From the
Due to 3` end modifications, mammalian U6 small nuclear RNA
(snRNA) is heterogeneous in size. The major form terminates with five U
residues and a 2`,3`-cyclic phosphate, but multiple RNAs containing up
to 12 U residues have a 3`-OH end. They are labeled in the presence of
[
Splicing of mRNA precursors occurs in a large ribonucleoprotein
complex called the spliceosome. The reaction requires five U
snRNAs
As a result of post-transcriptional 3` end modifications, metazoan
U6 snRNA is heterogeneous(10, 11) . In addition to a
major form terminating with five Us and a 2`,3`-cyclic
phosphate(12) , multiple minor RNAs exhibit, at their 3` end, an
oligouridylate stretch of variable length. All are part of the known U6
snRNA-containing particles, U4
We report here that the hnRNP C protein, belonging to the
family of proteins exhibiting a RNP consensus sequence (RNP-CS),
interacts with a subset of U6 snRNAs characterized by their relatively
long 3`-oligouridylate tail. Binding occurs at this tail since the
protein becomes cross-linked upon irradiation with UV light of nuclear
extracts containing 3` end-labeled U6 snRNAs. Significantly, the hnRNP
C protein, either purified from HeLa cells or produced as recombinant
protein from Escherichia coli, induces the release of
elongated U6 snRNAs from U4
Since U4 and U6 snRNAs become
separated within the spliceosome before the first step of splicing
takes place(33) , and since the hnRNP C protein is supposed to
be involved in splicing(14) , it is tempting to hypothesize that
U4
A U6 snRNA-hnRNP C protein interaction functional in splicing
raises the question of how elongated U6 snRNAs are generated. They
appear from shorter forms upon incubation of the extracts with an
excess of UTP, as well as with ATP under splicing conditions and are
present in all of the U6 snRNA-containing complexes(13) . In
fact, we can now add that preexisting elongated U6 snRNAs exist as well
and are assembled into multi-snRNP complexes. This was ascertained by
the finding that some labeled U6 snRNAs with long uridylate tail have
left their label when subjected to
The hypothesis of a U6 snRNA-hnRNP C
protein interaction related to the splicing mechanism is also
compatible with the recent proposal that the hnRNP C protein, as well
as other hnRNP proteins, have RNA chaperonine activity (Ref. 35 and,
for a review, see Ref. 36). It is thought that these proteins promote
association or dissociation of trans-acting factors in modulating the
pre-mRNA conformation. It is therefore tempting to speculate that the
hnRNP C protein acts similarly on the U6 snRNA conformation to disrupt
U4
We thank D. Portman and G. Dreyfuss for providing
recombinant hnRNP C protein and K94 mutant and helpful comments on the
manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-
P]UTP by the terminal uridylyl
transferase activity present in HeLa cell nuclear extracts. That these
forms all enter the U6 snRNA-containing particles, U4
U6,
U4
U5
U6, and the spliceosome, has been demonstrated
previously. Here, we report an interaction between the heterogeneous
nuclear ribonucleoprotein (hnRNP) C protein, an abundant nuclear
pre-mRNA binding protein, and the U6 snRNAs that have the longest
uridylate stretches. This U6 snRNA subset is free of any one of the
other snRNPs, since anti-Sm antibodies failed to immunoprecipitate
hnRNP C protein. Furthermore, isolated U4
U6 snRNPs containing U6
snRNAs with long oligouridylate stretches are disrupted upon binding of
hnRNP C protein either purified from HeLa cells or produced as
recombinant protein from Escherichia coli. In view of
these data and our previous proposal that the U6 snRNA active in
splicing has 3`-OH end, we discuss a model where the hnRNP C protein
has a decisive function in the catalytic activation of the spliceosome
by allowing the release of U4 snRNP.
(
)packaged into four individual snRNPs
(U1, U2, U4
U6, and U5) and an as yet undetermined number of
non-snRNP proteins. Spliceosome assembly involves the ordered
interaction of U1 and U2 snRNPs through RNA base pairing with the
5`-splice site and the sequence around the branch point, respectively,
and then the entry of the other snRNAs in the form of
U4
U6
U5 tri-snRNP (for a recent review, see Ref. 1). It is
known that U4 and U6 snRNAs are base-paired through two intermolecular
helices (stem I and stem II) forming an evolutionary conserved
secondary structure, the so-called Y structure(2) . This
structure is thought to occur in both U4
U6 and U4
U6
U5
complexes but to be disrupted once the U4
U6
U5 tri-snRNP has
entered the spliceosome. Indeed, compelling results in both yeast and
mammalian systems have led to the conclusion that U6 snRNA forms new
base-pairing interactions with U2 snRNA and the pre-mRNA in the
spliceosome (3, 4, 5, 6, 7) to
yield a tripartite structure reminiscent of that formed in domains 5
and 6 of group II introns(1, 8) . Extensive base pairing
between U4 and U6 is an obstacle to the formation of this tripartite
structure, and the U4
U6 snRNP is disrupted in the spliceosome
before the splicing intermediates and products appear(9) . The
step(s) leading to the release of U4 snRNP is (are) not understood.
U6, U4
U5
U6, and the
spliceosome. The finding that U6 snRNA with a 2`,3`-cyclic phosphate
end is generated within the spliceosome as a consequence of pre-mRNA
splicing (13) led us to propose that U6 snRNAs with elongatable
ends are the active forms in splicing. As a first step toward the
elucidation of the function of U6 snRNA elongation in splicing, we have
obtained evidence that the hnRNP C protein, a potential partner of the
splicing reaction(14) , is bound exclusively to the U6 snRNAs
having the longest oligouridylate tails, and we have demonstrated that
hnRNP C induces disruption of base-paired U4
U6 snRNAs in isolated
U4
U6 snRNPs.
Materials
Micrococcal nuclease, RNase A-and
protein A-Sepharose 4B were from Pharmacia Biotech Inc. RNase H was
from Life Technologies, Inc. The anti-C hnRNP (4F4), the anti-A1 hnRNP
(4B10), and the anti-U hnRNP (3G6) antibodies and the recombinant hnRNP
C1 protein and K94 mutant were generous gifts from Dr. G. Dreyfuss
(Howard Hughes Medical Institute, Philadelphia, PA). The other
antibodies used were the monoclonal anti-Sm Y12, and a patient serum of
SSb specificity (anti-La antibodies). All other chemicals were of
analytical grade.
Extracts and Oligodeoxynucleotide-directed Cleavage of
snRNAs
HeLa cell nuclear extracts were prepared by the method of
Dignam et al.(15) except that triethanolamine buffer
was used instead of HEPES. The DEAE-Sepharose-cleared extract was
according to Hinterberger et al.(16) . A
U4U6-enriched preparation was obtained from this extract by
centrifugation in a glycerol gradient as described
previously(13) . The hnRNP C protein was prepared according to
Pinol-Roma and Dreyfuss(17) . Oligodeoxynucleotide cleavage of
snRNAs was as described previously(18) . Oligonucleotides were
complementary to U4 (nucleotides 65-85), U5 (nucleotides
69-87) and U6 (nucleotides 77-95).
U6 snRNA Labeling and UV Cross-linking
Endogeneous
U6 snRNAs were 3` end-labeled by incubating 15 µl of nuclear
extract with 30 µCi of [-
P]UTP in a
final volume of 25 µl containing 3.2 mM MgCl
,
1 mM ATP, and 20 mM creatine phosphate for 30 min at
30 °C(13) . For UV cross-linking experiments(18) ,
the reactions were kept on ice for 10 min and then irradiated for 10
min with an UV transilluminator at 254 nm (7 milliwatts/cm
on the surface of the filter). The distance between the samples
and the filter was 9 cm. The samples were finally digested with RNase A
(0.6 µg/ml) for 60 min at 37 °C before being precipitated by 5
volumes of acetone and electrophoresed in a 10% SDS-polyacrylamide gel
followed by autoradiography on Kodak XAR films. RNase A digestion of
immunoprecipitated samples was carried out directly on protein
A-Sepharose beads after they were washed with NET 2 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Nonidet P-40, 0.5
mM dithiothreitol). Elution of the beads was in 50 µl of
Laemli buffer.
Glycerol Gradient Centrifugation and Detection of the
snRNAs
200-µl reactions containing 3` end-labeled U6 snRNAs
were loaded onto 4 ml of 10-30% glycerol gradients made in 50
mM Tris-glycine buffer (pH 8.8). Centrifugation was in a SW 60
Beckman rotor at 41,000 rpm for 3 h at 4 °C. A total of 18
fractions of 220 µl were recovered from the top. The RNAs present
in each fraction were extracted with phenol after proteinase K (4
mg/ml) digestion and separated in a 10% polyacrylamide, 8 M urea gel. Labeled RNAs were first detected by autoradiography, and
then the gel was electroblotted to Hybond membranes under the same
conditions as described by Blencowe et al.(19) . The
membranes were UV-treated for 30 s, baked 1 h at 80 °C, and then
prehybridized for 2 h before hybridization at 42 °C according to
Church and Gilbert(20) . DNA probes complementary to U4, U6, and
U5 snRNAs were the same as those used in the
oligodeoxynucleotide-directed cleavage experiments. Those complementary
to U1 and U2 corresponded to nucleotides 1-15. All were 5`
end-labeled with T4 polynucleotide kinase in the presence of
[-
P]ATP. Hybridizations were in the same
buffer at 42 °C. One wash of 5 min in 2
SSC and then three
washes of 15 min each in 1
SSC, 0.1% SDS were performed at room
temperature before autoradiography.
Immunoprecipitations
Assays to detect
immunoprecipitated protein adducts after UV cross-linking (see above)
and to identify those of the 3` end-labeled U6 snRNAs that are
immunoprecipitated were performed directly with 30 µl of a 3`
end-labeling reaction or from 100 µl after fractionation of this
same reaction in glycerol gradients. In all cases, antibodies were
pre-bound to protein A-Sepharose in NET 2 buffer as described
previously(21) . The samples were added to 50 µl of antibody
bound to protein A-Sepharose, adjusted to 200 µl, and incubated
with gentle agitation for 1 h at 4 °C. After four washes with 1 ml
of NET 2 buffer, bound material either was analyzed for protein adducts
if UV cross-linked or digested with proteinase K for RNA detection.
Released RNAs were extracted with phenol, separated by electrophoresis
in 10% polyacrylamide-urea gels in Tris borate/EDTA (TBE) buffer, and
finally detected by autoradiography.
hnRNP C Protein Interacts with U6 snRNAs with the
Longest Oligouridylate Stretches
Due to an endogenous terminal
uridylyl transferase, incubation of HeLa cell nuclear extracts under
splicing conditions in the presence of
[-
P]UTP leads to preferential 3`
end-labeling of U6 snRNA as well as other RNAs of smaller size (Ref. 13
and see ``Experimental Procedures''). Such an assay reveals
the presence of multiple U6 snRNAs, which differ by the length of their
oligo(U) tail. All are assembled into U6, U4
U6, and
U4
U6
U5 snRNPs(13) . UV cross-linking experiments
were done to determine whether spliceosomal proteins interact with the
oligouridylate stretch of the U6 snRNAs exhibiting a 3`
(U)
-OH end. Nuclear extracts were incubated in the
presence of [
-
P]UTP and irradiated at 254
nm. After digestion by RNase A, the presence of protein adducts was
examined by electrophoresis in a SDS-polyacrylamide gel. Two proteins,
one of about 50 kDa and a second one forming a doublet in the range of
42 kDa, were detected (Fig. 1, lane2). On the
basis of previously published data(22) , we suspected that the
50-kDa protein adduct represented the La antigen. In agreement with
this prediction, this protein adduct was immunoprecipitated with
anti-La antibodies (lane4). Two criteria led us to
suggest that the second protein adduct might be the hnRNP C protein:
its apparent size and the fact that the hnRNP C protein is known to
bind avidly to poly(U) stretches(23, 24) . This was
verified by the finding that anti-C antibodies precipitate the protein
adduct (lane6). Since some other RNAs are labeled
under the conditions used(13) , it was necessary to identify the
small RNAs that transferred their label to the La and C proteins. The
extracts incubated with [
-
P]UTP were
immunoprecipitated as described above. Fig. 2A shows
that anti-La, anti-Sm, and anti-C antibodies (lanes4-6, respectively) immunoprecipitated labeled RNAs
of a size compatible with U6 snRNA. Although we demonstrated previously
by sequencing that the RNAs indicated U6 in lanes1and 2 (controls) are authentic(13) , we
carried out a complementary assay based on oligodeoxynucleotide
directed cleavage by RNase H to be sure. As shown in Fig. 2B, among the three oligos used, only the antisense
U6 oligo was efficient in directing cleavage of the RNAs precipitated
by anti-C antibodies, thus confirming that they are U6 snRNAs. In Fig. 2A, one can see that anti-La, in contrast to
anti-Sm and anti-C antibodies, precipitated many other RNAs of smaller
size than U6. They most likely represent La-bound RNA polymerase III
transcripts(25) , which no doubt contributed to labeling the La
protein in the cross-linking assay (Fig. 1). A comparison of the
U6 snRNAs precipitated by each class of antibodies (Fig. 2A, lanes4-6) shows that
they are not identical in length. It is known that they uniquely differ
by the length of the added oligo(U) tail(13) . Those recovered
by anti-La antibodies have a short oligo(U) tail and are probably newly
synthesized U6 snRNAs(26) . Those recovered by anti-Sm
antibodies are much more heterogeneous, comprising U6 snRNAs having
oligo(U) tails containing up to 12 U residues. Finally, those recovered
by anti-C antibodies appear to correspond to those with the longest
oligo(U) tail among all the anti-Sm-precipitated U6 snRNAs, indicating
that a minimum size (at least 8 residues) is required for the oligo(U)
tail-hnRNP C protein interaction. Moreover, the possibility that the
interaction between the hnRNP C protein and this subset of U6 snRNAs
might be indirect, through some association with another hnRNP
component, is unlikely because antibodies directed against hnRNP A1 and
U proteins fail to precipitate U6 snRNAs from total nuclear extracts (lanes7 and 8).
Figure 1:
Identification of U6 snRNA bound
proteins by UV light-induced RNA-protein cross-linking. Reactions
containing 15 µl of nuclear extract were incubated for 30 min at 30
°C in the presence of 30 µCi of
[-
P]UTP in a final volume of 25 µl
containing 3.2 mM MgCl
, 1 mM ATP, and 20
mM creatine phosphate and then experimented, but not the
control, to irradiation with UV light. Lane1,
control, no irradiation; lane2, irradiation for 10
min with UV light and digested for 60 min at 37 °C with RNase A; lanes4-6, UV light-irradiated and
immunoprecipitated with anti-La, anti-Sm (Y12), and anti-C hnRNP (4F4)
antibodies, respectively, before RNase A digestion; lane3, the same assay using protein A-Sepharose beads without
antibodies. The proteins were separated in a 10% SDS-polyacrylamide gel
and revealed by autoradiography on XAR films. That the cross-linked
product in lane4 moved slightly faster than its
equivalent in lane2 is due to the presence of
comigrating IgG from the serum used as source of
antibody.
Figure 2:
Identification of a subset of U6 snRNAs
immunoprecipitated by the anti-hnRNP C protein antibody. A,
standard reactions containing 3` end-labeled U6 snRNA as described in
Fig. 1 were subjected to immunoprecipitation with anti-La (lane4), anti-Sm (Y12) (lane5), anti-C
(4F4) (lane6), anti-A1 (4B10) (lane7), and anti-U (3G6) (lane8)
antibodies. Lane3, a control immunoprecipitation
assay without antibodies. Total labeled RNAs are shown in lanes1 and 2. Conditions of labeling were either in
the presence (lane2) or in the absence (lane1) of ATP and creatine phosphate. The RNAs were extracted
and separated in a 10% polyacrylamide urea gel. B, reactions
as in A were immunoprecipitated by anti-C antibodies (4F4),
and then the resulting samples were subjected to
oligodeoxynucleotide-directed cleavage by RNase H. The oligonucleotides
were complementary to U4 (lane3), U6 (lane4), and U5 (lane5). Controls were
direct analysis of nonimmunoprecipitated (lane1),
and immunoprecipitated but not cleaved (lane2)
samples.
A striking result seen
in Fig. 1(lane5) is that anti-Sm antibodies
failed to precipitate any protein adduct, suggesting that the U6 snRNAs
interacting with either the La or the hnRNP C protein are free of U4
and U5 snRNAs. Concerning the U6-La interaction, the result agrees with
previously published data, i.e. those U6 snRNAs in U4U6
complexes are not bound to the La protein despite having a La-binding
site at their 3` ends(26) . To verify that U6 snRNAs with long
oligo(U) tails are free of U4 and U5 snRNAs when interacting with the
hnRNP C protein, we subjected a nuclear extract containing 3`
end-labeled U6 snRNAs to glycerol gradient centrifugation under
conditions allowing for separation of free U6 from U4
U6 and
U4
U6
U5 snRNPs (see ``Experimental Procedures'')
and looked for immunoprecipitated snRNAs in the resulting fractions
using anti-Sm and anti-hnRNP C protein antibodies. As expected, the
result (Fig. 3, A and B) was that those labeled
U6 snRNAs present in anti-C precipitates exclusively migrate as free U6
snRNPs, while those in anti-Sm precipitates are associated with U4
(U4
U6 snRNPs) and U4
U5 (U4
U6
U5 snRNPs). The
Northern blot shown in Fig. 3C reveals how the major U2
to U6 snRNAs distribute through the gradient.
Figure 3:
Glycerol gradient fractionation of U
snRNPs. Identical reactions to those described in Fig. 1 (150 µl)
were loaded onto a 10-30% glycerol gradient (see
``Experimental Procedures''). 3` end-labeled U6 snRNAs
present in each fraction were immunoprecipitated by anti-Sm (A) and 4F4 (B) antibodies. The Northern blot in C shows the anti-Sm precipitated U2 to U6 snRNAs through the
gradient. The bands designed U1x are detected by the U1 probe and are,
therefore, degraded forms of U1 snRNA.
From these results, we
conclude that the hnRNP C protein present in nuclear extracts interacts
directly with those of the U6 snRNAs terminating with the longest
oligouridylate tail. Moreover, such an U6 snRNA-hnRNP C protein
interaction does not exist when these U6 snRNAs are engaged with U4 and
U5 snRNAs to form U4U6 and U4
U6
U5 snRNPs.
hnRNP C Protein Induces Disruption in Vitro of
Base-paired U4-U6 snRNAs
The above results raised the question
of whether the association of the hnRNP C protein with free U6 snRNPs
is a cause or a consequence of U4U6 snRNP disruption. We
performed a complementation assay using purified hnRNP C protein and
U4
U6 snRNPs whose U6 snRNAs were elongated and labeled at their
3` end. To do this, a prerequisite was to prepare an extract
sufficiently enriched with U4
U6 snRNPs compared with free U6 and
U4
U5
U6 snRNPs. This was possible starting with a nuclear
extract that has been cleared by DEAE-Sepharose chromatography as
described previously(16) . Indeed, most of its U4 and U6 snRNAs
are in U4
U6 snRNPs(27) . It has TUTase activity and
contains the same elongatable forms of U6 snRNA as an extract active in
splicing. It also contains U6 snRNA with 2`,3`-cyclic phosphate end
(13). Moreover, this extract turned out to be virtually free of hnRNP C
protein on the basis of immunoblots assays (results not shown), thus
allowing the effect of exogeneously added hnRNP C protein to be
monitored. This cleared nuclear extract was first incubated in the
presence of [
-
P]UTP to label U6 snRNAs with
3`-OH end, and then the U4
U6 snRNPs were purified by
centrifugation through a 10-30% glycerol gradient (see
``Experimental Procedures''). These purified particles
contained all of the forms of U4
U6 snRNPs, i.e. those
with 3` end-labeled U6 snRNAs as well as those containing U6 snRNAs
ending with 2`,3` cyclic phosphate. The assay was carried out as
follows. First, the amount of hnRNP C protein added was calculated to
reconstitute approximately the U6 snRNA/hnRNP C ratio that exists in a
splicing extract. Second, the reactions with or without hnRNP C protein
added were subjected to immunoprecipitation by anti-C and anti-Sm
antibodies. Third, immunoprecipitated materials were analyzed for U6
snRNA content by polyacrylamide-urea gels (Fig. 4, A and B). Addition of hnRNP C protein led to precipitation of
labeled U6 snRNAs by anti-C antibodies (Fig. 4A, compare lanes5 and 6), while the precipitation of
these same U6 snRNAs by anti-Sm antibodies was largely decreased (Fig. 4A, compare lanes1 and 2). In contrast, as shown by Northern blots, hnRNP C protein
has no effect on the major form of U4
U6 snRNPs containing U6
snRNAs terminating with a 2`,3`-cyclic phosphate (Fig. 4B). It seems clear, therefore, that binding of
hnRNP C protein only occurs in U4
U6 snRNPs whose U6 snRNAs have
an elongated 3` end and that base pairing of U4 to U6 snRNAs is not an
obstacle for hnRNP C binding. Finally, disruption of base-paired U4 and
U6 snRNAs in these U4
U6 snRNPs provides the better explanation
for the low immunoprecipitation of labeled U6 snRNAs by anti-Sm
antibodies. To verify this, the following experiment was performed. The
above reactions with or without hnRNP C protein were centrifuged
through glycerol gradients to separate free U6 from U4
U6 snRNPs (Fig. 5). Probing for U4 and U6 snRNAs throughout the gradient
shows that these snRNAs still comigrate if hnRNP C was added to
U4
U6 snRNPs before centrifugation (Fig. 5A,
compare upper and lowerpanels). This was
expected since in the immunoprecipitation assay shown in Fig. 4B, all U6 snRNAs detected with the probe remained
precipitable by anti-Sm antibodies. The 3` end-labeled U6 snRNAs, due
to their low abundance, were not detected by the probe. They had a
slower sedimentation when the U4
U6 snRNPs were incubated with the
purified hnRNP C than without (Fig. 5B, compare the twofirstpanels).
Figure 4:
hnRNP C protein induces in vitro disruption of U4U6 snRNPs containing 3` end labeled U6
snRNA. A, U4
U6 snRNPs containing 3` end-labeled U6 snRNA
were isolated by glycerol gradient centrifugation from a cleared
nuclear extract (see ``Experimental Procedures''). They were
incubated for 30 min at 30 °C in the absence (lanes2, 3, 5, and 7) or the
presence (lanes1, 4, and 6) of 10
nM of purified hnRNP C protein. The reactions (200 µl)
were subjected to immunoprecipitations by anti-Sm (lanes1 and 2) and anti-C (lanes5 and 6) antibodies and analyzed in a 10% polyacrylamide-urea gel. Lanes3 and 4, controls treated on protein
A-Sepharose beads without antibodies. Lane7, a
reaction without immunoprecipitation in order to indicate the input
RNAs. B, the same gel as in A was transferred to
hybond N
membrane and then probed with U4 and U6
antisense oligodeoxynucleotide probes. The 3`-end labeled U6 snRNAs
cannot be seen here because they are much less radioactive than the
probes. It is noteworthy that U4 snRNA appeared as a doublet in this
gel, differently from the experiment shown in Fig.
4.
Figure 5:
Analysis
of disrupted U4U6 snRNPs by centrifugation in glycerol gradients.
Identical reactions to those described in Fig. 3 (200 µl) were
loaded onto a 10-30% glycerol gradient (see ``Experimental
Procedures''). After fractionation from the top, the RNAs were
extracted from each fraction and separated in a 10% polyacrylamide-urea
gel. A shows the distribution of U4 and U6 snRNAs detected
with the oligodeoxynucleotide probes. The autoradiographies were
scanned by densitometry (leftpanels). For each
snRNA, the values express a percentage of the sum of absorbancies
throughout the gradient. B shows the distribution of 3`
end-labeled U6 snRNAs from reactions with or without hnRNP C protein (upperpanel). Lowerpanel shows
the distribution of 3` end-labeled U6 snRNAs when the reactions were
carried out in the presence of either recombinant C1 or K94 mutant in
place of purified hnRNP C protein. The concentrations of protein used
are indicated. Densitometry scanning of the autoradiographies was as
above.
As the above
experiment did not rule out the possibility that the dissociating
activity could be contributed by some contaminant of the hnRNP C
protein preparation, the same assay was carried out using a recombinant
hnRNP C1 protein. This protein again dissociated U4U6 snRNPs
containing 3` end-labeled and elongated U6 snRNAs despite being 50-fold
less active than the purified hnRNP C protein (Fig. 5B, thirdpanel). It is possible that post-translational
modifications (i.e. phosphorylations) and/or the presence of
C2 isoform in the preparation from HeLa cells could account for this
difference.
Specificity of the Dissociation Activity of hnRNP C
Protein
It is true to say that U4U6 snRNPs containing
elongated U6 snRNAs are a weak part of the total U4
U6 particles.
Besides, the hnRNP C protein is a very abundant nuclear protein and is
one of the most avid pre-mRNA binding proteins in HeLa cell extracts.
It is known to bind to oligo(U) stretches in natural
RNAs(23, 24, 28, 29) . It was therefore
crucial to discover whether binding of hnRNP C protein to elongated U6
snRNAs followed by U4
U6 snRNP disruption is specific. We first
determined the stoechiometry of the reaction of dissociation. To do
this, different concentrations of purified hnRNP C protein were added
to labeled U4
U6 snRNPs, and the dissociation was monitored by
centrifugation in glycerol gradients (not shown). This led us to
consider that dissociation is effective when four molecules of purified
protein are added to one snRNA. It was assumed that 5% of the U6 snRNAs
have a 3`-OH elongatable end. We have also tested the dissociating
activity of the so-called K94 deletion mutant of hnRNP C protein. It
has the same RNP binding domain as the hnRNP C protein and it binds to
poly(U), but it lacks a large part of the hnRNP C1 protein sequence in
the side of the carboxyl terminus(30) . Used at the same
concentration as the hnRNP C1 protein in our complementation assay, it
failed to induce dissociation of U4
U6 snRNPs (Fig. 5B, lastpanel). However this
mutant protein binds to U4
U6 snRNPs containing 3` end-labeled U6
snRNAs since, upon UV cross-linking of a reaction, it became labeled as
did the hnRNP C protein (results not shown). It appears, therefore,
that something in the sequence other than the canonical RBD of hnRNP C1
is required to induce dissociation. Other experiments using mutated
proteins will be required to more precisely define the domain of the
hnRNP C protein, which is implied in dissociation of U4
U6 snRNPs.
Finally, other recombinant proteins with RNP-CS domains, namely U2AF,
known to bind tenaciously to poly(U) (31) and SF2
ASF were
tested in our complementation assay. As expected, they failed to
disrupt U4
U6 snRNPs (results not shown).
U6 snRNPs. Two points must be
underlined. First, the fact that the hnRNP C protein might bind to an
oligouridylate tail comprising from 8 to 12 residues agrees with
previously published data that the shortest uridine oligoribonucleotide
that binds efficiently to the hnRNP C protein is r(U)
(32).
Second, the disruption of U4
U6 snRNPs seems to be specific of the
hnRNP C protein since a mutated protein containing the RNP-CS domain
alone, as well as other factors with RNP-CS (U2AF, SF2
ASF) failed
to disrupt U4
U6 snRNPs.
U6 snRNP disruption induced by the hnRNP C protein is what
happens during splicing. Obviously, such a scenario implies that U6
snRNA functions in splicing with an elongatable 3`-OH end. Although
most of the U6 snRNAs in mammalian cells have a 2`,3`-cyclic phosphate
end(12) , the presence of U6 snRNAs with 3`-OH end within the
spliceosome is indubitable. Indeed, the five snRNAs involved in
splicing are 3` end-labeled by cytidine 3`,5`-bisphosphate when
extracted from mammalian affinity-selected spliceosomes(34) .
Also, we have previously obtained evidence that elongated U6 snRNAs are
present within the spliceosome and that a 2`,3`-cyclic phosphate at the
end of U6 snRNA is a consequence of splicing instead of being a
requirement for it(13) . Finally, it is known that in organisms
U6 snRNAs consist of forms with different 3` end groups(12) .
For example, all U6 snRNAs have 3`-OH end in T.
brucei, while in man and other mammals the >p, -OH ratio is
9:1.
-elimination (results not
shown). We have discussed already the possibility that elongatable U6
snRNAs might be generated from molecules terminating by a 2`,3`-cyclic
phosphate also present in U4
U6 and U4
U5
U6
complexes(13) . It remains to be understood why most of U6
snRNAs terminate by a 2`,3`-cyclic phosphate end in mammals and several
other species(12) . It could impair U6 snRNA to be bound to the
hnRNP C protein before the interaction is required. Similarly, once the
splicing reaction is accomplished, it could preserve U6 snRNA from
untimely association with the hnRNP C protein, which would lead to the
lack of U4-U6 snRNA interaction, therefore leaving U6 snRNA unavailable
for a new round of splicing.
U6 snRNP and promote U6-U2 base pairing. At this stage, it is
interesting to compare the annealing and the disruption of U4
U6
snRNAs in the yeast system and our proposal of a spliceosomal U6
snRNA-hnRNP C protein interaction occurring in mammals. In yeast, it
has been proposed that PRP24, a U6-specific binding protein with
RNP-CS, directs stabilization of U6 snRNP in a free form and promotes
reannealing of U6 with U4 through the formation of a transient
intermediate PRP24
U4
U6(37) . At first glance, this
is more compatible with hnRNP C protein having an annealing activity
than inducing U4
U6 snRNP disruption. However, it has been
proposed that PRP24 could have another role in the U4
U6 cycle in
concert with PRP28, a protein of the DEAD box
family(38, 39) . Once disruption of base-paired
U4
U6 has occurred, PRP24 could serve to stabilize the unwound
form of U6 snRNA(40) . This seems to be very similar to what we
propose for the function of the U6 snRNA-bound hnRNP C protein. If the
hnRNP C protein bound to U6 snRNA has in mammals the same stabilizing
function as the PRP24 in yeast, what could be the mammalian equivalent
of PRP28? Keeping in mind that ATP is not required to obtain hnRNP C
protein-induced disruption of U4
U6 snRNPs, one can envisage at
least two possibilities. Oligomerization of hnRNP C isoforms could lead
to a complex having both PRP24 and PRP28-like activities. As a matter
of fact, it is known that the hnRNP C protein is part of a tetrameric
structure containing three C1 and one C2 isoforms(41) .
Alternatively, one of the specific U4
U6 snRNP proteins (42) could have PRP28-like activity, becoming functional only
when the hnRNP C protein has bound to the oligouridylate tail.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.