(Received for publication, October 23, 1995)
From the
Six different protein factors are required for the specific
cleavage and polyadenylation of pre-mRNA in mammals. Whereas four of
them have been purified and most of their components cloned, cleavage
factor I (CF I
) and cleavage factor II
(CF II
) remained poorly characterized. We report here
the separation of CF I
from CF II
and the
purification of CF I
to near homogeneity. Three
polypeptides of 68, 59, and 25 kDa copurify with CF I
activity. All three polypeptides can be UV cross-linked to a
cleavage and polyadenylation substrate in the presence of a large
excess of unspecific competitor RNA, but not to a splicing-only
substrate. No additional protein factor is required for the binding of
CF I
to pre-mRNA. Gel retardation experiments confirmed the
results obtained by UV cross-linking. In addition, we could show that
CF I
stabilizes the binding of the cleavage and
polyadenylation specificity factor (CPSF) to pre-mRNA and that CPSF and
CF I
together form a slower migrating complex with pre-mRNA
than the single protein factors. Cleavage stimulation factor (CstF) and
poly(A) polymerase (PAP) had no detectable effect on the binding of CF
I
to pre-mRNA. Furthermore, the CstF
CPSF
RNA as
well as the CstF
CPSF
PAP
RNA complex are supershifted
and stabilized upon the addition of CF I
.
The 3` ends of almost all mRNAs in eukaryotes are generated
posttranscriptionally in two tightly coupled steps. The primary
transcript is first cleaved endonucleolytically at the polyadenylation
site in the 3`-untranslated region. The upstream cleavage fragment is
subsequently polyadenylated, whereas the downstream cleavage fragment
is rapidly degraded. The two reaction steps can be uncoupled
experimentally and assayed separately. Investigation of the cleavage
reaction alone is possible by suppression of the
Mg-dependent polyadenylation of the upstream cleavage
fragment. No polyadenylation occurs, when the reaction is performed
either in the presence of EDTA and ATP or in the presence of MgCl
and cordycepin 5`-triphosphate (3`-dATP) which acts as a chain
terminator. The polyadenylation reaction can be investigated with
``precleaved'' RNA as substrate that ends at or near the
natural cleavage and polyadenylation site.
Both cleavage and polyadenylation are dependent on cis-acting elements in the pre-mRNA and on trans-acting protein factors (for reviews, see (1, 2, 3, 4) ). Two essential cis-acting elements have been described: one of them, the highly conserved polyadenylation signal AAUAAA, lies 10-30 nucleotides upstream of the cleavage and polyadenylation site, and the second one, a less conserved GU- or U-rich sequence element, is located 10-30 nucleotides downstream of the cleavage and polyadenylation site.
Six
different protein factors have been shown to be required for specific
cleavage and polyadenylation of pre-mRNA in
vitro(5, 6) . Three of them, cleavage stimulation
factor (CstF), ()cleavage factor I
(CF
I
) and cleavage factor II
(CF II
;
abbreviations for CF I
and CF II
according
to(3) ) are involved in the cleavage reaction only, while the
cleavage and polyadenylation specificity factor (CPSF) and poly(A)
polymerase (PAP) are necessary for both steps. Poly(A)-binding protein
II (PAB II) acts as a stimulatory factor for poly(A) tail elongation.
CstF, CPSF, PAP, and PAB II have been studied extensively, are well
characterized, and most of their components have been cloned.
CstF consists of three subunits. Its 64-kDa subunit was shown to interact with the GU- or U-rich downstream sequence element(7, 8, 9, 10, 11) . CPSF consists of three or four subunits (12, 13, 14, 15) and binds specifically to the polyadenylation signal AAUAAA(12, 16) . CstF and CPSF, together with a pre-mRNA substrate, form a stable complex (17) and are thought to confer specificity to the 3` end processing reaction. The approximate region in which cleavage will occur is defined by the relative positions of the AAUAAA and the GU- or U-rich elements, and the precise site of cleavage is then determined by a preference for a local nucleotide sequence within this region(18) . CPSF remains bound to the hexanucleotide AAUAAA after cleavage has occurred and enables PAP to elongate specifically the upstream cleavage fragment to a poly(a) tail length of 250 nucleotides in the presence of PAB II (19) .
The endonuclease could not be identified so far. Good
candidates are the two poorly characterized cleavage factors CF I and CF II
, which are required only for the first step
of the reaction.
We report here the separation of CF I from CF II
and the purification of CF I
to near homogeneity. Each of the three polypeptides copurifying
with CF I
activity can be UV cross-linked to a cleavage and
polyadenylation pre-mRNA substrate and gel retardation experiments
showed that CF I
binds to pre-mRNA, even in the absence of
any of the other protein factors involved in 3` end processing of
pre-mRNA.
Plasmids pSV-L,
pSP6L3, and pSP6L31 were linearized with DraI, pSP6L3pre
with RsaI, and pBSAd1 with Sau3A I. Capped, uniformly
P-labeled RNAs were obtained by in vitro transcription of the linearized template DNAs with SP6 RNA
polymerase (Boehringer Mannheim GmbH; pSV-L, pSP6L3, pSP6L3
1,
pSP6L3pre) or T3 RNA polymerase (Stratagene; pBSAd1) in the presence of
m
G(5`)ppp(5`)G and [
-
P]UTP as
described(24, 25) , except that the UTP concentration
was 0.1 mM.
16 and 23 S rRNA and tRNA from Escherichia coli MRE 600 were purchased from Boehringer Mannheim GmbH.
Figure 1:
Separation of cleavage factors and
purification of cleavage factor I. The methods used to
separate CF I
from CF II
and to purify CF
I
are shown schematically. The gradients applied to the
columns and the salt steps in the preparation of the nuclear extracts
and the ammonium sulfate fractionation are indicated in brackets directly below each method, followed by the salt range in which
activity of the cleavage factors could be
detected.
HeLa cell nuclear extracts were prepared from 240 liters of freshly
harvested or frozen HeLa suspension cells as described (27) except that HeLa cells were grown to 4-6
10
cells/ml, and buffers A and C contained Tris
HCl
instead of Hepes
KOH. In addition, buffer C contained KCl instead
of NaCl and 0.5 mM phenylmethylsulfonyl fluoride, 0.4
µg/ml leupeptin hemisulfate, and 0.7 µg/ml pepstatin. High salt
buffer C was buffer C containing additionally 800 mM ammonium
sulfate. After the high salt extraction of the nuclei, the homogenate
was centrifuged for 1 h at 35,000 rpm (135,000
g
) in a Centrikon TFT 65.38 rotor (Kontron
Instruments). The nuclear extracts were stored frozen without dialysis.
312 ml of HeLa cell nuclear extract were allowed to thaw in 250 ml
of buffer without ammonium sulfate and subsequently diluted to the
conductivity of buffer containing 40 mM ammonium sulfate. 1460
ml (2.4 g of protein) of diluted nuclear extracts were spun for 30 min
at 8000 rpm (10,000 g
) in a Sorvall GSA
rotor, and the supernatant was applied to a DEAE-Sepharose fast flow
column (Pharmacia) of 5
12.5 cm equilibrated in 40 mM ammonium sulfate buffer. The column was washed with 1.4 column
volumes of the same buffer and developed with a gradient (5 column
volumes) of 40-300 mM ammonium sulfate at 340 ml/h.
Active fractions eluting between 90 and 150 mM ammonium
sulfate were pooled (300 ml) and dialyzed for 16 h against 2 1
liter of buffer containing 50 mM ammonium sulfate and 20
mM Hepes
KOH, pH 7.9, instead of Tris
HCl. Only 38%
of the DEAE-pool were carried through the following steps of the
purification. Therefore, 113 ml (147 mg of protein) were further
dialyzed for 3 h against 1 liter of 25 mM ammonium sulfate, 20
mM Hepes
KOH buffer, spun for 30 min at 9000 rpm (10,000
g
) in a Sorvall SS34 rotor, and loaded
onto an 8-ml Mono S HR 10/10 FPLC column (Pharmacia) at 1.1 ml/min. The
column was washed with 10 column volumes of the same buffer and eluted
with a gradient (40 column volumes) of 25-380 mM ammonium sulfate at 2 ml/min. CF I
activity eluted
between 90 and 190 mM ammonium sulfate and was pooled. 60 ml
(45 mg of protein) of this CF I
pool were adjusted to 1 M ammonium sulfate by addition of 2 M ammonium
sulfate, pH 7.9, stirred on ice for 85 min and centrifuged for 50 min
at 11,500 rpm (16,000
g
) in a Sorvall
SS34 rotor. The pellets were resuspended in 4.5 ml buffer without
ammonium sulfate and stirred on ice for 2.5 h. The mixture was spun for
30 min at 14,000 rpm (16,000
g
) in an
Eppendorf centrifuge rotor F-45-18-11, and the supernatant was adjusted
to 400 mM ammonium sulfate by addition of 2 M ammonium sulfate, pH 7.9. The solution was stirred on ice for 40
min, dialyzed against 500 ml of 450 mM ammonium sulfate buffer
for 4 h, and spun as above in an Eppendorf centrifuge. The supernatant
(5.5 ml, 11 mg) was applied to a 1-ml phenyl-Superose HR 5/5 FPLC
column (Pharmacia) equilibrated in the same buffer. The column was
washed with 10 column volumes of the 450 mM ammonium sulfate
buffer and developed with a gradient (15 column volumes) of 450 to 0
mM ammonium sulfate at 0.25 ml/min. CF I
fractions
eluting between 340 and 170 mM ammonium sulfate were pooled
(3.4 ml, 500 µg), dialyzed against 3
300 ml of 20 mM ammonium sulfate buffer for 3 h, and loaded onto a 1-ml Mono Q HR
5/5 FPLC column at 0.5 ml/min. The column was washed with 10 column
volumes of the dialysis buffer and developed with a gradient of
20-300 mM ammonium sulfate (40 column volumes) at 0.5
ml/min. CF I
eluted between 100 and 150 mM ammonium sulfate.
To quantitate cleavage reactions, gels were exposed to PhosphorImager screens for 1 h, the screens were scanned with a PhosphorImager 425 (Molecular Dynamics),and the ImageQuant program (version 3.3, Molecular Dynamics); the amount of precursor and upstream cleavage product was determined with the IPLab Gel software (version 1.5, Signal Analytics Corp.). The values obtained were corrected by subtraction of the background signal of the gel in a region where no radioactivity was detectable. The value for the upstream cleavage fragment was further corrected for the different uridine contents of precursor and product. Cleavage activity was calculated by dividing the amount of upstream cleavage product by the sum of upstream cleavage product and precursor and multiplying the ratio by the amount of precursor added to the reaction. 1 unit corresponds to 1 fmol of upstream cleavage product obtained during the incubation time and at the temperature indicated.
Figure 3:
Chromatography of CF I on Mono
Q. A, profile of the final Mono Q column of the preparation
summarized in Table 1. B, SDS-polyacrylamide gel
electrophoresis of Mono Q fractions. Aliquots of 2 µl of the
fractions indicated at the bottom were separated on two 10%
gels and stained with silver. The molecular masses of the size
standards in kilodaltons are indicated on the left. Arrowheads on the right indicate the three
polypeptides copurifying with CF I
activity. L,
load of the Mono Q column. C, cleavage of SV40 late pre-mRNA.
Assays were carried out as described under ``Experimental
Procedures'' for 85 min at 30 °C with 1 µl of the
fractions indicated at the bottom. Samples were analyzed on
two denaturing 6% (w/v) polyacrylamide gels. Sizes (in nucleotides) of
DNA size standards (lane M) are indicated on the left. The migration behavior of the SV40 late pre-mRNA
substrate and the upstream cleavage product are indicated on the right. R, SV40 pre-mRNA incubated without protein
fractions; -, SV40 pre-mRNA incubated in the presence of CstF,
CPSF, PAP, and crude CF II
; L, load of the Mono Q
column. D, gel retardation assay of Mono Q fractions. Aliquots
of 2 µl of the fractions indicated at the bottom were
preincubated with 7.5 fmol of uniformly
P-labeled L3
pre-mRNA in the presence of tRNA as unspecific competitor, and the
reactions were loaded directly on a native polyacrylamide/agarose
composite gel. The migration positions of the free pre-mRNA, and the
protein-RNA complexes are indicated at the left. R, RNA
incubated without protein fractions; L, load of the Mono Q
column. For details, see ``Experimental
Procedures.''
CF I was
purified from HeLa cell nuclear extracts by several fractionation
steps, as shown schematically in Fig. 1. Nuclear extracts from
HeLa cells turned out to be at least 50% more active when the nuclei
were extracted with 200 mM ammonium sulfate instead of 300
mM KCl. Since this effect might be due to a stabilization of
proteins by ammonium sulfate, all columns were eluted with ammonium
sulfate, even though already small concentrations of ammonium sulfate
(<30 mM) inhibit the cleavage reaction significantly (data
not shown).
When diluted nuclear extracts were applied to a
DEAE-Sepharose column, CstF and PAP did not bind to the column, whereas
CF I, CF II
, and CPSF bound and were eluted
with a salt gradient. CF I
and CF II
were
probably partially separated on this column, since a pool of the
fractions showing cleavage activity was more active than the single
fractions (data not shown). CF I
and CF II
were
separated in the next purification step, a Mono S column, as described
previously(5) . The profile of the Mono S column is shown in Fig. 2. All column fractions were assayed for CF I
and CF II
activity in the presence of CstF, CPSF,
PAP, and crude CF II
or crude CF I
,
respectively. Crude CF I
and CF II
used for
complementation in these assays were obtained from a 1-ml pilot Mono S
column and had been identified before by testing all possible fraction
combinations. The elution positions of CF I
and CF II
from the Mono S column described here are indicated by horizontal bars above the column profile (Fig. 2).
Figure 2:
Separation of cleavage factors on Mono S.
Profile of the Mono S column of the preparation summarized in Table 1. The elution positions of CF I and CF
II
are indicated qualitatively by horizontal bars.
For details, see ``Experimental
Procedures.''
By means of ammonium sulfate precipitation and two additional
chromatographic steps (see Fig. 1and ``Experimental
Procedures''), CF I was purified to near homogeneity.
The purification is summarized in Table 1. The quantitation of
cleavage reactions proved to be difficult for the first steps of the
purification. For obvious reasons, the calculation of activities gives
reliable numbers only after the separation of CF I
and CF
II
. The analysis of the separation of CF I
and
CF II
on Mono S was complicated for two reasons: first, a
nuclease interfered with the determination of CF I
activity
and second, maximal cleavage activity depended on the optimal ratio of
CF I
and CF II
at this stage of purification.
Only after further purification of CF I
did cleavage
activity depend linearly on the amounts of CF I
and CF
II
(data not shown). Therefore, the elution positions of CF
I
and CF II
are indicated only qualitatively in Fig. 2.
The profile of the final Mono Q column is shown in Fig. 3A. Three polypeptides with apparent molecular
masses of 68, 59, and 25 kDa copurified with CF I activity (Fig. 3, B and C). The fractions were also
tested for RNA binding by gel retardation (Fig. 3D, see
below). No RNA component could be detected in CF I
fractions of the Mono Q column after proteinase K treatment and
3` end labeling with [
-
P]cordycepin
5`-triphosphate and PAP (30) (data not shown). On this column,
the 68-kDa polypeptide eluted slightly later than the 59- and 25-kDa
polypeptides, and the staining was less intense. Independent
purifications over the same or different columns showed either the same
effect or resulted in exactly comigrating polypeptides with an
approximate equimolar ratio. Two-dimensional gel electrophoresis on a
nondenaturing polyacrylamide gel (31) in the first dimension
and a denaturing SDS-polyacrylamide gel in the second dimension showed
that the three polypeptides comigrated on the native gel (results not
shown). These and other data suggest that all three polypeptides are
part of CF I
(see below).
Figure 4:
All three polypeptides of CF I can be UV cross-linked to pre-mRNA substrates. 3` end processing
factors were incubated either separately or in different combinations
with 15 fmol of uniformly
P-labeled pre-mRNA as indicated
at the top and the bottom of the figures. After
preincubation for 15 min at 30 °C, UV cross-linking reactions were
carried out at room temperature in the presence of 2.5 µg of tRNA
as unspecific competitor. Samples were analyzed on a 10%
SDS-polyacrylamide gel. The molecular masses of
C-labeled
size standards in kilodaltons are indicated at the left. The
migration positions of the three polypeptides of CF I
, as
assessed by silver staining of the same gel, are indicated by arrowheads on both sides. RNA,
P-labeled
pre-mRNA irradiated in the absence of any protein factor. For details,
see ``Experimental Procedures'' and text. A,
autoradiograph of the silver-stained and dried gel. B, silver
staining of the same gel as in A.
UV Cross-linking of the 64-kDa subunit of CstF and of
the 30- and 160-kDa subunits of CPSF to RNA has been described
previously(7, 14, 16, 32, 33, 34) .
Under the conditions used here, only the cross-link of the 64-kDa
subunit of CstF to the cleavage and polyadenylation substrate L3 was
detectable and only in the presence of CPSF and PAP, which were shown
previously to stabilize the binding of CstF to RNA(13, 35) (compare lanes 2 with 3 and 6 with 7, respectively). The cross-linking efficiency of
the 64-kDa subunit of CstF was slightly reduced in the presence of heat
denatured rRNA, which was added to the reaction simultaneously with L3
pre-mRNA and tRNA as a less structured unspecific competitor (compare lanes 3 and 7). Weak cross-links of the 64-kDa
subunit of CstF could also be detected to the L31 substrate that
carries a point mutation in the AAUAAA polyadenylation signal and is
thus no longer able to bind CPSF (lane 11), and to the L3pre
RNA that ends one nucleotide upstream of the natural cleavage and
polyadenylation site of the L3 pre-mRNA and lacks the natural binding
site for CstF (L3pre; lane 15). The unspecific cross-linking
of CstF to RNA can probably be explained by the observation that CstF
alone has no strict sequence requirements for binding to RNA and that
CstF/RNA complexes are stabilized by CPSF and PAP, respectively. No
cross-link could be detected to the splicing substrate Ad1, which does
not contain a polyadenylation signal (lane 19).
The same
set of RNA substrates was used for UV cross-linking reactions with CF
I. The peak fraction of the Mono Q column shown in Fig. 3(fraction 37) was irradiated with UV light either alone or
in the presence of CstF, CPSF, and PAP. Surprisingly, all three
polypeptides of CF I
were cross-linked to the L3 pre-mRNA
in the absence of any other 3` end processing factor (lane 4).
The cross-links were assigned to the three polypeptides by
superimposing the autoradiograph and the silver-stained gel; the
migration positions of the 68-, 59-, and 25-kDa polypeptides of CF
I
on the silver-stained gel are indicated by arrowheads in Fig. 4. The 68- and 59-kDa polypeptides comigrated
exactly with signals detected on the autoradiograph, whereas the 25-kDa
polypeptide detected by silver staining migrated slightly faster than
the signal detected by autoradiography. This is probably due to
retardation of the cross-linked portion of the polypeptide by
covalently bound residual RNA nucleotides. The difference in the
migration behavior is detectable for small polypeptides like the 25-kDa
polypeptide but not for larger ones such as the 59- and 68-kDa
polypeptides of CF I
.
The signal of the cross-link of
the 68-kDa polypeptide is weaker than the signal of the 59-kDa
polypeptide. The cross-linking efficiency of proteins to RNA largely
depends on the amino acid composition of the RNA binding site and,
since the RNA substrate was labeled with
[-
P]UTP, also on the uridine content of the
protein binding site on the RNA. The cross-links of CF I
to
L3 pre-mRNA were neither reduced by the addition of rRNA as unspecific
competitor (lane 8) nor were they affected by a point mutation
in the AAUAAA polyadenylation signal (lane 12); however, they
were not detectable with the precleaved substrate L3pre (lane
16) nor with the splicing substrate Ad1 (lane 20). The
cross-linking efficiency was not enhanced by the addition of CstF,
CPSF, and PAP (compare lanes 4 with 5, 8 with 9, and 12 with 13).
In order to compare the
ability of all known 3` end processing factors to bind RNA in gel
retardation experiments, equal amounts (650 fmol) of CF I,
CstF, CPSF, and PAP were incubated separately with 7.5 fmol of
P-labeled L3 pre-mRNA and run on a native gel (Fig. 5). Even with this high excess of protein, only CPSF
formed a distinct complex with the pre-mRNA substrate (lane
4), whereas CF I
RNA and CstF
RNA complexes
formed short smears running above the free RNA (lanes 2 and 3). PAP did not shift the L3 pre-mRNA at all (lane
5). Normally, much smaller concentrations of CstF (30 fmol in 12.5
µl), CPSF (25 fmol in 12.5 µl), and PAP (120 fmol in 12.5
µl) were used in cleavage assays. To analyze the interactions of CF
I
, CstF, CPSF, and PAP with each other in the presence of
L3 pre-mRNA, gel retardation reactions were set up with the protein
concentrations used in cleavage assays and 650 fmol of CF I
(2 µl of the Mono Q fraction 34, Fig. 3). As expected,
no complex of CstF or PAP with L3 pre-mRNA could be detected (lanes
6 and 10) and the signal of the CPSF
RNA complex was
strongly reduced and less distinct than with larger amounts of protein (lane 8). Addition of CstF or PAP to CF I
did not
change the CF I
RNA complex (lanes 7 and 11), but CF I
, CPSF, and L3 pre-mRNA formed a
complex that migrated more slowly than the CPSF
RNA complex (lane 9). Furthermore, CPSF formed a slower migrating complex
in the presence of CstF (lane 12, complex A) which was
supershifted and stabilized upon the addition of PAP (lane 14,
complex C). The CstF
CPSF
RNA complex as well as the
CstF
CPSF
PAP
RNA complex were supershifted upon the
addition of CF I
, and the signal was significantly enhanced (lanes 13 and 15, complexes B and D, respectively). The CPSF
CF I
RNA
complex was already detectable in the presence of 150 fmol of CF
I
(0.5 µl of the Mono Q fraction 34, Fig. 3),
which corresponds to the amount used in the cleavage assay (Fig. 3C). 50 fmol of CF I
(0.15 µl of
the Mono Q fraction 34, Fig. 3) were sufficient to induce a
supershift of the CstF
CPSF
RNA and CstF
CPSF
PAP
RNA complexes. The signals increased in all three cases with
increasing amounts of CF I
(result not shown).
Figure 5:
Gel retardation assay of 3` end
processing factors with L3 pre-mRNA. Protein fractions indicated were
preincubated with 7.5 fmol of uniformly P-labeled L3
pre-mRNA in the presence of 1.25 µg of tRNA as unspecific
competitor and the reactions loaded directly on a native
polyacrylamide/agarose composite gel. The migration positions of the
free pre-mRNA and the protein-RNA complexes of lanes 12-15 are indicated at the left and the right,
respectively. For details, see ``Experimental Procedures''. Lane 1, RNA incubated without protein fractions; lane
2, 650 fmol of CF I
(2 µl of Mono Q fraction 34, Fig. 3); lane 3, 650 fmol of CstF; lane 4, 650
fmol of CPSF; lane 5, 650 fmol of PAP; lanes 6-15, 650
fmol of CF I
(2 µl of Mono Q fraction 34, Fig. 3), 30 fmol of CstF, 25 fmol of CPSF, 120 fmol of PAP in
the combinations indicated at the top. A,
CstF
CPSF
RNA complex in lane 12; B,
CstF
CPSF
CF I
RNA complex in lane
13; C, CstF
CPSF
PAP
RNA complex in lane 14; D, CstF
CPSF
PAP
CF
I
RNA complex in lane
15.
We report here the purification of CF I from HeLa
cell nuclear extracts. Three polypeptides of 68, 59, and 25 kDa
copurified with CF I
activity. Several lines of
experimental evidence support the notion that these polypeptides are
true subunits of CF I
. 1) All three polypeptides could be
UV cross-linked to the cleavage and polyadenylation substrate L3. 2)
The polypeptides formed a complex that was stable upon native gel
electrophoresis. 3) The three polypeptides copurified during different
purification procedures, although they were partially separated on
certain column matrixes. We, therefore, believe that all three
polypeptides are part of CF I
, but we cannot rule out at
this stage that the 59-kDa and/or 25-kDa polypeptide are degradation
products of the 68-kDa polypeptide that are still able to bind to RNA
and are at least partially active. Partial proteolysis of the 68-kDa
polypeptide would be one possible explanation for the observation that
the largest subunit was slightly less abundant than the other two
polypeptides in some CF I
preparations. Attempts to
reconstitute CF I
from the single polypeptides obtained by
elution from a SDS-polyacrylamide gel were unsuccessful. Thus, the true
composition of CF I
remains uncertain until cDNAs coding
for the polypeptides will be cloned.
The question whether CF I can bind to RNA either alone or in the presence of other 3` end
processing factors was addressed by two different methods, UV
cross-linking and gel retardation assays. Whereas by UV cross-linking
even weak interactions of proteins and RNA can be detected, and the RNA
binding polypeptide(s) can be identified on a SDS-polyacrylamide gel,
gel retardation assays can reveal protein-RNA interactions in solution.
Both methods allowed the detection of a CF I
RNA
complex in the absence of any other 3` end processing factor. UV
cross-links of CF I
could only be detected to the cleavage
and polyadenylation substrate L3, but not to the splicing substrate
Ad1. The preference of CF I
for L3 was in principle
confirmed by gel retardation assays, but the CF I
L3
shift could be competed by increasing amounts of Ad1, although less
efficiently than by L3 itself (data not shown). No cross-links of CF
I
to L3pre mRNA, which ends one nucleotide upstream of the
natural cleavage and polyadenylation site, could be detected. It is not
possible to decide from the experiments shown here where precisely CF
I
binds on L3 pre-mRNA. Further experiments are needed to
map the region on the pre-mRNA substrate that is bound by CF
I
.
Gel retardation assays with purified CstF, CPSF, PAP,
and crude cleavage factors, either alone or in different combinations,
and L3 pre-mRNA have been described
before(13, 16, 17, 19) . It was
shown that formation of the CPSFRNA complex depends on AAUAAA,
that the CPSF
RNA complex is stabilized by PAP, and that addition
of CstF causes the formation of a slower migrating complex. Under the
conditions used, neither a CstF
RNA nor a PAP
RNA complex
could be detected. These results are in good agreement with those
presented here, which were obtained with more highly purified
components. We further demonstrated the binding of CF I
to
L3 pre-mRNA in the absence of any other protein factor. Neither CstF
nor PAP had an influence on the binding of CF I
to L3
pre-mRNA, but the addition of CPSF led to the formation of a more
slowly migrating complex. Furthermore, CF I
stabilized the
CstF
CPSF
RNA as well as the
CstF
CPSF
PAP
RNA complex and resulted in slower
migrating complexes. In contrast, Gilmartin and Nevins (17) have reported previously that the addition of crude
cleavage factors abolished the formation of the CPSF
RNA complex
and destabilized the CstF
CPSF
RNA complex. The results
presented here were obtained in the absence of CF II
,
because this factor is presently only available as a relatively impure
fraction. The destabilization of the CPSF
RNA and the
CstF
CPSF
RNA complex by the cleavage factors (17) may thus be either an unspecific effect, since less
purified cleavage factors were used in these experiments, or it may
have been caused by CF II
.
The results obtained from the
UV cross-linking experiments and gel retardation assays are consistent
with the suggestion that CF I binds to pre-mRNA with a
certain degree of specificity and interacts with CPSF, but not with
CstF and PAP upon binding to RNA.
It remains to be shown which of
the protein factors essential for the cleavage reaction acts as the
actual endonuclease. This question can only be resolved after the other
cleavage factor, CF II, has been characterized. The
purification of all factors involved in cleavage and polyadenylation of
primary mRNA transcripts should allow it to study in more detail the
assembly of the components involved in 3` RNA processing, to
investigate the sequence requirements further, and to identify
regulatory factors that participate in the 3` end formation of pre-mRNA
in a well defined, fully reconstituted system.