IVS2 and mRNA bands was
~230 and ~110 nt, respectively. The lower panel is of
identical RNA samples as that of the upper panels but
hybridized with the IVS3/exon probe and run in different lanes. The
size of the spliced IVS3 and mRNA bands was ~330 and ~50 nt,
respectively (mature mRNA bands are not shown). All three
panels are from the same gel. C, Northern blot analysis
of the nuclear, nuclear wash, and cytoplasmic fractions. The three
blots were hybridized sequentially with the Pem E1-E4 (cDNA), U6
snRNA, and 18 S rRNA probes, which hybridized with bands of 1.0, <0.5, and 1.9 kb, respectively. ~5 µg of RNA was loaded in each
lane, except the overloaded 208F C and FRTL-5 NW
lanes and the underloaded FRTL-5 N lane, as shown by
18 S rRNA hybridization and methylene blue staining of 28 S and 18 S
RNA (see "Experimental Procedures").
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The observation that Pem introns accumulated in the
cytoplasm differed from what we had observed previously for the intron IVS1C
1 from the TCR-
gene. Although we had observed trace levels of IVS1C
1 in the
cytoplasm, the vast majority of this intron accumulated in the nucleus
(24). We considered the possibility that this difference between
Pem introns and IVS1C
1 resulted from experiment to experiment variation in the subcellular
fractionation protocol. To examine this possibility, we prepared
fractions from TCR-
- and Pem-expressing cells in
parallel. We found that, unlike Pem introns, the TCR-
intron IVS1C
1 (0.5 kb) accumulated at only
trace levels in the cytoplasmic fraction (F1) as compared with the
nuclear fractions (F3-F5) (Fig. 4F) (note that the A, B,
and pre-mRNA transcripts are described in Clement et al.
(24)). We concluded that a significant proportion of Pem
spliced introns (IVS1, IVS2, and IVS3) reside in the cytoplasmic fraction, whereas the spliced intron IVS1C
1, Pem precursor mRNAs, and TCR-
precursor mRNAs
accumulate predominantly in nuclear fractions. This indicates that
Pem introns are either uniquely exported to the cytoplasm
in vivo or they reside in a unique soluble nuclear fraction
that leaks out during RNA preparation in vitro.
We considered the possibility that the cytoplasmic accumulation of
Pem introns is a peculiarity of the fact that the stably transfected HeLa cell clone used for our analyses expresses high levels
of Pem from a heterologous promoter. Although plausible, we considered
it unlikely that high expression per se was sufficient for
cytoplasmic accumulation, as the TCR-
spliced intron
IVS1C
1 is expressed at high levels from a
heterologous promoter, but it does not accumulate in the cytoplasm of
HeLa cells (Fig. 4F; Ref. 24). Nevertheless, to examine this
issue directly, we elected to examine the localization of
Pem introns in cell lines that express only modest levels of
Pem. We used two criteria to select such cell lines as
follows: 1) they must express lower levels of rat Pem than
the stably transfected HeLa cell clone, and 2) they must express the
rat Pem gene from its normal endogenous site. After
screening several rat cell lines by Northern blot analysis, we
identified six cell lines from diverse origins that express modest
levels of Pem mature mRNA as follows: PS-1 prostate cells, FRTL-5 thyroid cells, McA-8994 liver cells, and the 208F, Rat-1/S, and Rat-1/R fibroblast cell lines. The levels of
Pem mRNA in all of these cell lines were at least 5-fold
less than that of the ptPem-HeLa cell clone when levels were normalized against the cyclophilin housekeeping gene (Fig. 5A). The
size of Pem mRNA expressed by these cell lines (~1 kb) was the
same as expressed in other rodent cell lines and normal tissues
(25-27, 29, 30, 32).
To assess the Pem intron levels in these cell lines, we
attempted to use Northern blot analysis but were unable to detect a
signal, even with previously unhybridized blots. This confirmed that
these cell lines expressed only low levels of Pem. To improve sensitivity, we turned to RPA. We generated and tested several different probes for the two larger introns (IVS2 and IVS3) in order to
identify those that gave the best signal. We found that the probes that
gave unambiguous results were ones containing 234-nt IVS2/106-nt
upstream exon and 333-nt IVS3/46-nt upstream exon, respectively. By
using these two probes, we examined spliced IVS2 and IVS3 levels in
nuclear (N), nuclear wash (NW), and cytoplasmic (C) fractions. The
nuclear wash was included to increase the purity of the nuclear
fraction; this step involved centrifuging the nuclear pellet in lysis
buffer containing the detergent sodium deoxycholate (see
"Experimental Procedures").
We found that spliced IVS2 and IVS3 were present in all three fractions
of PS-1, 208F, and FRTL-5 cells (Fig. 5B, upper and lower panels). The signals were weak because these cells
express Pem at modest levels. Nevertheless, the specificity
of the protected transcripts detected by RPA was demonstrated by our
observation that a rat T-cell line lacking Pem mRNA (based on
Northern blot analysis) failed to protect the bands ("T," Fig.
5B), whereas the ptPem-HeLa cell clone protected high levels
of the IVS2, IVS3, and mRNA bands (data not shown). To determine
whether there was significant nuclear contamination of the cytoplasmic
fraction, we assayed the levels of U6 small nuclear RNA (snRNA) by
Northern blot. We found that U6 snRNA was primarily restricted to the
nuclear fraction; little or none was detected in the nuclear wash or
cytoplasmic fractions of all six rat cell lines (Fig. 5C and
data not shown; note that although a weak cytoplasmic U6 snRNA signal
was seen in 208F cells, this lane was overloaded). As further evidence for purity, RPA showed that the levels of IVS2- and IVS3-containing pre-mRNA (~340- and ~380-nt protected bands, respectively) were >10-fold higher in the nuclear fraction than in the cytoplasmic fraction of ptPem-HeLa cells (data not shown). We were unable to assess
pre-mRNA levels in the six rodent cell lines, as the signal was
insufficiently above background for accurate quantitation, presumably
because these cell lines transcribe the Pem gene at a much
lower levels than does the ptPem-HeLa cell clone.
Table IV shows the ratio of spliced
intron-to-mRNA for four of the rat cell lines. The ratio of spliced
intron-to-mRNA was between 0.02 and 0.07 (1:50 to ~1:15 ratio) in
the nuclear fraction. This ratio went down to between 0.001 and 0.01 (1:1000-1:100 ratio) in both the nuclear wash and cytoplasmic
fractions. In part, this decreased ratio is the result of the much
higher steady-state level of mature mRNA in the nuclear wash and
cytoplasmic fractions than in the nuclear fraction (Fig.
5B). Similar results were obtained with the Rat-1/S and
Rat-1/R cell lines, but the levels of spliced IVS2 and IVS3 were too
low in most fractions to permit accurate quantitation. We conclude that
spliced IVS2 and IVS3 are present in both the nuclear and cytoplasmic
fractions of cell lines expressing the endogenous rat Pem
gene. The modest levels of spliced introns relative to mature mRNA
in the cytoplasmic fraction derives, at least in part, from the fact
that spliced IVS2 and IVS3 are much less stable than is mature Pem
mRNA in this fraction (see below).
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Table IV
The ratio of spliced Pem introns to mRNA in rat cell lines
The ratio of intron to mRNA was calculated by quantifying the IVS2,
IVS3, and mRNA-protected bands obtained from RPA (see text). The
values were normalized for both length and U content of the protected
bands.
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Half-life of Spliced Pem Introns in Vivo--
We determined the
half-life of the Pem introns by performing time course
experiments. Pem gene transcription was blocked by incubating the cells with tet; RNA was isolated at different time intervals, and the decrease in RNA levels after transcriptional blockade was analyzed by Northern blot analysis.
We first examined the half-life of IVS2, as it was easily detectable by
Northern blot analysis. As Fig.
6A shows, IVS2 levels decreased steadily after addition of tet. A line fit plot of the data
revealed that IVS2 decayed with first-order kinetics (Fig. 6B). Least square linear regression analysis of results from
three independent experiments showed that the mean half-life for IVS2 was 16.0 ± 4.2 min (Table III). IVS3 had an even longer
half-life; stability analysis of four independent experiments showed
that spliced IVS3 had an average half-life of 28.7 ± 5.2 min
(Fig. 7, C and D,
Table III). Stability analysis of IVS1 was more difficult to perform,
as spliced IVS1 was only barely detectable by Northern blot analysis,
as discussed earlier. However, when we used a double-labeled IVS1 probe
(see "Experimental Procedures"), we were able to determine its
half-life to be 9.4 min (Fig. 7, A and B, and
Table III). These intron half-lives are comparable to published
half-lives of c-myc and c-fos mRNAs,
determined using general transcriptional inhibitors such as actinomycin
D (45-47). Our own analysis using actinomycin D showed that
c-myc mRNA has a half-life of 18 min in HeLa cells (24).

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Fig. 6.
Half-life analysis of the spliced
Pem intron IVS2. A, Northern blot
analysis of total cellular RNA (10 µg) (electrophoresed in a 1.2%
agarose gel) isolated from the ptPem-transfected HeLa cell clone
incubated with tet for the times indicated. The blot was hybridized
sequentially with the indicated probes. B, line fit plot of
the time-dependent decay of Pem IVS2 based on
the data shown in A (values were corrected against
cyclophilin mRNA levels).
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Fig. 7.
Half-life analysis of the spliced
Pem introns IVS1 and IVS3. A and
C, Northern blot analysis of total cellular RNA (10 µg)
electrophoresed in a 1.2% agarose gel isolated from the
ptPem-transfected HeLa cell clone incubated with tet for the times
indicated. The blot was hybridized sequentially with the indicated
probes. B and D, line fit plot of the
time-dependent decay of IVS1 and IVS3, based on the data
from A and C (values were corrected against
cyclophilin mRNA levels).
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Although the Pem introns had half-lives similar to that of
c-myc mRNA, we found that they were much less stable
than mature Pem mRNA. m1 and m2, the major Pem mRNAs
expressed in the pt-Pem-transfected cells, exhibited a slow decrease in
levels after cessation of transcription that corresponded to half-lives
of greater than 1 h (Fig. 8). m3,
which our previous analysis had suggested is a 3' splicing intermediate
(Figs. 2 and 4), had a shorter half-life than that of m1 and m2
mRNAs (Fig. 8).

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Fig. 8.
Half-life analysis of mature Pem
mRNA. Northern blot analysis of total cellular RNA (10 µg) electrophoresed in a 1.2% agarose gel isolated from the
ptPem-transfected HeLa cell clone incubated with tet for the times
indicated. The blot was hybridized sequentially with the indicated
probes. The level of cyclophilin mRNA reflects the amount of RNA
loaded in each lane.
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Branch Point and Debranchability of Pem IVS2--
Our analysis of
the three introns from the coding region of the Pem gene
suggested that these spliced introns had some unexpected characteristics that did not conform to the commonly held view that
introns are degraded rapidly in the nucleus of cells (14, 22). Given
this, we assessed whether these introns are typical mammalian introns
in other respects. Table III shows that all three Pem
introns possess 5' and 3' splice sites that display a typical degree of
identity with the mammalian consensus sequences. The Shapiro and
Senpathy rodent scores for the Pem intron splice sites were
between 84 and 92%, which is in the normal range, as most mammalian
splice sites score >70% (48).
Because intron debranching is considered to be a limiting event that
dictates intron stability (18, 24, 49), we next investigated whether
Pem introns were unusual in this respect. We restricted our
analysis to spliced IVS2 for two reasons. First, we found that only
IVS2 accumulated at sufficient levels in vivo for branch
point and debranching analyses (the substrates used normally for these
assays are abundant introns generated by in vitro splicing
(16)). Second, IVS2 is sufficiently small to be analyzed by
polyacrylamide gel electrophoresis, which resolves linear and lariat molecules.
The in vivo branch point of IVS2 was determined by primer
extension analysis. By using oligonucleotide J (Table I), we found one
extension product that corresponded to a branch point at an A
nucleotide 55 nt from the 3' terminus of IVS2 (designated as an
"A" in the S100
lane in Fig. 9A).
This
55 branch point was located in a region that matched the
mammalian branch point consensus 5'-YNYURAY-3' (50) at 6 of
7 positions (the branch point nt is in bold). We also observed an
extension product that corresponded to a branch point at position
85
(designated as an "a" in the
S100
lane in Fig.
9A). This branch point region conformed to the consensus sequence at 5 of 7 positions, which is a typical degree of similarity for mammalian introns (50).

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Fig. 9.
Analysis of in vivo derived
Pem IVS2: branch point mapping and
debranchability. A, total cellular RNA (50 µg) from
the ptPem-transfected HeLa cell clone was subjected to primer extension
analysis with an oligonucleotide complementary to the extreme 3'
terminus of the Pem IVS2. Lanes 1-4 show results
of dideoxy sequencing reactions for the nucleotides shown. Lane
5 shows the primer extension products (a and
A; indicated with the arrows). Lane 6 shows the disappearance of the primer extension products after
debranching with S100 extracts. Lane 7 shows a primer
extension reaction with no template (NC). Below
the gel is the sequence of the 3' terminus of Pem IVS2, with
the two mapped branch point nucleotides (in bold) and the
mammalian branch point consensus sequence (R = A or G and Y = C or U) (50). B, Northern blot analysis of total cellular
RNA (20 µg) from the ptPem-transfected HeLa cell clone subjected to
in vitro debranching and electrophoresed in a 6%
polyacrylamide gel. S100 A and B represent two
independent debranching extracts. The lariat and linear forms of IVS2
are shown, as well as Pem m1 mRNA. Lane 4 shows the RNA molecular weight marker X174/HinfI.
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The branch point sequence of introns is known to be a major determinant
of their debranchability. Adenylate branch point nucleotides are
efficiently debranched (16), whereas nonconsensus branch points are
poorly debranched (51, 52). Because our mapping indicated that IVS2
lariats are generated in vivo using consensus adenylate
branch point nucleotides, this suggested that these intron lariats are
good substrates for the debranchase enzyme. To examine this issue
directly, we performed debranching analysis. Polyacrylamide gels were
used for Northern blot analysis because they resolve the lariat and
linear forms of introns (24). By using this approach, we found that
spliced IVS2 accumulated primarily in the lariat conformation in HeLa
cells. Two forms of IVS2 lariats were resolved (A and
a) that probably differed in their gel migration because
their loop sizes differed as a result of usage of the
55 or the
85
branch points, respectively (Fig. 9B). The evidence that
these two molecules were both lariats came from the finding that each
displayed a different migration in 6% polyacrylamide gels (Fig.
9B) and 4% polyacrylamide gels (data not shown). By contrast, the linear form of IVS2, as well as Pem mRNA (m1),
migrated identically with respect to linear molecular weight markers in 4 and 6% polyacrylamide gels.
When incubated with debranchase-containing (S100) extracts, the levels
of both IVS2 lariats decreased, whereas the level of the linear form of
IVS2 concomitantly increased (Fig. 9B). This conversion of
lariats into linear molecules occurred with two independent S100
preparations (lanes 2 and 3). Further evidence for efficient debranching came from finding that incubation with the
S100 debranchase extract prevented the generation of the
"a" and "A" primer extension products
(S100+ lane in Fig. 9A).
We conclude that Pem IVS2 is a typical mammalian intron
lariat that can be efficiently debranched.
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DISCUSSION |
Spliced introns constitute a major portion of the RNA metabolized
in vertebrate cells, yet little is known about their fate or stability.
In this report, we determined the stability and localization of the
three introns that are spliced from the coding region of the
Pem homeobox gene. We measured the half-lives of these
introns using a tet-regulated promoter (Figs. 6 and 7 and Table III).
This tet promoter approach has the advantage that only the
transcription of the test gene is shut off for half-life analysis, thus
minimizing the chance of artifacts. In contrast, most studies investigating RNA half-life have used general transcriptional inhibitors, such as actinomycin D or DRB, which not only block the
synthesis of the transcript under scrutiny but also all other mRNAs, including those encoding putative regulators of RNA
half-life (53, 54). By using this tet promoter approach, we found that the Pem introns IVS1, IVS2, and IVS3 had half-lives of 9, 16, and 29 min, respectively (Figs. 6 and 7 and Table III). This was surprising given that the prevailing view (without direct evidence) that introns have half-lives on the order of only seconds (14, 22). All
of the Pem introns had half-lives longer than that for the
TCR
intron IVS1C
1 (6 min), the only other
vertebrate intron whose half-life has been determined (24). The
Pem intron half-lives were comparable to those of unstable
mammalian mRNAs, such as those encoding c-myc and
c-fos (24, 45, 46). Little is known about the stability of
introns from other organisms. The S. cerevisiae actin intron
has a half-life of ~5 min, which is about one-third the average
half-life of yeast mature mRNA (49).
Studies in yeast suggest that lariat debranching is the rate-limiting
step in intron degradation. Mutation of the branch point sequence in an
actin intron inhibited its debranching and dramatically increased its
stability in S. cerevisiae (49). Mutant S. cerevisiae lacking debranchase activity accumulated high levels of
circular introns missing the lariat tail (18). Collectively, these data indicate that the circular portion of an intron lariat is relatively impervious to degradation, but when the circle is opened by
debranching, this permits rapid exonuclease digestion of the intron.
Consistent with this model, we found that most of Pem IVS2
that accumulated in vivo was in the lariat conformation
(Fig. 9B). If debranching is indeed rate-limiting for intron
degradation, then introns may differ in stability because of
differences in their intrinsic debranchability. We observed a wide
range of different intron half lives, as brief as 6 min for TCR
IVS1C
1 (24) and as long as 29 min for
Pem IVS3 (Fig. 7). We were not able to determine if the
differences in the stability of Pem introns were due to
differences in their debranchability, as only Pem IVS2 was
amenable to in vitro debranching analysis (IVS1 levels were
too low and IVS3 was too large for polyacrylamide gel analysis).
Factors other than debranchability may also regulate intron stability,
including intron length and their relative 5' to 3' position, as we
found that both of these factors correlated with intron half-life.
Another factor that may control intron stability is the ability to be
released from the spliceosome. Mutant S. cerevisiae have
been isolated that accumulate higher than normal levels of introns,
including mutants for PRP22, which accumulate intron lariats in the
spliceosome (55). If Pem introns vary in their ability to be
released from the spliceosome complex, this could be a factor that
dictates their half-lives.
Our subcellular fractionation analysis revealed that spliced
Pem introns are present in three of the four nuclear
fractions that we purified: the nuclear matrix, high salt-soluble, and
chromatin-associated fractions (Fig. 4). These same fractions were also
the ones containing Pem pre-mRNA, consistent with their
being the site of origin of the spliced Pem introns.
Consistent with our results, other investigators (56) have demonstrated
that transcription and RNA processing occur in the same nuclear
compartments, including the nuclear matrix. We observed few or no
spliced Pem introns or pre-mRNA in the nuclear
membrane-associated fraction. This is consistent with the available
evidence indicating that RNA splicing occurs in the nucleoplasm rather
than at the nuclear membrane (42-44). Because all three Pem
introns and the TCR
IVS1C
1 intron
exhibited a similar relative distribution in the four nuclear fractions, it appears that this nuclear distribution pattern is a
general one for spliced vertebrate introns.
A surprise was our observation that all three spliced Pem
introns were in the cytoplasmic fraction of HeLa and rat cell lines. For some of the Pem introns, we found more in the
cytoplasmic fraction than in the nuclear fractions. Several lines of
evidence suggested that this reflected their cytoplasmic localization
in vivo rather than leakage of nuclear RNA into the
cytoplasmic fraction during RNA isolation in vitro. First,
we performed cellular lysis under conditions that did not cause
noticeable nuclear disruption (see "Experimental Procedures").
Second, we found that U6 snRNA and precursor mRNAs (both from
Pem and TCR-
), which would be expected to be primarily in
the nucleus, were indeed found in the nuclear fractions and were
virtually excluded from the cytoplasmic fraction. Third, even the
largest Pem intron (2.4 kb) was in the cytoplasmic fraction,
indicating that preferential leakage of small RNAs was not responsible
for what we observed. Fourth, we found that, unlike the Pem
introns, the TCR
IVS1C
1 intron was
predominantly in the nuclear fraction, indicating that the cytoplasmic
localization of the Pem introns was gene-specific.
An apparent contradiction with the possibility that Pem
introns accumulate in the cytoplasm are the data from in
situ hybridization studies using intron probes. These in
situ studies have not detected intron-containing transcripts in
the cytoplasm; instead such transcripts were only detected near the
site of gene transcription in the nucleus, sometimes in the form of
"tracks" (35, 42, 43). Although this appears to differ with our own
observation, there are several explanations for this apparent
discrepancy. First, as described above, some introns may not share with
Pem introns the ability to accumulate at high levels in the
cytoplasm. Second, in situ hybridization analysis does not
distinguish between spliced introns and pre-mRNAs; thus it is not
clear whether spliced introns are even sufficiently abundant to be
detected by this technique. Third, the extraction conditions used to
prepare sections for in situ hybridization may not retain
spliced introns. Fourth, in situ hybridization only provides
good signals for transcripts that accumulate in specific
intracellular regions; it does not typically detect transcripts that
are either dispersed or only transiently present in a given region. For
this reason, newly transcribed pre-mRNAs that are localized at the
site of transcription are easily detectable by in situ
hybridization, whereas even highly abundant mature mRNAs typically
give a low, diffuse signal in the cytoplasm (42-44). Thus, unless
spliced introns are localized to very specific cytoplasmic sites after
their removal from pre-mRNA, they would not be expected to give a
signal above background by in situ hybridization. We
conclude that spliced introns may not be detectable by in
situ hybridization in the cytoplasm, even though our study
suggests that they can be present in higher total amounts in
the cytoplasm than in the nucleus.
Although our data are consistent with preferential export of spliced
Pem introns into the cytoplasm, an alternative explanation of our results is that spliced Pem introns never reach the
cytoplasm but rather accumulate in a unique detergent-soluble portion
of the nucleus. If this is the case, then this is a unique
characteristic of Pem introns, because the TCR
IVS1C
1 intron does not display this
characteristic nor do the introns examined from immunoglobulin-
and
adenovirus genes (19, 21). To our knowledge, the only other documented
case of an intron present at higher levels in the cytoplasmic fraction
than in the nuclear fraction is that of the latency-associated
transcript from herpes simplex virus type-1 (57-59). Although the
half-life of the latency-associated transcript has not been determined, this spliced intron appears to be relatively stable and may confer a
functional role during viral latency (60).
Why do spliced introns exhibit differences in their ability to
accumulate in the cytoplasmic fraction? One possibility is that their
relative 5'-to-3' position in pre-mRNA controls their ability to be
exported. This follows from the fact that 5' introns are typically
spliced cotranscriptionally, whereas 3' introns are often spliced after
transcription and polyadenylation (61-63). If efficient cytoplasmic
intron export were linked to transcription, this would explain the
higher cytoplasmic accumulation of 5' Pem introns than that
of 3' Pem introns. This is reasonable given that RNA
processing is linked to transcription (64-66). Another possibility is
that smaller introns undergo cytoplasmic export more readily than do
larger exons, either as a result of passive diffusion or active
transport. This hypothesis is supported by the correlation between the
size of Pem introns and their nuclear-to-cytoplasmic ratio.
However, contradicting this simple hypothesis was our finding that the
TCR
intron IVS1C
1 had the highest nuclear-to-cytoplasmic level ratio, despite being similar in size (0.5 kb) to the mid-sized Pem intron IVS2 (0.6 kb).
How could spliced introns reach the cytoplasm? The breakdown of the
nuclear membrane at mitosis may provide one portal of entry to the
cytoplasm. Other possible routes of entry are either passive diffusion
or assisted export through nuclear pores. Regardless of the precise
mechanism, our data suggest that the pathway of intron export to the
cytoplasm differs from that of mRNA cytoplasmic export. First,
Pem introns were found at only trace levels in the nuclear
membrane fraction, whereas mRNAs accumulated at high levels in this
fraction (Fig. 4). This suggests that introns only transiently
associate with the nuclear membrane during cytoplasmic export. Further
evidence that the intron and mRNA transport pathways differ is the
observation that the intron IVS1C
1 and
TCR-
mRNA did not cofractionate when HeLa nuclear extracts were
separated on sucrose gradients (24). However, a caveat to this
interpretation is that since the data from both the sucrose gradient
nuclear fractions and subcellular fractions reflect steady-state concentrations of RNA, we cannot definitely distinguish between sites
of transport and sites of degradation.
What are the functional consequences of the accumulation and
degradation of spliced introns? We predict that if there were degradation of introns in the cytoplasm, then this would have an impact
on the cytoplasmic decay of mature mRNAs. Cytoplasmic introns
undergoing degradation would sequester ribonucleases and associated
decay-promoting factors away from mRNAs, thus permitting cytoplasmic mRNAs to accumulate to higher steady-state levels. Similarly, the decay of introns in the nucleus would play a protective role for nuclear pre-mRNAs and mRNAs. The rate of nuclear
intron decay may also dictate the amount of ribonucleotides and
splicing factors available for further rounds of transcription and RNA splicing, respectively. We conclude that intron metabolism has the
potential to influence gene expression at several different levels in
higher eukaryotic cells.