A multisubunit 3' end processing factor from yeast containing poly(A) polymerase and homologues of the subunits of mammalian cleavage and polyadenylation specificity factor.
ABSTRACT Polyadenylation is the second step in 3' end formation of most eukaryotic mRNAs. In Saccharomyces cerevisiae, this step requires three trans-acting factors: poly(A) polymerase (Pap1p), cleavage factor I (CF I) and polyadenylation factor I (PF I). Here, we describe the purification and subunit composition of a multiprotein complex containing Pap1p and PF I activities. PF I-Pap1p was purified to homogeneity by complementation of extracts mutant in the Fip1p subunit of PF I. In addition to Fip1p and Pap1p, the factor comprises homologues of all four subunits of mammalian cleavage and polyadenylation specificity factor (CPSF), as well as Ptalp, which previously has been implicated in pre-tRNA processing, and several as yet uncharacterized proteins. As expected for a PF I subunit, pta1-1 mutant extracts are deficient for polyadenylation in vitro. PF I also appears to be functionally related to CPSF, as it polyadenylates a substrate RNA more efficiently than Pap1p alone. Possibly, the observed interaction of the complex with RNA tethers Pap1p to its substrate.
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ABSTRACT: 3′-Ends of almost all eukaryotic mRNAs are generated by endonucleolytic cleavage and addition of a poly(A) tail. In mammalian cells, the reaction depends on the sequence AAUAAA upstream of the cleavage site, a degenerate GU-rich sequence element downstream of the cleavage site and stimulatory sequences upstream of AAUAAA. Six factors have been identified that carry out the two reactions. With a single exception, they have been purified to homogeneity and cDNAs for 11 subunits have been cloned. Some of the cooperative RNA-protein and protein-protein interactions within the processing complex have been analyzed, but many details, including the identity of the endonuclease, remain unknown. Several examples of regulated polyadenylation are being analyzed at the molecular level. In the yeast Saccharomyces cerevisiae, sequences directing cleavage and polyadenylation are more degenerate than in metazoans, and a downstream element has not been identified. The list of processing factors may be complete now with approximately a dozen polypeptides, but their functions in the reaction are largely unknown. 3′-Processing is known to be coupled to transcription. This connection is thought to involve interactions of processing factors with the mRNA cap as well as with RNA polymerase II.FEMS microbiology reviews 01/1999; 23(3):277-295. · 10.96 Impact Factor
The EMBO Journal Vol.16 No.15 pp.4727–4737, 1997
A multisubunit 3? end processing factor from yeast
containing poly(A) polymerase and homologues of
the subunits of mammalian cleavage and
polyadenylation specificity factor
et al., 1989; Ru ¨egsegger et al., 1996). After cleavage, the
poly(A) tail is added by PAP in a processive event that
requires CPSF and a poly(A)-binding protein (PAB II)
(Wahle, 1991; Bienroth et al., 1993). The genes encoding
PAP, PAB II and the subunits of CPSF and CstF have
been cloned and sequenced (reviewed in Wahle and Keller,
1996; see also Jenny et al., 1996; Barabino et al., 1997).
The observation that RNA sequences directing 3? end
formation are far more redundant and degenerate in yeast
than they are in mammals led to the view that the factors
that specify the mRNA 3? end are distinct. However, more
recent findings confirm a fundamental structural and
functional conservation between the 3? end processing
machineries of these distantly related eukaryotes. Exhaust-
ive analyses of the sequences required for accurate 3? end
formation of the iso-1-cytochrome c (CYC1) mRNA has
led to the identification of two essential elements, both
located upstream of the cleavage site. These are a ‘posi-
tioning element’ close to the cleavage site and a distal
‘efficiency element’ (Guo and Sherman, 1996, and refer-
ences therein). The sequence AAUAAA can function as
the positioning element, but other sequences work as well.
Fractionation of trans-acting 3? end processing factors
was pioneered by Chen and Moore (1992). These authors
separated yeast whole-cell extracts into four chromato-
graphic fractions: poly(A) polymerase (Pap1p), which is
encoded by PAP1 (Lingner et al., 1991a,b), CF I and
CF II, and polyadenylation factor I (PF I). In reconstituted
in vitro systems, cleavage requires CF I and CF II, whereas
polyadenylation occurs upon combination of CF I, Pap1p
and PF I. The first genes besides PAP1 that were demon-
strated to encode subunits of a yeast 3? end processing
factor were RNA14 and RNA15 (L.Minvielle-Sebastia
et al., 1994). Rna14p and Rna15p are components of CF I,
and mutations in either gene abolish both cleavage and
polyadenylation activity (Minvielle-Sebastia et al., 1994).
Further purification revealed that CF I can be separated
into two activities (CF IA and CF IB) that are both
required for cleavage and polyadenylation (Kessler et al.,
1996). We independently have purified CF IA and found
that five polypeptides co-fractionate with the activity. They
include Rna14p, Rna15p, Pcf11p (Amrani et al., 1997), a
new protein called Clp1p (L.Minvielle-Sebastia et al.,
unpublished data) and remarkably, the major poly(A)-
binding protein Pab1p (Minvielle-Sebastia et al., 1997).
PCF11 initially was found in a two-hybrid screen designed
to identify proteins interacting with Rna14p and Rna15p
(Amrani et al., 1997). RNA14, RNA15 and PCF11 also
interact genetically, as combinations of temperature-
sensitive mutations in either of them are synergistically
lethal (Minvielle-Sebastia et al., 1994; Amrani et al.,
1997). The Rna15p subunit of purified CF IA contacts the
pre-mRNA, albeit with unknown specificity. The protein
has an RNA-binding domain with similarity to that of the
Pascal J.Preker1, Martin Ohnacker,
Lionel Minvielle-Sebastia and Walter Keller2
Department of Cell Biology, Biozentrum, University of Basel,
Klingelbergstrasse 70, CH-4056 Basel, Switzerland
1Present address: Department of Biochemistry and Biophysics,
UCSF School of Medicine, San Francisco, CA 94143-0448, USA
Polyadenylation is the second step in 3? end formation
of most eukaryotic mRNAs. In Saccharomyces cere-
visiae, this step requires three trans-acting factors:
poly(A) polymerase (Pap1p), cleavage factor I (CF I)
and polyadenylation factor I (PF I). Here, we describe
the purification and subunit composition of a multi-
protein complex containing Pap1p and PF I activities.
PF I–Pap1p was purified to homogeneity by comple-
mentation of extracts mutant in the Fip1p subunit of
PF I. In addition to Fip1p and Pap1p, the factor
comprises homologues of all four subunits of mamma-
lian cleavage and polyadenylation specificity factor
(CPSF), as well as Pta1p, which previously has been
implicated in pre-tRNA processing, and several as yet
uncharacterized proteins. As expected for a PF I
subunit, pta1-1 mutant extracts are deficient for poly-
adenylationinvitro. PFIalsoappears tobefunctionally
related to CPSF, as it polyadenylates a substrate RNA
more efficiently than Pap1p alone. Possibly, the
observed interaction of the complex with RNA tethers
Pap1p to its substrate.
Keywords: polyadenylation factor I/poly(A) polymerase
holoenzyme/pre-messenger RNA 3? end processing/yeast
The formation of the 3? ends of eukaryotic mRNAs occurs
in two tightly coupled steps and requires a complex set
of cis- and trans-acting factors. In mammals, the cleavage
and polyadenylation specificity factor (CPSF) is a key
component in this process, as it is required for both steps,
endonucleolytic cleavage of the primary transcript and
polyadenylation of the upstream cleavage product by the
enzyme poly(A) polymerase (PAP). CPSF is a multimeric
protein complex (Bienroth et al., 1991) that binds the
conserved hexanucleotide sequence AAUAAA upstream
of the cleavage site, by virtue of its largest subunit (Keller
et al., 1991; Murthy and Manley, 1995). CPSF binding
occurs cooperatively with the cleavage stimulation factor
(CstF), which recognizes a less well-defined U- or GU-
rich sequence downstream of the cleavage site through its
64 kDa subunit (Takagaki et al., 1992; MacDonald et al.,
1994). In addition, cleavage requires the presence of PAP
and two cleavage factors, CF Imand CF IIm(Takagaki
© Oxford University Press
P.J.Preker et al.
64 kDa subunit of mammalian CstF. Moreover, Rna14p
is significantly related to the 77 kDa subunit of CstF
(Takagaki and Manley, 1994).
A screen for proteins that interact with Pap1p in vivo
led to the identification of Fip1p as an essential component
of PF I (Preker et al., 1995). Two additional subunits of
PF I were identified, initially based on their homology to
subunits of mammalian CPSF (Chanfreau et al., 1996;
Jenny et al., 1996; Barabino et al., 1997). Ysh1p (yeast
73 kDa homologue) and Yth1p (yeast 30 kDa homologue)
are 53 and 40% identical to the 73 and 30 kDa subunits
of bovine CPSF, respectively (hence their names). Surpris-
ingly, Cft1p, a putative homologue of the largest subunit
of CPSF, has been reported to be a subunit of CF II
(Stumpf and Domdey, 1996). Thus, homologues of differ-
ent subunits of CPSF, which is required for both cleavage
and polyadenylation, appear to be associated with two
separate yeast factors that are required for either of the
two activities only.
Here we report the purification of a multimeric complex
required for pre-mRNA polyadenylation in yeast and the
identification of seven polypeptides associated with its
activity. These include the putative homologues of all four
subunits of CPSF, as well as Fip1p, Pap1p and Pta1p, a
protein that previously has been implicated in pre-tRNA
maturation (O’Connor and Peebles, 1992). All subunits
characterized to date are essential, underscoring the
important role of this factor in gene expression.
Fig. 1. Purification of yeast polyadenylation factor I. (A) Schematic
representation of strain PJP14 made for PF I purification. The
chromosomal FIP1 gene is replaced by a double-tagged allele
provided on a centromeric plasmid. The sequence of the HA1 epitope
is printed in bold. Centromere sequences are represented by open
circles. (B) Purification of PF I by conventional and metal chelate
affinity chromatography. Protein concentrations were determined with
a colorimetric assay (Bio-Rad) with bovine serum albumin (BSA) as a
standard. One unit of PF I is defined as the amount of protein that
polyadenylates 1 fmol of the 5? product generated by cleavage of the
CYC1 pre-mRNA in a fip1-1 mutant extract per minute. Assays
contained 20 fmol of32P-labelled substrate, and the amount of
polyadenylated end product was calculated with IPLab Gel software
(version 1.5, Signal Analytics Corporation) after scanning of gels on a
PhosphorImager 425 (Molecular Dynamics). All the values obtained
were corrected by subtracting the background value determined from
an empty lane. Because the crude extract and the ammonium sulfate-
fractionated extract are competent for polyadenylation by themselves,
they are not amenable to quantification. (C) Immunopurification of
PF I. A sample from the Mono S pool containing partially purified
PF I was pre-adsorbed to protein A–Sepharose and subsequently
subjected to immunoprecipitation with a control antibody (rabbit
anti-mouse immunoglobins; lane 5), the monoclonal antibody 12CA5,
specific for the HA1-peptide (lane 7), or a polyclonal antibody to
Fip1p (lane 9). Bound proteins were eluted from the pre-adsorbtion
resin (lane labelled no ab) and the antibody resins by boiling in
sample buffer. The eluate was analysed by electrophoresis on an
SDS–polyacrylamide (9%) gel and silver staining. A sample of each
antibody was loaded in adjacent lanes. Lane 1, 20 µl of the input
fraction; lane 2, 20 µl of the supernatant (sn) after precipitation with
affinity-purified antibodies to Fip1p. Major polypeptides in the eluates
are labelled according to their apparent molecular mass. The
polyclonal antibodies contain exogenous BSA. The positions of the
light and heavy chains of the immunoglobins and of molecular mass
standards (M) are indicated; sizes are in kilodaltons.
Purification of the multimeric PF I complex
To facilitate purification of PF I, a strain (PJP14) was
constructed in which a disruption of the chromosomal
FIP1 gene was rescued by expression of a tagged form
of Fip1p provided on a plasmid. The tag was fused in-
frame to the amino-terminus of Fip1p and consisted of
the influenza haemagglutinin HA1 epitope followed by
six consecutive histidine residues (Figure 1A). The tag
did not adversely affect cell growth or polyadenylation
activity, and, thus, PF I function, in cell-free extracts.
Ammonium sulfate-fractionated extract obtained from
500 g of PJP14 cells was first subjected to chromatography
on a Macro-Prep Q column. CF I- and PF I-containing
fractions were identified by their ability to complement
the 3? end processing defect of rna14-1 and fip1-1 mutant
extracts, respectively (Minvielle-Sebastia et al., 1994;
Preker et al., 1995). The two activities eluted separately
at ~150 and 270 mM salt, respectively. PF I activity was
purified further by chromatography on Blue–Sepharose
and heparin–Sepharose columns. Western blot analysis of
the complementing fractions confirmed that Fip1p always
co-purified with PF I activity. The pool of activity from
the heparin–Sepharose column was loaded on a Ni2?-
nitrilotriacetic acid (NTA)–agarose column for affinity
purification of the His6-tagged protein complex. This step
resulted in a 5-fold purification only (Figure 1B), possibly
because the six histidine residues, located between the
HA1 tag and the Fip1p sequence, are not easily accessible.
PF I activity was purified further by chromatography on
a Mono S column. From the Macro-Prep Q column to
this step, recovery of activity was ~70% and PF I was
purified ~90-fold (Figure 1B).
Fip1p and its associated proteins were immunoprecipit-
ated from a PF I fraction of the Mono S column. Anti-
HA1 or anti-Fip1p antibodies were immobilized on protein
Yeast poly(A) polymerase holoenzyme
A–resin and mixed with a sample of the PF I fraction.
After extensive washing, bound proteins were eluted and
separated by SDS–PAGE (Figure 1C, lanes 7 and 9). The
anti-HA1 and anti-Fip1p antibodies immunoprecipitated a
virtually identical set of proteins. None of these proteins
was retained in control experiments with an unrelated
antibody (lane 5) nor with antibodies to Fip1p that had
been pre-adsorbed to the antigen prior to immunoprecipit-
in the eluate precluded detection of any proteins with Mrs
of ~50 and 30 kDa on the silver-stained gel. Pre-treatment
of the input fraction with RNase A did not affect the
composition of proteins in the immunoprecipitate, sug-
gesting that the integrity of the complex does not require
a similar set of proteins was also found in a distinct
purification scheme that did not involve immunoaffinity
purification (data not shown). The supernatant and the
eluate of the anti-HA1 antibody precipitation were probed
on blots with the anti-Fip1p antibody and vice versa. In
that the antibodies had adsorbed the protein completely
(data not shown).
To recover PF I in its native form, Mono S fractions
were loaded on an anti-HA1 antibody column, and PF I
was eluted with competing HA1 peptide (Field et al., 1988;
Keys et al., 1994). No signal was seen if immunoblots
of the eluate were probed with antibodies to mouse
immunoglobins, indicating that the anti-HA1 antibody had
not desorbed from the column in detectable amounts (data
not shown). The immunopurified fractions were analysed
for their protein content by gel electrophoresis on an
SDS–polyacrylamide gel and silver staining (Figure 2B,
lane 2). The five largest proteins with Mrs of 150, 105,
100, 85 and 64 kDa were similar to proteins observed in
the previous immunoprecipitation experiments (Figure
1C). Other major polypeptides had apparent Mrs of 58, 55,
53, 36 and 35 kDa. To confirm that the immunoprecipitated
proteins co-migrate with PF I activity, a fraction of the
eluate was subjected to chromatography on a Mini Q
column. The composition of the complex did not change
due to chromatography after immunopurification, indicat-
ing that the factor has been purified to near homogeneity.
All polypeptides were recovered at apparently equimolar
ratios with the exception of the 55 (see below) and the
58 kDa subunits. Fractions were assayed for their ability
to restore polyadenylation of fip1-1 mutant extract (Figure
2C). Polyadenylation activity co-eluted with the protein
peak in fractions 24–26 (Figure 2B, lanes 5 and 6 and C,
lanes 9 and 10). In addition, polyadenylation of the CYC1
pre-cleaved precursor could be obtained upon combination
of partially purified CF I (Chen and Moore, 1992) and
purified PF I from the same Mini Q column fractions
(Figure 2D, lanes 4 and 7–16). No exogenous poly(A)
polymerase was required. The presence of the poly(A)
polymerase in PF I fractions explained this surprising
result (see below).
Although the immunopurification step only recovered
a fraction of the PF I activity in the eluate, this step may
afford another 20- to 50-fold purification as judged by
polyacrylamide gel electrophoresis (Figure 1C).
Fip1p, Pap1p, Pta1p and homologues of the four
subunits of CPSF are associated with PF I activity
Protein fractions of the final Mini Q column were tested
for cross-reactivity with several antibodies. Antibodies to
Fip1p detecteda protein thatcorresponded toa polypeptide
of 55 kDa. The apparent absence of stoichiometry of
Fip1p is reproducible and may be due to the inherently
poor staining of Fip1p with silver or partial degradation
of the protein.
Affinity-purified antibodies to Pap1p detected a protein
of 64 kDain active fractions (Figure 2A). Thisis surprising
because previous studies have indicated that Pap1p and
PF I elute from a Mono Q column as separate factors
(Chen and Moore, 1992). That the 64 kDa protein detected
by Western blot analysis is Pap1p was confirmed by
several experiments. First, immunoprecipitation with the
anti-Pap1p antibodies precipitated the same set of proteins
as the anti-Fip1p antibodies (data not shown). Second,
(Lingner et al., 1991a) had an electrophoretic mobility
indistinguishable from that of the 64 kDa subunit of PF I
(data not shown). Third, in the absence of other factors,
the PF I fractions unspecifically polyadenylated the CYC1
full-length and pre-cleaved pre-mRNA substrates (Figure
2C and D, lanes 5, respectively) or a poly(A) primer (data
not shown). As observed with the authentic enzyme,
poly(A) is elongated much more efficiently in the presence
of manganese ions than in the presence of magnesium
ions (data not shown). Finally, the notion that Pap1p is a
component of PF I is consistent with the observation
that specific polyadenylation of a pre-cleaved pre-mRNA
substrate can be reconstituted from purified PF I–Pap1p
and partially purified CF I from a Mono Q column (Chen
and Moore, 1992) in the absence of exogenous Pap1p
(see Figure 2D). Remarkably, small amounts of the 64 kDa
subunit began to elute from fraction 10 of the Mini Q
column, ahead of the PF I peak (Figure 2B, lane 3; and
data not shown). However, the Pap1p peak coincided with
that of the other PF I subunits and thus with PF I activity.
A yeast homologue of the 73 kDa subunit of the
mammalian CPSF recently has been cloned and shown to
be a subunit of PF I (Jenny et al., 1996). Ysh1p has a
coding capacity for a 87 kDa protein (note that the same
gene was cloned independently as BRR5 by Chanfreau
a polypeptide of 100 kDa in fractions containing purified
PF I (Figure 2A).
In the same way that we characterized Ysh1p, the
cloning of the 30 kDa subunit of CPSF allowed us to
identify a yeast protein of 26 kDa which showed 40%
identity with the mammalian protein. It was therefore
called Yth1p (Barabino et al., 1977). Extracts prepared
from a yth1 mutant strain show normal cleavage but
are deficient in polyadenylation (Barabino et al., 1997).
Although proteins smaller than 28 kDa are not shown
on the protein gel in Figure 2B, no additional major
polypeptides were observed on gels that resolved proteins
as small as ~15 kDa (data not shown). However, antibodies
against Yth1p clearly recognized a band of the expected
size (26 kDa) in fractions containing PF I activity (Figure
3B, lanes 2 and 3; Barabino et al., 1997). As observed
with Fip1p and the 58 kDa protein, it is possible that
Yth1p does not stain well with silver. Nevertheless, results
P.J.Preker et al.
obtained with yth1 mutant extracts clearly showed that
this polypeptide behaves as an actual subunit of PF I.
Microsequences of proteolytic fragments of the 85, 105
and 150 kDa subunits of PF I were obtained (see Materials
and methods). Five peptides derived from the 150 kDa
subunit are identical to a predicted protein of 153 kDa
encoded by an open reading frame on chromosome IV
(systematic name: YDR301w, SwissProt accession No.
S61187). Three peptides from the 105 kDa protein match
a predicted protein of 96 kDa (YLR115w, S64952).
Strikingly, these proteins are 23.5 and 24.4% identical to
the 160 and 100 kDa subunits of CPSF (Jenny et al.,
1994; Jenny and Keller, 1995; Murthy and Manley, 1995),
respectively. Although less obvious than for Ysh1p and
Yth1p, these homologies are statistically significant [error
probabilities P ?6.3e-23 (Altschul et al., 1994)] and lend
strong support to the hypothesis that the two genes encode
actual subunits of PF I. The 105 kDa protein is also
significantly related to Ysh1p and to CPSF-73 (Jenny
et al., 1996). The two genes were therefore tentatively
named YHH1 (yeast 160 kDa homologue 1) and YDH1
(yeast double homologue 1). While this manuscript was
in preparation, a report appeared showing that Yhh1p is
required for pre-mRNA 3? end formation in yeast (Stumpf
and Domdey, 1996). Based on immunodepletion experi-
ments, the authors concluded that the protein is a subunit
of CF II and thus named it cleavage factor two 1 protein
(Cft1p; see Discussion). Therefore, we will hereafter refer
to the yeast homologue of CPSF-160 as ‘Yhh1p/Cft1p’.
As expected, a polyclonal antibody raised against the
carboxy-terminus of Yhh1p/Cft1p (Stumpf and Domdey,
1996) recognized the 150 kDa subunit of PF I in peak
fractions of the Mini Q column (see Figure 3B).
Standard genetic analyses demonstrated that YDH1 and
YHH1/CFT1 are essential for cell viability (see Materials
and methods). YDH1, cloned from an S.cerevisiae genomic
plasmid bank, could rescue a disruption of that gene when
provided on a single-copy plasmid. We repeatedly failed
to clone YHH1/CFT1 from the same library by using a
probe corresponding to amino acids 1–379 of the protein.
Possibly, this gene is detrimental to bacterial growth.
Fig. 2. PF I is a multiprotein complex containing Pap1p, Pta1p, Fip1p
and homologues to CPSF subunits. (A) Western blot analysis across
the PF I activity peak of the Mini Q column. Proteins were detected
with polyclonal antibodies to Ysh1p, Pap1p and Fip1p. (B) Subunit
composition of PF I. Aliquots (20 µl) of the Mini Q column fractions
indicated at the top of the panel were separated on an SDS–
polyacrylamide (9%) gel and visualized by silver staining. Proteins
that correspond in size to proteins detected by immunoblotting (A) or
that have been identified by peptide sequencing are indicated. Other
major polypeptides are designated according to their apparent
molecular mass. (C) PF I activity of the Mini Q fractions shown in
(B). Of the indicated Mini Q fractions, 4 µl of each were mixed with
3 µl of an ammonium sulfate-fractionated fip1-1 extract and
32P-labelled CYC1 pre-mRNA under standard reaction conditions at
30°C for 45 min. Hatched and open boxes represent the 5? and 3?
portions of the precursor, respectively. Control reactions were done in
parallel with the following components: lane 1, dialysis buffer (G-50);
lane 2, 3 µl of a wild-type extract; lane 3, 3 µl of fip1-1 extract; lane
4, as lane 3, but containing 3 µl of a crude PF I fraction from the
Macro-Prep Q column; lane 5, 4 µl of the peak fraction (# 24) from
the Mini Q column; lane 6, 3 µl of the load fraction and 3 µl of fip1-1
extract. (D) Reconstitution of specific polyadenylation activity with
partially purified CF I and PF I fractions of the Mini Q column. For
assays, 4 µl of the PF I fractions shown in (B) and 3 µl of partially
purified CF I were incubated with the CYC1 pre-cleaved transcript
(represented by a hatched box) under standard reaction conditions.
Lane 1, unreacted CYC1 pre-cleaved; lane 2, 3 µl of a wild-type
extract; lane 3, 3 µl of CF I only; lane 4, 3 µl of CF I and 4 µl of
Mini Q fraction 24; lane 5, 4 µl of Mini Q fraction 24 only; lane 6,
3 µl of CF I and 4 µl of the load fraction. In (C) and (D) the reaction
products were resolved on a 5% polyacrylamide–8.3 M urea gel and
exposed to X-ray film for 12 h. Markers (M) are HpaIII-digested
pBR322 DNA. Their sizes, in number of nucleotides, and positions are
indicated on the right.
Yeast poly(A) polymerase holoenzyme
nuclease activity (O’Connor and Peebles, 1992). To test
whether the PTA1 gene product is also involved in 3? end
formation, extracts from a pta1-1 mutant strain were
assayed for their ability to cleave and polyadenylate CYC1
pre-mRNA substrates in vitro. Whereas cleavage activity
was normal, the extracts failed to polyadenylate both the
upstream cleavage product (Figure 3A, lane 6) and a pre-
cleaved CYC1 RNA (data not shown). In contrast, extracts
from the pta1-1 strain transformed with the wild-type
gene borne on a single-copy plasmid polyadenylated the
substrate RNA with efficiency comparable with the wild-
activity could be restored efficiently by addition of extracts
mutant in CF I, which on their own neither cleave nor
polyadenylate (compare lanes 5 and 8), but not by extracts
of mutants in the Fip1p subunit of PF I (lane 7). It thus
appears that Pta1p and Fip1p are tightly associated in a
complex required for polyadenylation. Most importantly,
extracts by addition of PF I from the Mono S column
(Figure 3A, lane 11) or the Mini Q column (data not
shown). Differences in the length of the polyadenylated
products after complementation of fip1-1 and pta1-1
extracts were not reproducible (lanes 8, 10 and 11).
The complementing fractions contained Fip1p, Yth1p,
Ysh1p, Yhh1p/Cft1p (Figure 3B, lanes 2 and 3) and Pap1p
CF II/CF IB fractions do not contain any of these proteins
(lanes 1 and 4; see Discussion). In addition, the anti-Fip1p
antibody detected a faster migrating protein species (lane
3), referred to here as Fip1p*. Fip1p* had been observed
throughout the entire purification, but its abundance
relative to the full-length protein varied. Because this
species is also recognized by the anti-HA1 antibody, it
probably represents a C-terminally truncated form of
Fig. 3. Mutant pta1-1 extracts are deficient for polyadenylation and
can be complemented by PF I fractions containing Fip1p and
homologues to CPSF subunits. (A) The in vitro phenotype of pta1-1
mutant extracts is similar to that of fip1-1 mutant extracts. Three to
six µl of extract or buffer and 1 µl of a Mono S fraction containing
PF I activity were combined with32P-labelled CYC1 substrate RNA as
indicated at the top of each lane, and incubated under standard
reaction conditions for 40 min. Reaction products were separated by
denaturing gel electrophoresis, and the gel was exposed to film, as
described in the legend to Figure 2. Lower case italicized labels at the
top refer to mutant extracts. PF I refers to the partially purified
protein. Markers (M) are as in Figure 2. (B) The complementing PF I
fractions contain Fip1p, Yhh1p/Cft1p, Ysh1p and Yth1p. PF I
fractions (lanes 2 and 3) and CF II/CF IB fractions (lanes 1 and 4;
hydroxyapatite column, Minvielle-Sebastia et al., 1997) were
electrophoresed on an SDS–polyacrylamide (10%) gel, blotted to
membrane and probed with antibodies to Yhh1p/Cft1p, Ysh1p and
Yth1p (lanes 1 and 2). After stripping, the blot was reprobed with
antibodies to Ysh1p, Yth1p and Fip1p (lanes 3 and 4). A polypeptide
with a higher electrophoretic mobility was reproducibly detected with
antibodies to Fip1p. This protein is probably a proteolytic form of
Fip1p and was therefore denoted Fip1p* (see text).
PF I modulates the polyadenylation activity of
Pap1p associated with it
The purified PF I–Pap1p complex polyadenylates RNA in
the absence of other factors and without specificity for a
genuine pre-mRNA 3? end (Figures 2C, lane 5, and 3A,
lane 9). We compared the kinetics of poly(A) addition
with CYC1 pre-mRNA by either the PF I–Pap1p complex
or Pap1p alone. Time course experiments were done with
immunoaffinity-purified PF I, containing ~10 ng of Pap1p
(final concentration ~3 nM), or the same amount of
the recombinant enzyme (Figure 4). Recombinant Pap1p
elongated the primer at a slow and uniform rate to a final
average tail length of ~200 adenosine residues after 1 h.
In stark contrast, polyadenylation by Pap1p in the PF I
complex was fast. After 4 min, poly(A) tails were ~400
residues long. After 32 min, no further elongation could
be detected owing to the limited resolution capacity of
the gel. Another remarkable difference is that, in contrast
to what was observed with Pap1p alone, only a minor
portion of the RNA was used as a substrate by the poly(A)
polymerase holoenzyme (Figure 4, compare lanes 6 and
12). Two possible explanations may account for this result.
First, the RNA is polyadenylated more processively when
Pap1p is in a complex with the other components of PF I.
Second, a substrate RNA is elongated preferentially once
it has received a minimal number of adenosine residues,
Peptide microsequencing of the 85 kDa subunit of PF I
revealed that this protein is the product of the essential
gene PTA1 (coding capacity 88 kDa). PTA1 initially was
defined by a conditional growth mutation, pta1-1, that
causes the accumulation of unspliced pre-tRNAs in vivo
(O’Connor and Peebles, 1992). All 10 intron-containing
tRNA families are affected. Surprisingly, extracts prepared
from pta1-1 cells have normal pre-tRNA splicing endo-
P.J.Preker et al.
polyadenylate a pre-cleaved pre-mRNA substrate when
combined with partially purified CF I, with no additional
poly(A) polymerase. Our results differ from those of
earlier work indicating that PF I and Pap1p are two
separable factors, each one required individually to restore
specific polyadenylation activity (Chen and Moore, 1992).
We do not know if these discrepancies are attributable to
variations in the protocols used for extract preparation
and biochemical fractionation or might reflect the different
genetic backgrounds of the strains used in these studies.
Remarkably, the previously reported purification of Pap1p
from yeast did not result in the identification of any
additional polypeptides (Lingner et al., 1991b). Because
this purification relied solely on the unspecific polyadenyl-
ation activity of the enzyme, any polyadenylation activity
associated with PF I may have escaped detection. Early
work of Haff and Keller (1975) demonstrated that Pap1p
activity of yeast cell extracts can be separated into distinct
peaks by chromatography on DEAE-cellulose. A minor
portion of the activity eluted at high salt concentrations
after the major peak of activity. Possibly, this portion
corresponded to Pap1p in a complex with PF I.
Of the other polypeptides that co-purified with PF I
activity, one was identified by Western blot analysis as
Ysh1p, a protein that is highly homologous to mammalian
CPSF-73 (Chanfreau et al., 1996; Jenny et al., 1996).
Also, Western blot analysis confirmed the presence of
Yth1p (the homologue of CPSF 30 kDa subunit) in this
factor even though no polypeptide with an Mr?35 kDa
was visible in highly purified PF I preparations. The
biochemical characteristics of Ysh1p and Yth1p are con-
sistent with these proteins being genuine subunits of
PF I. Antibodies directed against the recombinant proteins
depleted PF I activity from whole-cell extracts or PF I
fractions. In both cases, activity could be restored by
adding back purified PF I (Jenny et al., 1996; Barabino
et al., 1997). Moreover, extracts from conditional ysh1/
brr5 or yth1 mutants are deficient for polyadenylation
but not for cleavage (Chanfreau et al., 1996; Barabino
et al., 1997).
The two largest subunits associated with PF I activity
are significantly related over the entire sequence length to
the 160 and 105 kDa subunits of CPSF, respectively.
Therefore, it is highly likely that these proteins play an
essential role in 3? end formation as well. Yhh1p/Cft1p,
the CPSF-160 homologue, recently has been reported to
be a subunit of CF II (Stumpf and Domdey, 1996). The
authors showed that antibodies to Cft1p immunodeplete
cleavage and polyadenylation activity from wild-type
extracts. The two activities could be complemented by
addition of Mono Q fractions containing CF II and PF I
activity, respectively. In Western blot experiments, Cft1p
was found associated with CF II activity. However, the
two activities largely overlapped in the column fractions
used for complementation (Stumpf and Domdey, 1996).
Although we cannot rigorously rule out the possibility that
the CPSF-related polypeptides are involved in cleavage
in vivo, we found no evidence that this is the case in our
reconstituted in vitro system. The fractions required for
cleavage, purified CF IA and partially purified CF II/
CF IB (Minvielle-Sebastia et al., 1997), do not contain
detectable amounts of Yth1p, Ysh1p or Yhh1p (Figure
3B). Moreover, cleavage can be reconstituted efficiently
Fig. 4. Poly(A) polymerase activity is modulated by other components
of PF I. Pap1p activity was assessed in standard reaction mixtures
containing (in 50 µl) 100 fmol of32P-labelled CYC1 pre-mRNA and
either 200 fmol of immunoaffinity-purified PF I–Pap1p (lanes 1–6) or
200 fmol (13 U) of recombinant Pap1p (lanes 7–12). Aliquots (8 µl)
were withdrawn from each tube (0 min time point), and the remainder
was pre-warmed briefly to 30°C. The reaction was started by addition
of ATP to a final concentration of 0.5 mM. Samples (8 µl) were
removed at the time points indicated at the top and diluted into
proteinase K digestion buffer (Minvielle-Sebastia et al., 1994). The
RNA was extracted from the aliquots and analysed on a denaturing
5% polyacrylamide gel. The size of DNA markers (M) in number of
nucleotides is indicated on the left.
as in mammals where CPSF and PAB II stimulate the
activity of the poly(A) polymerase after oligoadenylation
of the precursor (Wahle, 1991; Bienroth et al., 1993).
Because the authentic poly(A) polymerase and the recom-
binant enzyme purified from E.coli have indistinguishable
biochemical and enzymatic properties (Lingner et al.,
1991a), we consider it unlikely that the differences are
attributable to an intrinsic property of the recombinant
enzyme. When combined with purified CF IA and CF IB/
CF II fractions, the PF I–Pap1p complex was fully active
in the specific cleavage and polyadenylation reaction with
CYC1 pre-mRNA (Minvielle-Sebastia et al., 1997). It also
specifically polyadenylated a pre-cleaved RNA substrate
when combined with partially purified CF I (see
Complexity of yeast 3? end processing factors
PF I was purified from a strain expressing epitope-tagged
Fip1p. In addition to Fip1p, PF I comprises Pap1p, Pta1p
and, at a minimum, eight other protein subunits. The
finding that Fip1p is stably associated in a complex agrees
with the observations that recombinant Fip1p was neither
able to substitute for PF I in the reconstituted in vitro
system nor to complement polyadenylation-deficient fip1
extracts (P.J.Preker, unpublished results).
The purified PF I–Pap1p complex can specifically
Yeast poly(A) polymerase holoenzyme
without PF I. In any case, the observation that immuno-
depletion with antibodies to Pap1p or PF I subunits, such
as Fip1p and Ysh1p/Brr5p, also impairs cleavage to
various degrees (Minvielle-Sebastia et al., 1994; Preker
et al., 1995; Chanfreau et al., 1996) suggests that cleavage
factors and PF I may be directly associated and that some
aspects of this association may be maintained in vitro.
The finding that the 85 kDa subunit of PF I is identical
to Pta1p (O’Connor and Peebles, 1992) adds yet another
layer of complexity to the mechanism of yeast 3? end
formation. In vitro, pta1-1 mutants have a 3? end pro-
cessing defect very similar to that of fip1-1, ysh1/brr5 or
yth1-1 mutants. Pta1p has been implicated in pre-tRNA
splicing, because pta1-1 mutants accumulate all 10 end-
trimmed, intron-containing pre-tRNA families in vivo
(O’Connor and Peebles, 1992). However, the mutant
exhibits no pre-tRNA splicing defect in vitro. Similar
phenotypes are common to a number of other mutants,
such as nucleoporin mutants (Simos et al., 1996) and the
pleiotropicrna1-1mutant (Hopperetal.,1978). Ithasbeen
speculated that pre-tRNA processing might be coupled to
mRNA export (Simos et al., 1996). Likewise, Pta1p
(and so, possibly, other components of PF I) may have
overlapping functions in both tRNA and mRNA matur-
ation. Alternatively, the accumulation of unspliced pre-
tRNAs in pta1-1 mutants might be a secondary effect of
a reduced 3? end processing efficiency in these mutants.
To this end, we have used an in vivo assay based on loss
of suppressor tRNA activity (Simos et al., 1996) to test
whether mutants in 3? end processing factors are generally
impaired in tRNA processing. However, we failed to
detect any significant decrease in suppressor tRNA activity
in an rna14-1, rna15-1 or fip1-1 mutant background
(P.J.Preker, unpublished data).
We showed that purified PF I exhibits non-specific
polyadenylation activity on its own that is significantly
more efficient than that of Pap1p alone. A plausible
explanation of how the other subunits of PF I stimulate
the activity of Pap1p could be that the holoenzyme
contains an RNA-binding component that promotes its
interaction with the RNA substrate. In fact, immuno-
affinity-purified PF I can form a complex with the CYC1
pre-mRNA with a lower electrophoretic mobility in non-
denaturing gels (results not shown). In contrast, free Pap1p
The peak of the RNA-binding activity coincided with that
of PF I activity when fractions of the final Mini Q column
were assayed for complex formation. This indicates that
PF I contains at least one RNA-binding component. In
this respect, recombinant Yth1p has been shown to have
unspecific RNA-binding activity in vitro (Barabino et al.,
1997). Whether or not additional subunits of PF I are
binding to RNA remains to be determined. However, the
binding of PF I–Pap1p to the RNA is not specific for 3?
end processing-competent substrates because an RNA–
protein complex was also formed with a cyc1-512 mutant
RNA, which is not processed in vitro (results no shown).
Similarly, CF IA interacts with wild-type and mutant
pre-mRNAs (Kessler et al., 1996; L.Minvielle-Sebastia,
P.J.Preker, Y.Strahm and W.Keller, unpublished data).
Thus, sequence-specific RNA binding may require differ-
ent conditions and/or additional factors.
Evolutionary conservation of 3? end formation in
Recently, Murthy and Manley (1995) have reported that
CPSF-160 interacts with both bovine PAP and the 77 kDa
subunit of CstF. In view of the finding that the Fip1p
subunit of yeast PF I tethers Pap1p to the Rna14p subunit
of CF I (Preker et al., 1995), these authors speculated that
Fip1p might be the functional homologue of the CPSF
160 kDa subunit. An extension of this idea is that PF I
The sequence homologies of PF I components to the four
subunits of mammalian CPSF strengthens this hypothesis.
Sequence similarities have also been reported between the
Rna14p and Rna15p subunits of yeast CF I and, respect-
ively, the 77 and 64 kDa subunits of mammalian CstF
(Takagaki and Manley, 1994). Thus, 3? end processing
factors appear to be conserved between yeast and higher
eukaryotes, at least at the amino acid sequence level.
Obvious differences exist in the function of related genes
in either system. Whereas mammalian CstF only particip-
ates in the cleavage reaction, CF I is essential for both
cleavage and polyadenylation in yeast. On the other hand,
CPSF is required for both steps in 3? end formation,
whereas PF I is dispensable for the initial cleavage step.
Similarly, poly(A) polymerase is required for cleavage in
the mammalian, but not in the yeast system. The apparent
discrepancies between sequence and functional homology
might be rationalized in part by the fact that the inventory
of the factors is still incomplete and that the detailed
molecular mechanism of how the known protein factors
act in the reaction is largely unknown. In addition, in
contrast to the mammalian situation, it is unclear how
CF I and PF I are arranged on the RNA substrate. Both
factors bind RNA in vitro but their sites of interaction
have not yet been mapped. It could be that the interaction
of PF I–PAP1p with the pre-mRNA is not specific or
strong enough and that only the simultaneous binding of
the factor to the RNA and to the already bound CF I leads
to the formation of a stable and specific polyadenylation
complex via the Fip1p–Rna14p protein–protein contact.
In a model for the assembly of a yeast polyadenylation
complex, Pap1p is tethered to the RNA primer by at least
two distinct recognition events (see Figure 5). First, direct
binding of PF I–Pap1p to the efficiency element of the
upstream cleavage fragment involves Yth1p and possibly
other components of PF I. Second, the Fip1p subunit of
PF I acts as a bridge between Pap1p and CF IA, which
binds the pre-mRNA through Rna15p, possibly onto the
positioning element. The binding of CF I to the positioning
ally. However, as the positioning element determines the
site of endonucleolytic cleavage, and as PF I is dispensable
for the cleavage reaction, it is reasonable to assume that
CF I binds to the positioning element. Likewise, PF I may
bind to the efficiency element. Specific recognition of the
by the assembly of a complex network of RNA–protein
and protein–protein interactions.
In mammals, the requirement for CPSF is reflected by
the specific interaction of its 30 and 160 kDa subunits with
the almost invariant 3? end processing signal AAUAAA
(Keller et al., 1991; Murthy and Manley, 1995). Gel
retardation assays indicate that yeast PF I binds to pre-
P.J.Preker et al.
Materials and methods
All buffers used for PF I purification contained 0.5 mM dithiothreitol
(DTT), unless otherwise noted, and a cocktail of protease inhibitors
(0.5 mM phenylmethylsulfonyl fluoride, 0.7 µg/ml pepstatin and
0.4 µg/ml leupeptin hemisulfate). Buffer B: 20 mM HEPES–KOH
pH 7.0, 1.5 mM Mg acetate and 10 mM K acetate; buffer D: 20 mM
Tris–HCl pH 8.0, 0.5 mM EDTA, 20% glycerol and KCl at the millimolar
HEPES–KOH pH 7.4 instead of Tris–HCl and 1 mM β-mercaptoethanol
instead of DTT; buffer G: as buffer F, but with K acetate instead of KCl
and 0.02% NP-40; buffer N: 20 mM HEPES–KOH pH 7.4, 20% glycerol,
50 mM K acetate, 0.02% NP-40 and 1 mM β-mercaptoethanol; buffer
I: 20 mM Tris–HCl pH 8.3, 0.5 mM EDTA, 0.05% NP-40 and K acetate
concentrations as indicated.
Fig. 5. Schematic representation of the yeast polyadenylation complex.
The binding of CF I and PF I–Pap1p to the positioning and efficiency
elements, respectively, is hypothetical. CF I and PF I–Pap1p subunits
are shaded in light and dark grey, respectively. Pab1p is associated
loosely with CF IA and is thus not considered as an actual subunit of
any of these factors (Minvielle-Sebastia et al., 1997). Protein–protein
interaction between polypeptides of the two factors is indicated by a
Plasmids, S.cerevisiae strains and disruption of YDH1 and
Standard cloning procedures were performed as described (Sambrook
et al., 1989). Polymerase chain reaction (PCR) amplifications were done
by the manufacturer) supplemented with 50 pmol of each primer and
100 ng of plasmid DNA or 200 ng of genomic yeast DNA. Yeast media
and standard genetic manipulations were as described elsewhere (Guthrie
and Fink, 1991).
A plasmid for expression of double-tagged Fip1p in yeast was
constructed as follows: the coding sequence of FIP1 was amplified from
a genomic clone by PCR utilizing a 5? primer (5?-GGGGCGGCCGCCA-
GCTCCAGTGAAGACG-3?) and a 3? primer (5?-GGGCCTTAGGGT-
CATTTCGAATTTTG-3?). The resulting fragment was restricted with
NotI (underlined) and HindIII and cloned in the corresponding sites of
pHH1, a vector designed for the expression of HA1-His6-tagged proteins
in yeast (L.Minvielle-Sebastia, unpublished), yielding plasmid pIA96.
The 0.3 kb XhoI (blunt ended)–SacI fragment, carrying the CYC1
promoter, the double tag and the first codon of FIP1 following the
initiation codon, was isolated from this plasmid and inserted into pIA22
(CEN4-TRP1-FIP1; Preker et al., 1995) restricted with PstI (blunt ended)
and SacI. Fip1p encoded by the resulting plasmid (pIA97) contains an
additional 19 amino acids (MYPYDVPDYAHHHHHHAAA) at its
amino-terminus (the HA1 epitope peptide is underlined). This vector
was transformed into strain PJP24 [relevant genotype: fip1::LEU2 trp1
ura3-52 pIA24 (CEN4-URA3-FIP1)] and the residual URA3-marked
plasmid was eliminated on synthetic complete medium containing
5-fluoroorotic acid (5-FOA), yielding PJP14.
For cloning of YDH1, an ~330 bp fragment of the amino-terminal
part of the coding region was amplified by PCR on yeast genomic DNA
and used as a probe to screen an S.cerevisiae genomic plasmid bank
(Pick, 1995). Three independent clones were obtained that contained the
entire YDH1 open reading frame and its 5?- and 3?-regulatory elements.
A 3.3 kb PmlI–ApaI fragment containing YDH1 was subcloned into
SmaI–ApaI-restricted pBlueskriptKS– (Stratagene) to yield plasmid
pIA111. The shuttle vector pIA115 was generated by insertion of a
BamHI–KpnI fragment from pIA111 into the same sites of plasmid
pFL38 (CEN4-URA3; Bonneaud et al., 1991).
Strains heterozygous for a deletion of YDH1 and YHH1/CFT1 were
generated by PCR-mediated, single step disruption of the respective
open reading frames (Baudin et al., 1993). For that, a BglII fragment of
plasmid pFL38 containing the TRP1 marker was amplified with two
primers that contained 40–45 bp homologous to sequences flanking the
gene of interest at their ends. PCR products were transformed into the
diploid strain BMA41-2N (MATa/MATα ura3-1/ura3-1 ∆trp1/∆trp1
ade2-1/ade2-1 leu2-3,112/leu2-3,112 his3-11,15/his3-11,15; Baudin-
Baillieu et al., 1997). Transformants were selected on medium lacking
tryptophan, and integration of the marker at the correct loci was verified
by Southern blotting. Upon sporulation and tetrad dissection, no more
than two spores gave rise to growing colonies at 22°C, and all viable
clones were tryptophan auxotrophs. The deletion of YDH1 could be
rescued by transformation of the diploid strain with plasmid pIA115
(CEN4-URA3-YDH1) prior to sporulation. In this case, all tryptophan
heterotrophs were sensitive to 5-FOA.
A TRP1-marked plasmid (pIA114) for the expression of wild-type
PTA1 in yeast was generated by subcloning a ~5.3 kb HindIII fragment
from YCpPTA1 (O’Connor and Peebles, 1992) into the HindIII site of
YCplac22∆BglII/∆SacI (Preker et al., 1995).
LM113 is a pta1-1 mutant segregant of a backcross of POC8-23d
(pta1-1 MATa ade2-1 leu2-∆1 lys2-801 trp1-∆101 ura3-52; O’Connor
mRNAs in vitro (data not shown). Strikingly, both the
recombinant 30 kDa subunit of CPSF and Yth1p do
interact with homopolymeric RNA in vitro (Barabino
et al., 1997). As mentioned above, direct binding of the
PF I–Pap1p holoenzyme to RNA would probably increase
the processivity of polyadenylation and might thus explain
the more rapid polyadenylation exerted by the complex
as compared with Pap1p alone (Figure 4). In mammals,
PAP is activated by two factors, PAB II and CPSF (Wahle,
1991; Bienroth et al., 1993). These factors increase the
processivity of mammalian PAP to various extents. The
sequence similarities between yeast PF I and CPSF suggest
that some of the PF I subunits may act similarly on Pap1p.
This issue should be addressed by a careful analysis of
the kinetic parameters of the PF I–Pap1p complex. The
PF I–Pap1p complex extends poly(A) tails far beyond the
length observed in vivo. Normal polyadenylation can be
reproduced by the addition of purified CF IA and partially
purified CF II/CF IB, implying that these fractions contain
an activity that controls the length of the poly(A) tail
synthesized by PF I–Pap1p (Minvielle-Sebastia et al.,
1997). We characterized the different polypeptides co-
purifying with CF IA activity as Rna14p, Rna15p, Pcf11p
(Amrani et al., 1997), a new protein called Clp1p
(L.Minvielle-Sebastia et al., unpublished data) and, inter-
estingly, Pab1p (Minvielle-Sebastia et al., 1997). We
showed that Pab1p is required for the synthesis of normal
poly(A) tails, and might thus represent the activity that
controls the length of the poly(A) tail synthesized by
PF I–Pap1p. Possibly, Pab1p acts directly on Pap1p by
inhibiting its activity once the poly(A) tails have reached
the normal length. The tentative arrangement of the factors
involved in the polyadenylation reaction is depicted in the
model shown in Figure 5.
similarity to Pta1p or Fip1p was found in the databanks.
However, putative Pta1p and Fip1p homologues do exist
in the fission yeast Schizosaccharomyces pombe. It thus
remains an open question whether the PF I subunits other
than Pap1p and those related to CPSF are unique to lower
eukaryotes, or whether their mammalian relatives have
merely not yet been discovered.
Yeast poly(A) polymerase holoenzyme
and Peebles, 1992) to W303 (MATα ade2-1 his3-11,15 leu2-3,112
trp1-1 ura3-1; R.Rothstein, Columbia University, New York). The
temperature-sensitivity of LM113 can be complemented by transform-
ation with pIA114 (CEN4-TRP1-PTA1). Other strains and their relevant
genotypes are LM88 (rna14-1 ade2-1 his3-11,15 leu2-3,112 trp1-1
ura3-1; Minvielle-Sebastia et al., 1994) and LM96 (fip1-1 ade2-1 his3-
11,15 leu2-3,112 trp1-1 ura3-1). LM96 was obtained by integration of
the temperature–formamide-sensitive fip1-1 mutant allele (Preker et al.,
1995) in place of the wild-type FIP1 allele.
600 µl of protein A–Sepharose CL-4B with dimethyl pimelimidate as
cross-linking reagent according to Harlow and Lane (1988). The slurry
was loaded in a column (1 cm diameter) and equilibrated in buffer
G-300. Of the Mono S pool, 20% (2 ml, 1.2 mg of protein) was injected
at a rate of 0.02 ml/min. The flow trough was reloaded at a rate of
0.04 ml/min, and the column was washed with buffer G-300 until the
OD280of the eluate was stable. The affinity beads were transferred to a
5 ml centrifuge tube with 3 ml of the above buffer, recovered by
centrifugation and suspended in two volumes of buffer G-300 containing
HA1 epitope peptide (Berkeley Antibody Co., Richmond, CA) at a
concentration of 100 µM. Elution was done for 10–20 min at
24–30°C with occasional mixing. The supernatant was recovered after
centrifugation, and the elution step was repeated two or three times. The
resin was regenerated by washing with 2 ml of glycine–HCl pH 2.8 and
4 ml of buffer G-300, and re-used twice without detectable loss in
The eluate from one of these columns was dialysed against buffer
D-50 and loaded on a Mini Q PC 3.2/3 column (bed volume 240 µl)
equilibrated in loading buffer at a rate of 0.1 ml/min. The column was
washed with 3 ml of buffer D-50 and connected to a 4.8 ml gradient
from 50 to 500 mM KCl in buffer D. PF I activity was recovered at
~290 mM KCl.
Cell culturing and extract preparation
Saccharomyces cerevisiae strain PJP14 was cultured in YPD medium
(1% yeast extract, 2% peptone and 2% dextrose) supplemented with
adenine hemisulfate (20 mg/l) and ampicillin (50 mg/l). Five times 10 l
of medium were inoculated with an exponentially growing culture and
vigorously aerated. The temperature of the culture was ~28°C and the
generation time was ~3 h. At an OD600of 3–4, 200 g of dry dextrose
were added. At an OD600of ~6, cells were harvested by centrifugation
(3000 r.p.m., 10 min) in a Cryofuge 6000 (Heraeus Sepatech) at
room temperature and converted into spheroplasts by treatment with
Zymolyase-100T (Seikagaku, Tokyo, Japan) according to Butler et al.
(1990). All subsequent steps were done on ice or at 4°C. The spheroplasts
were resuspended in 200 ml of buffer B per 10 l of initial culture volume
and lysed by 20 strokes in a 60 ml Dounce homogenizer with a tight-
fitting plunger (type S; B.Braun Biotech). The lysate was clarified by
centrifugation (10 000 r.p.m., 15 min) in a Sorvall GSA rotor. The
supernatant was brought to 200 mM K acetate and stirred gently for
30 min. The cell debris was removed by centrifugation at 290 000 gmax
for 90 min in a Kontron TFT 45.94 rotor. The supernatant was removed
with a pipette, taking care to avoid the flaky white layer at the top of
the tubes, and adjusted to 40% saturation by the addition of solid
ammonium sulfate. After incubation overnight, precipitated proteins
were pelleted by centrifugation (10 000 r.p.m., 20 min) in a Sorvall
GSA rotor and the pellet was resuspended in buffer D-50 to a final
protein concentration of ~15 mg/ml. Typically, the precipitate contained
20–25% of the total protein present in the crude extract. The residual
ammonium sulfate was removed by extensive dialysis against two
changes of buffer D-50. The dialysate was diluted with buffer D-0 to
adjust the conductivity to that of the dialysis buffer and clarified by
centrifugation (10 000 r.p.m., 20 min) in a Sorvall GSA rotor.
Small-scale extracts were prepared from strains grown in 0.5 l of
YPD at 30°C on a shaker and processed as described by Butler et al.
(1990) except that buffers were as for the large-scale extracts (see above).
3? end processing assays
Substrate RNAs were produced as run-off transcripts with pG4-CYC1
or pG4-CYC1pre as templates that had been linearized with EcoRI or
NdeI, respectively (Preker et al., 1995).
During PF I purification, 100 µl samples from every other column
fraction were dialysed against buffer G-50, and PF I-containing fractions
were detected by their ability to restore polyadenylation activity of
fip1-1 mutant extracts prepared from LM96 cells as described (Preker
et al., 1995).
Antibodies, Western blotting and immunoprecipitations
Polyclonal antibodies directed against purified recombinant Pap1p and
Fip1p were affinity purified on immobilized proteins (Preker et al.,
1995). Rabbit antisera to Cft1p (Stumpf and Domdey, 1996), Ysh1p
(Jenny et al., 1994) and Yth1p (Barabino et al., 1997) were gifts from
the respective authors.
Immunoblotting assays were done by standard procedures (Sambrook
et al., 1989). Monoclonal antibody 12CA5 specific for the HA1 epitope
(Boehringer Mannheim) was used at a dilution of 2 µg/ml in buffer
(20 mM Tris–HCl pH 7.5, 150 mM NaCl and 0.05% Tween-20).
Polyclonal antibodies were diluted in the above buffer containing 5%
anti-rabbit and rabbit anti-mouse immunoglobins; DAKO, Denmark)
were diluted 1:2000 and 1:1000, respectively. Detection was done with
the enhanced chemiluminescence kit according to the protocol provided
by the manufacturer (Amersham).
For co-immunoprecipitation experiments, 80 µl from a Mono S peak
fraction were first pre-adsorbed to 400 µl of a 15% protein A–Sepharose
slurry in buffer I-300, to remove proteins that bind to the affinity resin
unspecifically. Following incubation on a roller at 4°C for 30 min, the
resin was pelleted and the supernatant was recovered. Monoclonal
antibody (10 µg) or 20–40 µl of polyclonal antiserum were coupled to
protein A–Sepharose in buffer I-300. After extensive washing with the
above buffer, the antibody beads were combined with 300 µl of the pre-
adsorbed PF I fraction and binding was performed on a roller for 2 h at
4°C. The resin was pelleted by centrifugation in a microfuge (5000
r.p.m., 30 s) and washed four times with 1 ml of buffer I-300 at room
temperature. Alternatively, the antibody beads were washed three times
with buffer I-100, twice with the same buffer containing 1.2 M K acetate,
and once with buffer I-100. After the final wash, bound proteins were
eluted in 40 µl of sample buffer for 5 min at 90°C and resolved by
electrophoresis on SDS–polyacrylamide gels.
Purification of PF I
All manipulations were done at 0–4°C. Column material was purchased
from Pharmacia, Bio-Rad and Qiagen. Columns were run at one column
volume per hour, except for fast protein liquid chromatography (FPLC)
columns. Fractions were frozen in liquid nitrogen and stored at –70°C.
Ammonium sulfate-fractionated extract was applied to a 430 ml
Macro-Prep high Q anion exchange column equilibrated in buffer D-50.
The column was washed with one column volume of the above buffer
and developed with a 2.4 l gradient of buffers D-50 to D-500. PF I
activity desorbed between 200 and 330 mM KCl. Peak fractions were
pooled and dialysed for 3 h against 4 l of buffer D-0 and for 2 h against
2 l of buffer D-50. The dialysate was loaded on a 100 ml Blue–Sepharose
column that had been equilibrated in buffer D-80. The column was
washed with 80 ml of buffer D-80 and eluted with an eight column
volume gradient from 80 to 500 mM KCl in buffer D. Fractions
containing PF I activity were dialysed for 4 h against two changes of
buffer F-0 and applied to a 100 ml heparin–Sepharose CL-6B column
equilibrated with buffer F-100. After washing with one column volume
of buffer F-100, PF I activity was recovered at ~420 mM KCl in a
400 ml gradient of buffers F-100 to F-800. Mg acetate and NP-40 were
added to the eluate to final concentrations of 2 mM and 0.02% (v/v),
respectively, before loading on a 4 ml Ni2?-NTA–agarose column
equilibrated in buffer N-50. The column was washed with 4 vols of
buffer N-50 and developed with a gradient (12 column volumes) from
0 to 250 mM imidazole in buffer N-50. PF I activity eluted in a broad
peak between 30 and 170 mM imidazole. The pool of activity was
loaded directly on a 1 ml Mono S HR 5/5 column equilibrated with
buffer G-100 at a flow rate of 0.3 ml/min. The column was washed with
2 ml of the above buffer and developed with a gradient from 100 to
500 mM K acetate in buffer G over 20 column volumes. PF I was
recovered between 160 and 400 mM K acetate.
An immunoaffinity column was prepared by covalently coupling
200 µg of monoclonal antibody 12CA5 (Boehringer Mannheim) to
Amino acid sequencing of PF I components
Immunopurified PF I from three runs of the anti-HA1 affinity column
(corresponding to 30 l of starting cell culture) was pooled and proteins
were precipitated by the addition of trichloroacetic acid to a final
concentration of 17% (w/v). The PF I pool contained ~100 µg/ml of the
HA1 nonapeptide and we believe that this served as a carrier for
precipitation of the polypeptides. After centrifugation at 4°C in a
microfuge at maximal speed for 1 h, pellets were washed subsequently
with ice-cold 80 and 100% acetone, air dried and resuspended in 250 µl
of protein sample buffer. The amount of PF I was estimated by
comparison of a sample run on a polyacrylamide gel with known
P.J.Preker et al.
amounts of Pap1p run on the same gel. The following steps were done
by the TopLab company (Mu ¨nchen, Germany). Of the 150, 105 and
85 kDa subunits of PF I, 40–60 pmol each were excised from a
polyacrylamide gel and digested in situ with endoproteinase Lys-C.
Peptides were eluted from the gel slices, separated by reverse phase
chromatography, and microsequenced with an automated sequencer
(Porton 3600; Beckman Instruments, Fullerton). The sequences obtained
from the 150 kDa subunit were NIIDIQFLK, FHGLITDIGLIPQK,
SNIYYIQMEAEGRLLI, LVXAGNTTISK and VIGYDENVPXAEG-
FQSGILLINP. The sequences from the 105 kDa subunit were SYG-
TVVDFTMFLPDDS, NLN(S/N)QYSGFSGT(G/E)EAENFDNLD and
GALSIGDVRLAQLK. From the 85 kDa subunit the sequence obtained
Amino acid sequences were compared with the yeast protein database
at MIPS, Martinsried. For further sequence analysis, the BLASTp
program (Altschul et al., 1994) at the Baylor College of Medicine
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Received on March 18, 1997; revised on May 5, 1997
Note added in proof
While this manuscript was under review, it was reported that the
homologues of the three large subunits of CPSF are present in the
purified yeast CF II factor [Zhao,J., Kessler,M.M. and Moore,C.L. (1997)
Cleavage factor II of Saccharomyces cerevisiae contains homologues to
subunits of the mammalian cleavage/polyadenylation specificity factor
and exhibits sequence-specific, ATP-dependent interaction with precursor
RNA. J. Biol. Chem., 272, 10831–10838]. This is in contrast to our
findings reported here. The discrepancy could perhaps be explained by
assuming that a complex consisting of PF I/PAPp and CF II exists in
subcomplexes, one of which has CF II activity and the other PF I/PAP
activity. The CPSF homologues might form a core of polypeptides
contained in both subcomplexes. The fact that we did not detect these
proteins in our partially purified CF II fractions (Figure 3B, lanes 1 and
4) may be because the assay for CF II activity is more sensitive than
the detection of proteins in the Western blot.