From the U526-Laboratoire de Virologie, Faculté
de Médecine, Avenue de Valombrose, 06107 Nice cedex 2, France,
the
Institut de Génétique Moléculaire de
Montpellier, 34293 Montpellier Cedex 5, France, the ** Department of
Anatomy and Structural Biology and Cell Biology, Albert Einstein
College of Medicine, Bronx, New York 10461, and the
§ Laboratoire de Transport Nucléocytoplasmique,
Institut Jacques Monod, 2 place Jussieu, 75251 Paris cedex 5, France
Received for publication, January 18, 2001, and in revised form, April 10, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Determining the cis-acting elements
controlling nuclear export of RNA is critical, because they specify
which RNA will be selected for transport. We have characterized
the nuclear export motif of the adenoviral VA1 RNA, a small cytoplasmic
RNA transcribed by RNA polymerase III. Using a large panel of
VA1 mutants in both transfected COS cells and injected
Xenopus oocytes, we showed that the terminal stem of VA1 is
necessary and sufficient for its export. Surprisingly, we found that
the nucleotide sequence within the terminal stem is not important.
Rather, the salient features of this motif are its length and its
relative position within the RNA. Such stems thus define a novel and
degenerate cytoplasmic localization motif that we termed the minihelix.
This motif is found in a variety of polymerase III transcripts, and cross-competition analysis in Xenopus oocytes revealed that
export of one such RNA, like hY1 RNA, is specifically competed by VA1 or artificial minihelix. Taken together these results show that the minihelix defines a new cis-acting export element and
that this motif could be exported via a novel and specific nuclear export pathway.
Correct intracellular localization of RNA is essential for its
function and can be utilized by the cell to regulate gene expression. Cytoplasmic or nuclear localization of RNA results from a balance between two opposing mechanisms, nuclear retention and transport through the nuclear pores (1-3). Analyses of RNA export in
Xenopus oocytes have shown that the export machinery relies
on saturable factors (4-6), and cross-competition experiments have
revealed the existence of only a few transport pathways, roughly
corresponding to major functional classes of RNA: rRNA, mRNA,
snRNA, and tRNA.
Many essential transport factors have now been identified, such as Ran,
exportins of the importin Despite our knowledge of export mechanisms, the actual RNA elements
that promote export are still not well characterized. These elements
are however critical because they are responsible for the specificity
of transport; they are the initial trigger of the export process, and
they determine which RNA will be selected from the nuclear RNA
population. To date, the best example characterized is that of snRNAs,
for which the export determinant was shown to consist of the 5'-cap
structure and is thus identical for all the RNAs of this family (16). A
common export determinant probably also exists for tRNAs because Xpo-t
recognizes a conserved feature of the structure that is formed by
acceptor and T stems (11, 17, 18). The export determinants of other
classes of RNA are not known. In the case of mRNAs, a variety of
sequences could be involved in addition to the 5'-cap and the poly(A)
tail (1, 19-21). Indeed, many mRNA-binding proteins shuttle
between the nucleus and the cytoplasm (22, 23, 24) and could be
adaptors between mRNA and the export machinery.
As suggested by these examples, it is likely that cytoplasmic RNAs of
different classes bear specific cis-acting export elements, many of them remaining uncharacterized. In the present study, we
focused on polymerase III transcripts (pol
III),1 and we have analyzed
in detail the adenovirus VA1 RNA (VA1). VA1 is a small viral RNA, which
accumulates in large amounts in the cytoplasm of adenovirus-infected
cells (25, 26). Its function is to inhibit the
double-stranded-dependent kinase, PKR, which otherwise
blocks translation of the viral mRNAs (27). Our study led us to
identify a new cis-acting RNA export motif that we termed the minihelix motif. Interestingly, this motif is encountered in a
large family of small viral and cellular transcripts, which all are
transcribed by pol III.
Plasmid Constructions--
The sequences of U1 Recombinant Proteins--
Purified recombinant
Schizosaccharomyces pombe Rna1p and RanBP1 were provided by
A. Wittinghofer (Max Planck Institute, Dormund, Germany). RanQ69L-GTP
was expressed and purified essentially as described (54) using an
expression vector provided by C. Dingwall (Stony Brook,
N.Y.).
Cell Culture--
Monkey COS1 cells were grown at 37 °C in
Dulbecco's modified Eagle's medium containing 10% calf fetal serum.
Cells were transformed by the calcium-phosphate co-precipitation
procedure and analyzed 24 h after transfection (31).
In Vitro Transcriptions--
Transcription reactions were
performed on polymerase chain reaction products using the Ampliscribe
T7 transcription kit (Epicentre Technologies) (see also Ref. 30).
Labeled RNAs were synthesized by adding 80 µCi of
[32P]UTP (3000 Ci/mmol), and unincorporated nucleotides
were removed by gel filtration. Capped U1 Oocyte Injection--
Nuclear injections were performed in
Xenopus oocytes as previously described (32), with a total
volume of 20 nl of RNA mixture per nucleus. To control nuclear
injection, samples were mixed with trypan blue (0.5 mg/ml). After
nuclear injection, oocytes were incubated at 19 °C for the indicated
time in modified bath medium, then transferred into ice-cold 1%
trichloroacetic acid. After manual dissection, only oocytes with blue
nuclei were used for further analysis. Nuclear and cytoplasmic
fractions were homogenized in solubilization buffer (50 mM
Tris-HCl, pH 7.5, 5 mM EDTA, 300 mM NaCl, and
0.5% SDS). Samples were then shaken for 30 min at 4 °C before
digestion by proteinase K (2 mg/ml) for 30 min at 56 °C. RNAs were
purified using conventional molecular biology techniques (phenol
extraction and ethanol concentration) then analyzed on denaturing
polyacrylamide gels and autoradiographed. For each sample, five oocytes
were pooled and 2 oocyte equivalents were loaded by lane. As artificial
minihelices are highly structured RNAs, they migrate faster than
expected and Stem17 (90 nt) migrates faster than tRNAPhe
(76 nt) unless gels are prerun for 30 min at 25 watts before loading.
When indicated results were quantified using the Bioprint acquisition
system and Bioprofil program (Vilbert Lourmat, France).
In Situ Hybridization--
In situ hybridization was
performed as previously described (30, 33) with digoxigenin-labeled RNA
probes and revealed with alkaline phosphatase-conjugated antibodies.
The Terminal Stem of VA1 Determines Its Intracellular
Localization--
VA1 has been previously dissected into three
different functional domains (Refs. 25, 34-36 and Fig.
1A): an apical stem-loop, required for PKR binding; a central domain, responsible for PKR inhibition; and a terminal stem, which brings together the 5'- and
3'-ends of the RNA. In previous studies, several VA1 mutants, having
well defined secondary structure, had been generated (30, 34-36). The
mutations were introduced in the apical or central domains but
maintained an intact terminal stem (Fig. 1B). Intracellular localization of these VA1 mutants was analyzed in transiently transfected COS1 cells using in situ hybridizations. These
mutant RNAs (dl2, dl4, dl7, VA
To test whether the terminal stem was necessary for export, we created
a new set of mutants. Mutations were inserted into a VA1 derivative
(VA
Changes in either the export process itself, or in RNA stability, can
affect the steady-state cellular localization of RNA. Indeed, some
mutations could selectively impair VA1 stability in the cytoplasm and
to result in a global change of nucleocytoplasmic partitioning.
Steady-state expression levels, of all the tested VA1 RNA mutants, were
however very similar in different cell types (293, COS1) as
demonstrated by Northern blot analysis of transiently transfected cells
(data not shown). We also measured VA1 RNA mutant half-life in 293 cells treated with actinomycin D (Table I). With the exception of
Mut10, which was significantly less stable (half-life: 40 min),
half-lives of all the VA1 mutants were very similar, independent of the
nuclear or the cytoplasmic localization of the mutant. Half-lives of
these RNAs were similar to that of the parental VA1 ranging between 2.5 and 6.5 h (Table I). Thus, it appeared that the terminal stem was
required for export per se and not for cytoplasmic RNA stability.
The Terminal Stem of VA1 Is a Nuclear Export Element--
To
unambiguously prove that the VA1 terminal stem was a
cis-acting export element, we turned to the model of
Xenopus oocyte, which allows direct kinetic analyses of
transport. Different radiolabeled RNAs were injected into oocyte
nuclei, and their nucleocytoplasmic distribution was analyzed at
different times after injection (Fig. 3A). Consistent with earlier
findings, tRNAphe and hY1 RNA were gradually exported to
the cytoplasm, whereas a U6
Previous studies in Xenopus oocytes have shown that all
known RNA export pathways can be specifically and independently
saturated by an excess of substrate RNA. As shown in Fig.
4, co-injection of an excess of VA1
completely inhibited the nuclear export of radiolabeled VA1
(lanes 5 and 6 versus lanes 2 and
3). The competition was specific because the export of
U1 Terminal Stems of Artificial Sequences Are Exported to Cell
Cytoplasm--
A comparative analysis of all VA1 sequences from human
and simian adenoviruses available in databases showed that the overall structure of the VA1 terminal stem was well conserved. This stem was
characterized by base pairing of the 5'- and 3'-RNA termini over 20 bases and the presence of two mismatches regularly spaced (Fig.
1A and Ref. 25). To determine whether the mismatches were important for export, we converted them into base pairs (Mut1-3, Fig.
2A). After transfection in COS1 cells, these mutants
localized in the cytoplasm as efficiently as VA1 (Fig. 2B).
A further analysis of VA1 sequences failed to reveal a particular
consensus motif in the sequence of the terminal stem (data not shown).
This suggested that the primary sequence of the stem was not important,
as long as its structure was conserved. To positively prove this point, we created several artificial RNAs (Fig.
5A). These RNAs were derived
from the LacZ sequence, and they were chosen to show no similarity to
the primary sequence of VA1. Even though they contained no natural
sequences, they were designed to form a terminal stem mimicking that of
VA1 at the structural level. In a first step, two RNAs were generated,
that formed a terminal stem of either 20 or 17 bases (Fig.
5A, Stem20 and Stem17). Because the
VA1 gene contains an intragenic promoter (40), another pol
III promoter, the human U6 promoter, was used to express Stem20 and
Stem17 in COS1 cells. As shown in Fig. 5B, Stem20 and Stem17
efficiently accumulated in the cytoplasm of transfected cells. In
addition, injection of Stem20 and Stem17 in Xenopus oocyte
nuclei showed that these artificial RNAs were also exported in this
system, similar to VA
These results demonstrated that artificial RNAs displaying a terminal
stem with structural features similar to the one of VA1 were localized
in the cytoplasm of both mammalian cells and Xenopus
oocytes. Remarkably, no specific sequences appeared to be required
within the terminal stem, they thus represented a highly degenerate
cytoplasmic localization motif that we termed the minihelix.
Furthermore, cross-competition experiments indicated that artificial
terminal stems and VA1 likely shared the same export pathway.
Structural Requirements in Minihelices for RNA Export--
The
apparent lack of requirement for a specific sequence in the minihelix
motif is paradoxical, because many RNAs that contain helices of similar
length are not exported. To delineate the specific features of the
minihelix motif, we constructed new mutants, derived from Stem17 and
Stem20, to analyze their localization in COS1 cells.
First, a conserved feature of minihelices was the length of the stem.
We thus created a set of artificial RNAs, which shortened the stem
gradually from 20 to 12 bases (Fig. 5A). A stem of 12 bases
was not exported, whereas a stem of 14 bases gave an intermediate phenotype (Fig. 5B).
A second characteristic of minihelices was base pairing of the RNA
5'-end, i.e. its first nucleotides with nucleotides close to
the 3'-end (Fig. 1A). This suggested that the precise
position of the RNA 5'- and 3'-ends could be critical for minihelix
function. As shown in Fig. 5, a mutation designed to disrupt the first
base pair of Stem20 (Fig. 5A, MM1) was sufficient
to significantly decrease Stem20 export (Fig. 5B and Table
I). Furthermore, mispairing of the first three base pairs completely
blocked Stem20 export (Fig. 5B, MM3; and Table I). These
results indicated that the RNA 5'-end had to be part of the minihelix
in order to promote RNA export. Another set of mutants were designed to
maintain the 5'-terminal base pairing, whereas the RNA 3'-end was
displaced further downstream from the base of the stem. Insertion of 8 bases had little effect on export (Fig. 5B, Xt8), but an
18-base insertion blocked it completely (Fig. 5B, Xt18).
This indicated that the minihelix should not only contain the RNA
5'-end, but should also have the 3'-end in its vicinity.
The last characteristic of the minihelix that we analyzed was its
tolerance to bending. Indeed, mismatches such as the ones found in the
terminal stem of VA1 allow stacking of the neighboring helices (41),
but with flexibility and bending at the junction (42). As shown
in Fig. 5A, we inserted bulges of 3 or 6 As in the middle of
the stem of Stem20, because previous in vitro work predicted
that these would create bends of 60° and 90°, respectively (42).
The two mutant RNAs were still exported (Fig. 5B, Stem-3A and Stem-6A), indicating that artificial stems could
tolerate strong bends and still be competent for export.
Altogether, these results showed that the minihelix had to meet some
requirements in order to be a cytoplasmic localization motif. Beginning
with the RNA 5'-end, the RNA 3'-end should be close to the base of the
stem, and its length should be longer or equal to 14 nucleotides. To
confirm that the effects we observed were due to variations in export
efficiencies and not RNA stability, the expression levels and
half-lives of several mutants were compared and showed no difference
that could account for their opposite localization (Table I).
Furthermore, mutants that had a short stem (Stem12), or that disrupted
the base pairing of the RNA 5'-end (MM3), were not exported in
Xenopus oocytes either (Fig. 3B).
Minihelix-containing RNAs are exported via a common export pathway. In
order to investigate the relations between minihelices and other export
pathways, we performed additional competition experiments in
Xenopus oocytes. Among cellular RNAs, hY1 and tRNAs were of
particular interest because these RNAs were both transcribed by pol III
and displayed a terminal stem very similar to the one of VA1 (Fig. 7
and data not shown). As shown in Fig. 4, co-injection of an excess of
VA1, Stem 17 or hY1 specifically inhibited the export of both VA1,
Stem17, and hY1 (Fig. 4,). These cross-competitions were specific
because none of these RNAs were able to inhibit export of U1
Interestingly, export of VA1, Stem17, and hY1 was also partially
inhibited by an excess of tRNAPhe (Fig. 4, lane
18). However, export of tRNAPhe was unaffected by an
excess of VA1 (lane 6), Stem17 (lane 12), or hY1
(lane 15), whereas it was inhibited by the same amount of
tRNAPhe (lane 18). These results were consistent
with earlier findings, because inhibition of hY1 export by excess of
tRNA, but not the reverse, has been previously described (39). Taken
together, this suggested that tRNAs may have some affinity for the
minihelix trans-acting factors (see below).
Nuclear Export of Minihelices Is a Ran-dependent
Process--
To further characterize the export pathway of
minihelices, we next analyzed whether RanGTP is required in this
process. For this purpose, a mixture of U1 Our analysis of VA1 export brings new insights into the mechanisms
controlling the nucleocytoplasmic partitioning of pol III RNAs. We have
discovered a novel and highly degenerate motif, the minihelix, that
promotes cytoplasmic localization of pol III RNAs in vertebrates. In
addition, the simplicity and the high degeneracy of this motif makes it
very suitable to vehicle therapeutic RNAs, such as ribozymes, antisense
or aptamers, into the cell cytoplasm.
The Minihelix Is a Novel RNA Export Motif, in Situ--
detection
of RNAs in transfected mammalian cells was used to show that the VA1
terminal stem is necessary and sufficient for cytoplasmic localization.
Measurements of RNA half-lives suggested that the terminal stem did not
selectively stabilize the RNA in the cytoplasm, but promoted RNA
export. This point was further tested using nuclear injections in
Xenopus oocytes. We found that indeed VA1 was gradually
exported from the nucleus of Xenopus oocytes and that
mutations that disrupted the terminal stem precluded its export.
Furthermore, we showed that VA1 export could be specifically blocked by
saturating amount of VA1 RNA.
Remarkably, the VA1 terminal stem can be replaced by a randomly chosen
sequence without loss of activity. This observation demonstrates that
the terminal stem does not require a specific sequence to be competent
for the export. This point can be further reinforced by considering the
high level of sequence variability observed among a large panel of
adenovirus VA1 sequences. These stems, however, still required
particular features to promote export. Indeed, artificial RNAs that had
a stem shorter than 14 bases, or that disrupted the pairing of the RNA
5'-end, were unable to reach the cytoplasm and remained nuclear in
mammalian cells. Importantly, these RNAs were also not exported in
Xenopus. By testing a large panel of mutants, we found that
in order to function the terminal stem should start with the RNA
5'-end, should be longer than 14 nucleotides, and should have a 3-8
nucleotide long protruding 3'-end. This family of degenerated sequences
thus defines a novel cytoplasmic localization motif, which we refer to
as the minihelix motif.
An essential feature of the minihelix motif is its high degree of
degeneracy. It not only lacks any sequence requirement, but it can also
accommodate many distortions within the stem. The terminal stem of all
VA1 RNAs contains two mismatches, and several VA1 mutants that we have
generated contain slightly larger mismatches and, however, are still
exported (Fig. 2). Also, bulges of 3 and 6 As could be inserted within
the minihelix without preventing its export, when such bulges have been
shown to induce bends of 60 and 90°, respectively (42).
Our study did not address a sequence requirement for the last 3' bases,
unpaired Us in all our mutants because it was not possible to mutate
them since they acted as a pol III transcription termination signal.
However, in the case of the hY1, which bears a functional minihelix
motif (see below), these terminal Us promote nuclear retention via
binding to the La protein, and do not export (39).
Minihelices Require a Common Limiting Transporter--
By
injection of competitor RNAs in Xenopus oocytes, we have
characterized the minihelix export pathway. It was first observed that
Stem17, an artificial RNA displaying a minihelix, could specifically compete with VA1 for export, and reciprocally. This suggests that export of unrelated minihelices relies on the same limiting
transporter, which thus defines the minihelix pathway.
To determine the relationship between the minihelix and other export
pathways, we tested the effect of other competitors, hY1,
tRNAPhe and U1
The relationship between the minihelix and tRNA pathways appears to be
less clear. As previously observed for hY1, a tRNA competitor could
block export of VA1 and Stem17; however, an excess of VA1 or Stem17
could not block export of tRNAPhe. This could occur if
tRNAPhe could bind the minihelix transporter, whereas
minihelices would not bind the tRNA transporter. The main pathway for
tRNA export in Xenopus oocytes has been described to utilize
Xpo-t, the tRNA exportin and in vitro binding assay has
shown that human Xpo-t does not bind minihelices (11-13, 17). Thus,
the minihelix export pathway is likely to be distinct from the one
mediated by Xpo-t.
We have shown that minihelix export is blocked in the presence of the
dominant negative GTPase-deficient mutant of Ran, RanQ69L, or when
RanGAP1 or RanBP1 are mislocalized. This indicates that minihelix
export depends on Ran and suggests that this pathway involves an
importin- Natural RNAs Exported by the Minihelix Pathway--
The high
degeneracy of the minihelix motif raises the possibility that many pol
III RNAs, from viral or cellular origin, could be exported through this
export pathway. The precise definition of the minihelix motif suggests
that candidate RNAs could be identified only on the basis of their
structural features. As illustrated in Fig.
7, folding of the VA1 and VA2 homologs
from simian and chicken adenoviruses fits the minihelix motif (25),
suggesting that they could be exported by this pathway. The EBER and
HPV RNAs, found in Epstein-Barr and herpes papio viruses, are similar to the VA RNAs and also display a terminal stem (43, 44). This stem,
however, deviates from the minihelix consensus because the RNA 5'-end
is left unpaired. Interestingly, we have shown that this kind of
mutation reduces export, leading to an intermediary phenotype with the
presence of such RNA both in nucleus and cytoplasm. This is in fact the
intracellular distribution of the EBER RNAs (45), suggesting that an
inefficient export signal could be used to regulate the localization of
viral RNAs.
Several cellular RNAs transcribed by RNA pol III also fit the minihelix
consensus. For instance, the Y RNAs that are components of Ro
ribonucleoprotein particles and which form a family of small cytoplasmic RNAs conserved from worms to humans (Fig. 7 and Refs. 46,
47), have a minihelix motif. This structural similarity predicts that
these RNAs could be exported by the minihelix pathway, an idea that is
supported both by the fact that hY1 is a specific competitor for
minihelices (this study) and by the requirement of an hY1 minihelix for
export (39). Remarkably, a Y RNA that contains a minihelix has recently
been found in Eubacteria (48). This suggests that Y RNAs may be widely
distributed in the eukaryotic kingdom and that the minihelix pathway
could be conserved among many eukaryotes.
Another intriguing possibility is that the minihelix pathway could
export tRNAs. Indeed, a tRNA competitor could block minihelix export.
Furthermore, tRNAs are L-shaped molecules, topologically similar to
minihelices with a 90° bend, which are competent for export. The main
pathway for tRNA export in Xenopus oocytes is mediated by
the transporter Xpo-t, which does not recognize minihelices (11, 17).
However, deletion of the yeast homolog of Xpo-t is known to be not
lethal for the yeast, suggesting that tRNAs should be exported via an
alternative export pathway. This subsidiary pathway is currently being
characterized (49-51). Whereas export of tRNA by the minihelix pathway
is speculative at this point, it is an attractive hypothesis because
the high degeneracy of the motif would ensure export of all mature tRNA
species independent of their primary sequence and subtle variations in
three-dimensional architecture.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
family, and TAP (7-10). Exportins and
TAP are transporters that shuttle between the nucleus and the cytoplasm
to transport a new cargo at each cycle. They can bind their cargo
directly or via adaptor molecules. Ran is a small GTPase able to switch
between GDP- and GTP-bound states, Ran-GTP is formed in the nucleus,
whereas conversion into Ran-GDP is catalyzed in the cytoplasm. RCC1,
the Ran GDP exchange factor (RanGEF), is exclusively nuclear whereas
the Ran GTPase-activating protein (RanGAP) as well as the
co-stimulatory factors RanBP1 and RanBP2 are cytoplasmic or at the
cytoplasmic face of the nuclear pore complex. This is thought to
provide a gradient of Ran within the cell with RanGTP in the nucleus
and RanGDP in the cytoplasm. Exportins bind their substrates in a
RanGTP-dependent way and in the cytoplasm, stimulation of
GTP hydrolysis on Ran triggers dissociation of the cargo from its
receptor. A well characterized example of a Ran-dependent
export pathway is illustrated by the tRNA export. RanGTP, but not
RanGDP, can form, in the nucleus, a stable trimeric complex containing
Ran, tRNA, and the tRNA exportin, Xpo-t, (11-13). Following
translocation through the nuclear pores, the cytoplasmic
Ran-GTPase-activating proteins promote GTP hydrolysis and disassembly
of the complex (14, 15). The tRNA is thus released, and the other
factors can be recycled for another round of export.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Sm, U6
ss,
hY1, and tRNAPhe have been previously described (16, 28,
29). The different mutations introduced into the terminal stem of the
VA
IV gene were obtained through the specific annealing of two
complementary synthetic oligonucleotides and cloned into blunted
restriction sites of the VA
IV (EcoRV and
Eco47III) (30). Artificial sequences were obtained by
cloning polymerase chain reaction fragments downstream of the human U6 promoter. All constructs containing artificial sequences were derived
from the pU6+1 plasmid (31). All relevant sequences are shown in the
figures, except for the loop of the artificial RNAs (base number
2321-2364 of LacZ, starting from the ATG).
Sm RNA was transcribed in
the presence of 3 mM of m7G(5')pppG. The
sequence of the RNA injected into Xenopus oocytes corresponded exactly to the one expressed in mammalian cells, except
that the first three bases (GUC) were replaced by GGG to allow for an
efficient in vitro transcription. The complementary bases in
the RNA structure were also modified to compensate for these changes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IV, ls1, ls2, ls5) accumulated in
large amounts in the cytoplasm, similarly to the parental VA1 (Fig. 1C and see Table I for
quantifications). Thus, neither the apical domain nor the central
domain appeared to be required for the cytoplasmic localization of VA1.
Some sequences were, however, necessary, because a construct for which
the 3'-half of VA1 was replaced by some unrelated lacZ sequence
remained nuclear (Fig. 1C, VA-Z).
View larger version (78K):
[in a new window]
Fig. 1.
Nuclear export of VA1 does not require its
central and apical domains. A, sequence and secondary
structure of the Ad2 VA1 RNA. The apical, central, and terminal domains
are indicated by large brackets. B, the VA1
mutants. Apical, central, and terminal domains are figured using
black, gray, and white boxes,
respectively. The position of the deletions (dl) are
indicated by gaps, whereas linker scanning mutants
(ls) are pictured by hatched lines (52). The
VA IV mutant lacks the entire central domain (30). C,
intracellular localization of VA1 RNA mutants. COS1 cells were
transiently transfected with the indicated plasmids, and the
localization of the resulting RNA was analyzed by in situ
hybridization. Each field represents 100 µm and shows a
representative cell (see Table I for quantifications).
Quantification of the localization of the different constructs used in
this study and characterization of their intracellular stability
IV) that despite the lack of the central domain localized in the
cytoplasm (Refs. 30, 37 and Fig. 1C). As shown in Fig.
2A, some mutations introduced
mismatches within the stem (Mut4, Mut5, Mut6), whereas others
interrupted it (Mut8, Mut10). It was expected that the design of these
mutations might profoundly affect the secondary structure of the VA1
terminal stem. Mut4, Mut5, and Mut10 were not exported to the cytoplasm and remained mostly nuclear, while Mut6 and Mut8 showed an intermediary phenotype (Fig. 2B and see Table I for quantifications). It
thus appeared that mutations affecting the terminal stem structure had
profound effects on VA1 cytoplasmic localization.
View larger version (61K):
[in a new window]
Fig. 2.
Nuclear export of VA1 requires an intact
terminal stem. A, structures of the VA1 terminal stem
mutants: only the terminal stem is pictured (from base 1 to 33 and from
base 130 to 160, respectively, see Fig. 1), and the mutations are
indicated by asterisks. B, the VA1 terminal stem
is required for its export. COS1 cells were transiently transfected
with the indicated plasmids, and the localization of the resulting RNA
was analyzed by in situ hybridization. Each field represents
100 µm and shows a representative cell (see Table I for
quantifications).
ss RNA remained nuclear (6, 38, 39). VA1
and VA
IV were also gradually exported, and about half of the RNA was
detected in the cytoplasm 3 h after nuclear injection.
Subsequently, the export of several of the VA1 RNA mutants, previously
used in COS1 cells (see Fig. 2), was analyzed in Xenopus
oocyte (Fig. 3B). VA1 RNA mutants having no terminal stem
(Mut10) or a strong alteration of the stem structure (Mut5), and which
were shown to remain nuclear in COS1 cells (Fig. 2B), were
also not exported in Xenopus (Fig. 3B). This lack
of cytoplasmic accumulation did not result from a specific degradation
in the oocyte cytoplasm, because decay of the total injected RNA was
minimal and also similar for exported RNAs (Fig. 3B).
View larger version (51K):
[in a new window]
Fig. 3.
Analysis of RNA export in Xenopus
laevis oocytes. A, nuclear export of VA1 and
VA IV RNAs. A mixture of 1.5 fmol of 32P-labeled VA1,
VA
IV, hY1, U6
ss, and tRNAPhe was injected into oocyte
nuclei. After the indicated time at 19 °C, nuclear and cytoplasmic
RNA (N and C, respectively) were extracted and
analyzed by polyacrylamide gel electrophoresis in denaturing
conditions. B, terminal stems are required to export VA1 or
artificial minihelices. The structure of the different VA1 RNA mutants
used in this experiment (Mut10 and Mut5) is shown
in Fig. 2, and the artificial RNAs (Stem20 and
Stem17) are depicted in Fig. 5. 1.5 fmol of the indicated
radiolabeled RNAs were injected into oocyte nuclei. After the indicated
time at 19 °C, total (T), nuclear (N), and
cytoplasmic (C) RNA were treated as described in
A.
Sm was not affected whereas, conversely, an excess of U1
Sm did
not compete for VA1 export (lanes 8 and 9). Taken
together, these experiments showed that the VA1 terminal stem was
sufficient and necessary to promote the nuclear export of the VA1 both
in mammalian cells and Xenopus oocyte through a saturable
export pathway.
View larger version (87K):
[in a new window]
Fig. 4.
RNA export in the presence of
competitors. A mixture of 1.5 fmol of radiolabeled VA1, U1 Sm,
hY1, U6
ss, tRNAPhe, and Stem17 was co-injected into
oocyte nuclei in absence or in presence of 2.5 pmol of the indicated
competitor. After injection, oocytes were incubated for 3 h at
21 °C, total (T), nuclear (N), and cytoplasmic
(C) RNAs were then extracted and analyzed by polyacrylamide
gel electrophoresis under denaturing conditions.
IV (Figs. 3B and 4). Interestingly,
export of Stem17 appeared to be a saturable process because it was
blocked by an excess of Stem17 (Fig. 4, lane 12 versus
lane 3). Furthermore, export of Stem17 was also inhibited by an
excess of VA1 competitor (Fig. 4, lane 6 versus lane
3), and, reciprocally, export of VA1 was blocked by an excess of
Stem17 (Fig. 4, lane 12 versus lane 3). These
competitions were specific because export of U1
Sm was not
affected.
View larger version (68K):
[in a new window]
Fig. 5.
Terminal stems of random sequences promote
export of pol III RNA. A, structures of the artificial
RNAs. These RNAs were derived from LacZ, and formed a loop of about 40 nucleotides closed by a terminal stem. Only the sequence and structure
of the terminal stem are depicted. B, the intracellular
localization of the artificial RNAs. The RNAs were expressed from the
human U6 promoter, which is transcribed by pol III. COS1 cells were
transiently transfected with the indicated plasmids, and the
localization of the resulting RNA was analyzed by in situ
hybridization. Each field represents 100 µm and shows a
representative cell (see Table I for quantifications).
Sm
(lanes 8 and 9). Conversely, U1
Sm did not
compete in the export of VA1, Stem17, nor hY1. These results thus
indicate that VA1, Stem17, and hY1 use the same nucleocytoplasmic
export pathway, or at least a common limiting factor but do not share any export factor used for the nuclear export of U1.
Sm, VA
IV, U6
ss,
Stem20 and tRNAPhe was co-injected into the nucleus of
oocytes with either RanBP1 to reduce the nuclear concentration of
RanGTP or with RanGAP from S. pombe (Rna1p) to deplete
RanGTP from the nucleus (Fig. 6). As
previously reported (55) nuclear injection of RanBP1 prevented the
nuclear export of U1
Sm without affecting the export of tRNA (lanes 7-9 versus lanes 4-6). This experimental
condition also led to an inhibition of VA
IV and Stem20 transport. In
agreement with this result, nuclear injection of Rna1p completely
blocked nuclear export of U1
Sm, tRNAPhe, and minihelices
(lanes 10-12 versus lanes 4-6). These data
indicate that nuclear export of minihelix-containing RNAs depends on
nuclear RanGTP. To determine whether this export pathway requires GTP hydrolysis by Ran, we used a dominant-negative, GTPase-deficient mutant
of Ran, RanQ69L. Nuclear injection of RanQ69L partially inhibited the
nuclear export of U1
Sm, VA
IV, and Stem20 but did not affect tRNA
transport (lanes 13-14 versus lanes 4-6)
suggesting that nuclear export of minihelices likely depends on GTP
hydrolysis by Ran.
View larger version (52K):
[in a new window]
Fig. 6.
Nuclear export of minihelices is a
Ran-dependent process. A mixture of
32P-labeled U1 sm, VA
IV, U6
ss, Stem20, and
tRNAPhe was co-injected into oocyte nuclei in the absence
(lanes 1-6) or in the presence of RanBP1 (lanes
7-9), Rna1p (lanes 10-12), or RanQ69L (lanes
13-15) at the indicated concentrations in a total injection
volume of 20 nl. After 0 (lanes 1-3) or 3 h
(lanes 4-15) at 19 °C, total (T), nuclear
(N), and cytoplasmic (C) RNAs were extracted and
analyzed by polyacrylamide gel electrophoresis under denaturing
conditions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Sm snRNA. Cross-competition occurred
between VA1, Stem17, and hYI RNAs, indicating that VA1 and hY1 RNAs
were likely to share the same nuclear export pathway or at least one
common trans-acting factor. In contrast, U1
Sm snRNA had
no inhibitory activity on the export of either VA1, Stem17, or hY1 RNA,
indicating that the minihelix pathway is distinct from that involving
snRNAs and thus does not appear to involve CRM1. This was in agreement
with previous results, which showed that hY1 was utilizing a pathway distinct from snRNA and leucine rich NES (39).
-related receptor. As a consequence, the minihelices
receptor is likely a different TAP, a receptor involved in mRNA
export that does not belong to the
-importin family and does not use
Ran to interact with cargoes.
View larger version (28K):
[in a new window]
Fig. 7.
Structure of RNA potentially exported by the
minihelix pathway. The structure of the minihelix of several small
cytoplasmic RNAs is pictured: adenovirus type 2 (VA2); Epstein-Barr
virus (EBER1 and EBER2); Herpesvirus papio (HPV1 and HPV2);
human Y1, Y3, Y4, and Y5 (hY1, hY3, hY4, and hY5);
Caenorhabditis Y RNA. Only the sequence of the minihelix is
shown, the rest of the RNA is schematized with a loop. The consensus
for the minihelix motif is also shown. Note that this consensus
tolerates bulges and mismatches. RNA sequences were retrieved from
GenBankTM and folded using mfold software (53).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Ian Mattaj and Michael Mathews for
providing us with U6ss and U1
Sm constructs and VA1 mutants,
respectively. We thank A. Wittinghofer for recombinant Rna1p and RanBP1
proteins. Special thanks are due to Magali Mailland for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by the Agence Nationale de Recherche sur le SIDA (ANRS), Sidaction, the Boris Vlasov Fundation (Monaco), ARC grant 9043, and National Institutes of Health Grants GM54887 and 57071.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.: 33 4-93-37-7678; Fax: 33 4-93-81-5484; E-mail: doglio@unice.fr.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M100493200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: pol III, polymerase III; nt, nucleotide.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Custodio, N.,
Carmo-Fonseca, M.,
Geraghty, F.,
Pereira, H. S.,
Grosveld, F.,
and Antoniou, M.
(1999)
EMBO J.
18,
2855-2866 |
2. | Legrain, P., and Rosbash, M. (1989) Cell 57, 573-583[Medline] [Order article via Infotrieve] |
3. | Schmidt-Zachmann, M. S., Dargemont, C., Kuhn, L. C., and Nigg, E. A. (1993) Cell 74, 493-504[Medline] [Order article via Infotrieve] |
4. | Bataille, N., Helser, T., and Fried, H. M. (1990) J. Cell Biol. 111, 1571-1582[Abstract] |
5. | Khanna-Gupta, A., and Ware, V. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1791-1795[Abstract] |
6. | Zasloff, M. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6436-6440[Abstract] |
7. | Dahlberg, J. E., and Lund, E. (1998) Curr. Opin. Cell Biol. 10, 400-408[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Izaurralde, E.,
and Adam, S.
(1998)
RNA
4,
351-364 |
9. | Mattaj, I. W., and Englmeier, L. (1998) Annu. Rev. Biochem. 67, 265-306[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Stutz, F.,
and Rosbash, M.
(1998)
Genes Dev.
12,
3303-3319 |
11. | Arts, G. J., Fornerod, M., and Mattaj, I. W. (1998) Curr. Biol. 8, 305-314[Medline] [Order article via Infotrieve] |
12. |
Hellmuth, K.,
Lau, D. M.,
Bischoff, F. R.,
Kunzler, M.,
Hurt, E.,
and Simos, G.
(1998)
Mol. Cell. Biol.
18,
6374-6386 |
13. | Kutay, U., Lipowsky, G., Izaurralde, E., Bischoff, F. R., Schwarzmaier, P., Hartmann, E., and Gorlich, D. (1998) Mol. Cell 1, 359-369[Medline] [Order article via Infotrieve] |
14. | Hopper, A. K., Traglia, H. M., and Dunst, R. W. (1990) J. Cell Biol. 111, 309-321[Abstract] |
15. | Matunis, M. J., Coutavas, E., and Blobel, G. (1996) J. Cell Biol. 135, 1457-1470[Abstract] |
16. | Hamm, J., and Mattaj, I. W. (1990) Methods Enzymol. 181, 273-284[Medline] [Order article via Infotrieve] |
17. |
Arts, G. J.,
Kuersten, S.,
Romby, P.,
Ehresmann, B.,
and Mattaj, I. W.
(1998)
EMBO J.
17,
7430-7441 |
18. |
Lipowsky, G.,
Bischoff, F. R.,
Izaurralde, E.,
Kutay, U.,
Schafer, S.,
Gross, H. J.,
Beier, H.,
and Gorlich, D.
(1999)
RNA
5,
539-549 |
19. | Hamm, J., and Mattaj, I. W. (1990) Cell 63, 109-118[Medline] [Order article via Infotrieve] |
20. | Jarmolowski, A., Boelens, W. C., Izaurralde, E., and Mattaj, I. W. (1994) J. Cell Biol. 124, 627-635[Abstract] |
21. | Wickens, M. P., and Gurdon, J. B. (1983) J. Mol. Biol. 163, 1-26[Medline] [Order article via Infotrieve] |
22. |
Caceres, J. F.,
Screaton, G. R.,
and Krainer, A. R.
(1998)
Genes Dev.
12,
55-66 |
23. | Nakielny, S., and Dreyfuss, G. (1997) Curr. Opin. Cell Biol. 9, 420-429[CrossRef][Medline] [Order article via Infotrieve] |
24. | Pinol-Roma, S., and Dreyfuss, G. (1992) Nature 355, 730-732[CrossRef][Medline] [Order article via Infotrieve] |
25. | Ma, Y., and Mathews, M. B. (1996) J. Virol. 70, 5083-5099[Abstract] |
26. | Mathews, M. B., and Shenk, T. (1991) J. Virol. 65, 5657-5662[Medline] [Order article via Infotrieve] |
27. | O'Malley, R. P., Mariano, T. M., Siekierka, J., and Mathews, M. B. (1986) Cell 44, 391-400[Medline] [Order article via Infotrieve] |
28. | O'Brien, C. A., Margelot, K., and Wolin, S. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7250-7254[Abstract] |
29. | Roe, B. A., Anandaraj, M. P., Chia, L. S., Randerath, E., Gupta, R. C., and Randerath, K. (1975) Biochem. Biophys. Res. Commun. 66, 1097-1105[Medline] [Order article via Infotrieve] |
30. | Barcellini, C. S., Fenard, D., Bertrand, E., Singer, R. H., Lefebvre, J. C., and Doglio, A. (1998) Antisense Nucleic Acid Drug Dev. 8, 379-390[Medline] [Order article via Infotrieve] |
31. |
Bertrand, E.,
Castanotto, D.,
Zhou, C.,
Carbonnelle, C.,
Lee, N. S.,
Good, P.,
Chatterjee, S.,
Grange, T.,
Pictet, R.,
Kohn, D.,
Engelke, D.,
and Rossi, J. J.
(1997)
RNA
3,
75-88 |
32. | Terns, M. P., and Goldfarb, D. S. (1998) Methods Cell Biol. 53, 559-589[Medline] [Order article via Infotrieve] |
33. |
Samarsky, D. A.,
Fournier, M. J.,
Singer, R. H.,
and Bertrand, E.
(1998)
EMBO J.
17,
3747-3757 |
34. | Mellits, K. H., and Mathews, M. B. (1988) EMBO J. 7, 2849-2859[Abstract] |
35. | Mellits, K. H., Pe'ery, T., and Mathews, M. B. (1992) J. Virol. 66, 2369-2377[Abstract] |
36. | Pe'ery, T., Mellits, K. H., and Mathews, M. B. (1993) J. Virol. 67, 3534-3543[Abstract] |
37. | Cagnon, L., Cucchiarini, M., Lefebvre, J. C., and Doglio, A. (1995) J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 9, 349-358[Medline] [Order article via Infotrieve] |
38. | Hamm, J., and Mattaj, I. W. (1989) EMBO J. 8, 4179-4187[Abstract] |
39. | Simons, F. H., Rutjes, S. A., van Venrooij, W. J., and Pruijn, G. J. (1996) RNA 2, 264-273[Abstract] |
40. | Bhat, R. A., Metz, B., and Thimmappaya, B. (1983) Mol. Cell. Biol. 3, 1996-2005[Medline] [Order article via Infotrieve] |
41. | Kim, J., Walter, A. E., and Turner, D. H. (1996) Biochemistry 35, 13753-13761[CrossRef][Medline] [Order article via Infotrieve] |
42. | Zacharias, M., and Hagerman, P. J. (1995) J. Mol. Biol. 247, 486-500[CrossRef][Medline] [Order article via Infotrieve] |
43. | Howe, J. G., and Shu, M. D. (1988) J. Virol. 62, 2790-2798[Medline] [Order article via Infotrieve] |
44. | Rosa, M. D., Gottlieb, E., Lerner, M. R., and Steitz, J. A. (1981) Mol. Cell. Biol. 1, 785-796[Medline] [Order article via Infotrieve] |
45. | Schwemmle, M., Clemens, M. J., Hilse, K., Pfeifer, K., Troster, H., Muller, W. E., and Bachmann, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10292-10296[Abstract] |
46. | van Gelder, C. W., Thijssen, J. P., Klaassen, E. C., Sturchler, C., Krol, A., van Venrooij, W. J., and Pruijn, G. J. (1994) Nucleic Acids Res. 22, 2498-2506[Abstract] |
47. | Van Horn, D. J., Eisenberg, D., O'Brien, C. A., and Wolin, S. L. (1995) RNA 1, 293-303[Abstract] |
48. |
Chen, X.,
Quinn, A. M.,
and Wolin, S. L.
(2000)
Genes Dev.
14,
777-782 |
49. | Grosshans, H., Simos, G., and Hurt, E. (2000) J. Struct. Biol. 129, 288-294[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Sarkar, S.,
and Hopper, A. K.
(1998)
Mol. Biol. Cell
9,
3041-3055 |
51. |
Sarkar, S.,
Azad, A. K.,
and Hopper, A. K.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14366-14371 |
52. | Mellits, K. H., Kostura, M., and Mathews, M. B. (1990) Cell 61, 843-852[Medline] [Order article via Infotrieve] |
53. |
Zuker, M.,
and Jacobson, A. B.
(1998)
RNA
4,
669-679 |
54. | Klebe, C., Bishoff, F. R., Ponstingl, H., and Wittinghofer, A. (1995) Biochemistry 34, 639-647[Medline] [Order article via Infotrieve] |
55. |
Izaurralde, E.,
Kutay, U.,
Von Kobbe, C.,
Mattaj, I. W.,
and G![]() |