Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.

Abstract

Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including cleavage and polyadenylation at the 3'-end. Despite the characterization of many proteins that are required for the cleavage reaction, the identity of the endonuclease is not known. Recent analyses indicated that the 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF-73) might be the endonuclease for this and related reactions, although no direct data confirmed this. Here we report the crystal structures of human CPSF-73 at 2.1 A resolution, complexed with zinc ions and a sulphate that might mimic the phosphate group of the substrate, and the related yeast protein CPSF-100 (Ydh1) at 2.5 A resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-beta-lactamase domain and a novel beta-CASP (named for metallo-beta-lactamase, CPSF, Artemis, Snm1, Pso2) domain. The active site of CPSF-73, with two zinc ions, is located at the interface of the two domains. Purified recombinant CPSF-73 possesses RNA endonuclease activity, and mutations that disrupt zinc binding in the active site abolish this activity. Our studies provide the first direct experimental evidence that CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.

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LETTERS
Polyadenylation factor CPSF-73 is the pre-mRNA
3’-end-processing endonuclease
Corey R. Mandel1, Syuzo Kaneko1*, Hailong Zhang1*{, Damara Gebauer1{, Vasupradha Vethantham1,
James L. Manley1& Liang Tong1
Most eukaryotic messenger RNA precursors (pre-mRNAs)
undergo extensive maturational processing, including cleavage
and polyadenylation at the 39-end1–8. Despite the characterization
of many proteins that are required for the cleavage reaction, the
identity of the endonuclease is not known4,9,10. Recent analyses
indicatedthatthe73-kDasubunitofcleavageandpolyadenylation
specificityfactor(CPSF-73)mightbetheendonucleaseforthisand
relatedreactions10–15,althoughnodirectdataconfirmedthis.Here
we report the crystal structures of human CPSF-73 at 2.1A˚reso-
lution, complexed with zinc ions and a sulphate that might mimic
thephosphategroupofthesubstrate,andtherelatedyeastprotein
CPSF-100(Ydh1)at2.5A˚resolution.BothCPSF-73andCPSF-100
contain two domains, a metallo-b-lactamase domain and a novel
b-CASP (named for metallo-b-lactamase, CPSF, Artemis, Snm1,
Pso2) domain12. The active site of CPSF-73, with two zinc ions, is
located at the interface of the two domains. Purified recombinant
CPSF-73possessesRNAendonucleaseactivity,andmutationsthat
disrupt zinc binding in the active site abolish this activity. Our
studies provide the first direct experimental evidence that CPSF-
73 is the pre-mRNA 39-end-processing endonuclease.
CPSF-73 belongs to the metallo-b-lactamase superfamily of zinc-
dependent hydrolases11,12. Canonical metallo-b-lactamases contain
five signature sequence motifs—Asp (motif 1), His-X-His-X-Asp-
His (motif 2), His (motif 3), Asp (motif 4) and His (motif 5), most
of which are ligands to the two zinc ions in theiractive site. Sequence
conservation between CPSF-73 and the canonical metallo-b-lacta-
mases is limited to these signature motifs. Whereas the first four
motifs have been identified in the N-terminal segment of CPSF-73
(see Supplementary Fig. 1a, Supplementary Table 1), the fifth motif
was uncertain, with three candidates, A (Asp or Glu), B (His) and C
(His) (see Supplementary Fig. 1a), in the so-called b-CASP motif12.
Motif B was proposed to be equivalent to motif 5 in the canonical
metallo-b-lactamases. Another subunit of CPSF, CPSF-100, shares
sequence conservation (see Supplementary Fig. 1b) and a similar
domain architecture (see Supplementary Fig. 1a) with CPSF-73 but
lacks the putative zinc-binding residues.
To understand the roles of CPSF-73 and CPSF-100 in pre-mRNA
39-end processing, we determined the structures of human CPSF-73
(residues 1–460) and yeast CPSF-100 (residues 1–720) (the crystal-
lographic data are summarized in Supplementary Table 2). The two
structures obtained for CPSF-73 were crystallized in the absence or
presence of 0.5mM zinc (although both structures contained zinc
atoms;seebelow).Wediscoveredserendipitously thatinsituproteo-
lysis by a fungal protease is crucial for the crystallization of yeast
CPSF-100 (ref. 16).
The structure of CPSF-73 can be divided into two domains
(Fig. 1a). The amino-terminal residues (amino acids 1–208) form a
*These authors contributed equally to this work.
1DepartmentofBiologicalSciences,ColumbiaUniversity,NewYork,NewYork10027,USA.{Presentaddresses:ArrayBioPharmaInc.,Boulder,Colorado80301,USA(H.Z.);Johnson
& Johnson Pharmaceutical Research & Development LLC, San Diego, California 92121, USA (D.G.).
N
β2
626
β−CASP
domain
Metallo−β−lactamase
domain
C
β3
β4
β5
β6
β7
β8
β9
β10
β11
β12
β13
β14
β15
α1
α2
α3
α4
α5
α6
αA
αB
αC
αD
αE
αF
αG
βA
βB
βC
βD
βE
βF
βG
βH
βI
276
388
397
717
422
b
CPSF-100
N
β2
112
β−CASP
domain
Metallo−β−lactamase
domain
C
7
β3
β4
β5β6
β7
β8
β9
β10
β11
β12
β13
β14
β15
α1
α2
α3
α4
α5
α6
αA
αB
αC
αF
αG
βA
βB
βC
βD
βH
βE
βG
βI
122
300
288
459
a
CPSF-73
Figure 1 | Structures of human CPSF-73 and yeast CPSF-100 (Ydh1).
a,SchematicrepresentationofthestructureofhumanCPSF-73.Theb-strands
anda-helicesarelabelled,andthetwozincatomsintheactivesiteareshownas
grey spheres. The sulphate ion is shown as a stick model. b, Schematic
representation of the structure of yeast CPSF-100. The zinc atoms in the
CPSF-73 structure are shown for reference. See Supplementary Fig. 2 For
Schematic drawings of the metallo-b-lactamase domains of the two proteins.
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domain similar to the structure of canonical metallo-b-lactamases,
with a four-layered ab/ba architecture (see Supplementary Fig. 2a).
A close structural homologue of this domain is the L1 metallo-b-
lactamase from Stenotrophomonas maltophilia (see Supplementary
Fig. 2c)17, with a root mean squared (r.m.s.) distance of 3.3A˚for
167 equivalent Ca atoms, calculated using the program Dali18.
However, the sequence identity for these structurally equivalent resi-
dues is only 17%.
Despite the overall similarity to canonical metallo-b-lactamases,
the structure of this domain of CPSF-73 contains several distinctive
features.Mostremarkably,residues395–460,atthecarboxy-terminal
end of the expression construct (see Supplementary Fig. 1a), are also
partofthemetallo-b-lactamasedomain(Fig.1a).Theseresiduesadd
three strands (b13 to b15) to the central b-sandwich, and the last
residue of the structure is located next to the first residue (see
Supplementary Fig. 2a). These additional b-strands in the metallo-
b-lactamase fold are also seen in the structure of CPSF-100 (Fig. 1b,
Supplementary Fig. 2b; see below) and RNase Z (see Supplementary
Fig. 2d)19,20, indicating that they might be unique to RNA/DNA-
processing nucleases. This feature of the metallo-b-lactamase fold
is crucial for the activity of these proteins, as the loop after strand
b13providesoneoftheligands(motifC)tothezincionsintheactive
site(seeSupplementaryFig.2aandseebelow).Ther.m.s.distancefor
210 equivalent Ca atoms between CPSF-73and RNase Z is 2.6A˚, but
the sequence identity is only 18%. RNase Z is also distinct from
CPSF-73 in that it lacks the second domain (Fig. 1a, Supplemen-
tary Fig. 2d).
The second domain of CPSF-73 covers residues 209–394 (Supple-
mentary Fig. 1a), and contains a central, fully parallel, six-stranded
b-sheetthatissurroundedbyfivea-helicesonbothfaces(Fig.2a).In
addition, a small b-hairpin structure is on the surface of the domain.
This second domain can be considered as a cassette inserted into the
metallo-b-lactamase domain, between strand b12 and helix a5
(Fig. 1a). As it is related to the b-CASP motif identified earlier on
the basis of sequence analyses12, we have named it the b-CASP
domain (see Supplementary Fig. 1a).
The closest structural homologue of the b-CASP domain is the
nucleotide-binding fold (NBF, Supplementary Fig. 3), with a Z score
of 6 from Dali18. However, the b-CASP domain lacks the Walker A
motifforbindingnucleotidephosphategroups21(seeSupplementary
Information and Supplementary Fig. 3). Therefore, the b-CASP
domain seems to be a new example of the NBF superfold but is
unlikelytobindnucleotides.Thisdomainishighlyconservedamong
CPSF-73 proteins, and probably has important functions in regulat-
ing their activity (see below).
The overall structure of yeast CPSF-100 (Fig. 1b) is remarkably
similartothatofhumanCPSF-73,despitethelowdegreeofsequence
conservation between them (see Supplementary Fig. 1b). The r.m.s.
distance for 235 equivalent Ca atoms in the metallo-b-lactamase
domain (see Supplementary Fig. 2b) of the two structures is 2.2A˚
(with 21% sequence identity), and that for 154 equivalent Ca atoms
in the b-CASP domain is 2.3A˚(with 17% identity).
There are, however, significant differences in the structures of
CPSF-100 and CPSF-73 (see Supplementary Fig. 4). Most impor-
tantly, motifs for zinc binding in the metallo-b-lactamase domain
are missing in CPSF-100 (see Supplementary Fig. 2b), and therefore
this protein cannot bind zinc and is unlikely to possess nuclease
activity. In addition, the b-CASP domain of CPSF-100 (Fig. 2b) is
much larger than that of CPSF-73, but contains a highly flexible
segment(seeSupplementaryInformationandSupplementaryFig.5).
Clear electron density for two zinc ions (confirmed on the basis of
anomalous scattering data) was observed in the CPSF-73 active site
(seeSupplementaryFig.6a),regardlessofwhetherzincwaspresentin
the crystallization solution. Even in the absence of added zinc, the
zinc ions in the structure seem to have full occupancy as their tem-
perature factor values are comparable to those of their protein
ligands. This indicates that the active site of CPSF-73 might possess
extremely high affinity for zinc ions, making it impossible to remove
the zinc from the protein, consistent with the known resistance of 39
cleavage torelatively highconcentrations ofEDTA22.Twoadditional
zinc ions were observed on the surface of CPSF-73 in the crystal
grown in the presence of 0.5mM zinc (see Supplementary Infor-
mation and Supplementary Fig. 7).
The two zinc atoms in the active site are each bound in an octa-
hedral environment, with a hydroxide ion as one of the bridging
ligands (Fig. 3a, Supplementary Fig. 6b). The other bridging ligand
is the side-chain carboxylate oxygen atom of Asp179 from motif 4
(Fig.3a).His71,His73(motif2)andHis158(motif3)providethree
more ligands to the first zinc atom (Zn1), and Asp75, His76 (motif
2) and His418 (motif C, see below) are the ligands to Zn2 (Fig. 3a).
Finally, the two zinc ions are coordinated by oxygen atoms from a
sulphate ion (Fig. 3a), which was present at 300mM in the crystal-
lization solution and has clear electron density (Supplementary
Fig. 6a).
The structure of its active site strongly implies that CPSF-73 pos-
sesses hydrolase/nuclease activity. In canonical metallo-b-lacta-
mases, the hydroxide that bridges the two zinc ions is the
nucleophile for the hydrolysis reaction, and the substrate is directly
liganded to the zinc atoms23. In our structure, the hydroxide is
directly below the sulphate group (Fig. 3a), which is probably a good
mimic for the phosphate group of the pre-mRNA substrate at the
cleavage site. Therefore, the hydroxide is located at the perfect posi-
tion for an inline nucleophilic attack on the phosphate group to
initiate the nuclease reaction (Fig. 3a).
N
C
αA
αB
αC
αF
αG
βA
βB
βC
βD
βE
βG
βH
βI
288
209
300
a
N
C
αA
αB
αC
αD
αE
αF
αG
βA
βB
βC
βD
βE
βF
βG
βH
βI
626
422
276
388
397
219
268
661
b
Figure 2 | The b-CASP domain of CPSF-73 and CPSF-100. Schematic
drawings of the b-CASP domains of human CPSF-73 (a) and yeast
CPSF-100 (b).
158
71
73
75
76
396
Zn1
Zn2
2
5 (C)
179
4
204
418
B
A
3
a
160
84
86
88
89
225
Zn1
Zn2
2
5
Nucleophile
3
b
Figure 3 | The active site of CPSF-73. a, The zinc binding site for human
CPSF-73.Themotifsarelabelled,andthebridginghydroxideionisshownas
aredsphere.Ligandinginteractionsareindicatedbythinmagentalines,and
hydrogen-bonding interactions by thin red lines. The arrow indicates the
nucleophilic attack from the hydroxide ion. b, The zinc binding site in L1
metallo-b-lactamase17.
LETTERS
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Our analysis indicates that His396 (motif B, in the linker between
the b-CASP domain and the metallo-b-lactamase domain; see
Supplementary Fig. 2a) is the general acid for catalysis. This residue
ishydrogen-bondedtothesidechainofGlu204(motifA,lastresidue
ofstrandb12inthemetallo-b-lactamasedomain,seeSupplementary
Fig. 2a) as well as an oxygen atom of the sulphate (Fig. 3a). This
oxygen is located opposite to the direction of attack of the hydroxide
(Fig. 3a), and therefore probably represents the leaving group in the
hydrolysisreaction.Afterhydrolysis,theHis396sidechaincanfunc-
tion as the general acid to protonate the oxyanion in the reaction
product.ThispairofGlu-Hisresiduesisalsofoundinthestructureof
RNase Z, hydrogen-bonded to a phosphate group in the active site19.
In yeast, mutation of either of these residues is lethal13.
AlthoughanearliersequenceanalysisindicatedthatmotifBmight
be the best candidate for the zinc ligand in CPSF-73 (ref. 12), our
structure shows that the ligand is the His residue from motif C. This
residue is conserved among CPSF-73 proteins and its close homo-
logues(RC-68orInt11)24,25,butnotintheDNAnucleaseArtemis12,26
(see Supplementary Fig. 1b). Therefore, the Zn2 atom might have
a different coordination sphere in Artemis. The structure-based
sequence alignment also indicates a change in the assignment of
residues equivalent to motifs B and C in CPSF-100 (ref. 12; see
SupplementaryFig.1b),althoughneitherresidueisHisinCPSF-100.
The binding modes of the zinc ions in CPSF-73 (Fig. 3a) are
similar to that in RNase Z but distinct from those in canonical
metallo-b-lactamases (Fig. 3b; see Supplementary Information).
Another important difference between CPSF-73 and other metallo-
b-lactamases is the presence of the b-CASP domain. Although most
metallo-b-lactamases (Supplementary Fig. 2c), and RNase Z
(Supplementary Fig. 2d), have an open zinc-binding site, the active
site in CPSF-73 is located deep in the interface between the metallo-
b-lactamase and the b-CASP domains (Fig. 1a). In fact, the b-CASP
domainseverelyrestrictsaccesstotheactivesite(SupplementaryFig.
8), and the scissile phosphate group probably cannot reach the zinc
ions in the current structure. This indicates that a conformational
change in the enzyme might be required for pre-mRNA binding (see
Supplementary Information).
To show that CPSF-73 has endonuclease activity in vitro and to
provide direct evidence for its role in 39-end processing, we assessed
the nuclease activity of the bacterially expressed CPSF-73, together
with a derivative containing a double mutation (D75K/H76A) that
destroys two of the zinc ligands in motif 2. We used a 59-capped,
uniformly labelled RNA containing the SV40 late polyadenylation
site (SVL) as the substrate13,27. Using standard conditions for 39 pro-
cessingreactions,therewasevidenceforweakcleavagewiththewild-
type but not the mutant derivative (Supplementary Fig. 10a); a simi-
larly prepared CPSF-100 derivative was also inactive (results not
shown). Thisinduced us toattempt tooptimize conditions to obtain
more efficient cleavage.
Among the possibilities tested to increase nuclease activity was
preincubation with divalent cations. Our structural studies showed
that there are binding sites for additional Zn21on the surface (see
Supplementary Fig. 7), and it is possible that (non-specific) binding
of divalent cations to the protein surface could affect its activity.
Preincubation withZn21andallother divalentcationstested,except
Ca21, resulted in precipitation of the enzyme. Strikingly, after pre-
incubation with Ca21, the wild-type enzyme degraded the SVL sub-
strate almost entirely to oligonucleotides, whereas the mutant was
still inactive (Fig. 4a). Asimilar-sized RNA fromadenovirus was also
entirely degraded by wild-type but not mutant CPSF-73 (Fig. 4a).
Preincubation with Ca21, although required for enzyme activation
(results not shown), was unnecessary for catalysis. It was diluted to
50mM in the reactions and nuclease activity was not sensitive to the
addition of 5mM EGTA (results not shown). Furthermore, there is
no evidence that Ca21is required in authentic 39 processing, indi-
cating that its activating function is probably performed by other
components of the polyadenylation machinery. At lower CPSF-73
enzyme levels, partially degraded, higher-molecular-weight inter-
mediates were seen (see Supplementary Fig. 10b). Significant cleav-
age required relatively high concentrations (10ngml–1or ,200nM),
which we attribute to only a small fraction of the enzyme being
activated by the preincubation.
AlthoughourdataareconsistentwithpurifiedCPSF-73possessing
non-specific endonuclease activity, they do not conclusively rule out
that it might have exonuclease activity, as previously suggested14.
Indeed, there is evidence that Artemis has exonuclease activity28.
Althoughthepresenceofa59capontheRNAsanalysedabovelargely
excludes 59R39 exonuclease activity, we nonetheless compared the
ability of CPSF-73 to degrade 59 and 39 end-labelled RNA (Fig. 4b).
The two RNAs were degraded with comparable kinetics and gener-
ated similar patterns of intermediates, consistent only with endonu-
clease activity.
Theseresults,togetherwithpreviousdata13,14,establishCPSF-73as
a strong, sequence-independent endonuclease that functions with
specificity in 39-end processing of both polyadenylated and histone
pre-mRNAs by interacting with other factors. In addition, our data
indicate that the close sequence homologue of CPSF-73, RC-68 or
Int11, is probably the endonuclease in the 39-end processing of small
nuclear RNAs24.
METHODS
Proteinexpression,purificationandcrystallization.Residues1–460ofhuman
CPSF-73 were sub-cloned into the pET28a vector (Novagen) and overexpressed
in Escherichia coli at 20uC. The soluble protein was purified by nickel–agarose
a
SV40
pre-mRNA
(5’ capped)

Cleaved
products
Ad-L3
pre-mRNA
(5’ capped)

Cleaved
products
240
160
110
67
M
Wild type
30
Mutant
0 30
Pre Wild type
Mutant
CPSF73
(1 µg)
1 2 3 4 5
6 7 8
34
M
b
Cleaved
products
Cleaved
products
SV40 pre-mRNA
(3’ end labelled)
SV40 pre-mRNA
(5’ end labelled)
Pre 5 10 15 20 30 30
Pre 5 10 15 20 30 30
Time (min)
Time (min)
Mutant
Mutant
8 9
CPSF73
(500 ng)
CPSF73
(500 ng)
M
1 2 3 4 5 6 7 M
240
160
110
67
34
240
160
110
67
34
10 11 12 13 14
Pre 0
CPSF73
(1 µg)
Time (min)
160
110
67
34
190
Wild type
Wild type
Figure 4 | CPSF-73possessesendoribonucleaseactivity. a,Cleavageof59-
cappedSV40latepre-mRNA(SVL)wasperformedwithwild-type(lane3)or
mutant (lane 5) CPSF-73 at 30uC for 30min. Incubations for 0min are
shown in lanes 2 and 4. Cleavage of 59-capped adenovirus L3 pre-mRNA
(Ad-L3)isshowninlanes7and8.PrecursorRNAs(Pre)areshowninlanes1
and 6. M, MspI-digested pBR322DNA markers. b, Time-course analysis of
RNA cleavage by CPSF-73. Cleavage of 59 (lanes 2–6) and 39 (lanes 9–13)
end-labelled SVL pre-mRNAs was performed with wild-type CPSF-73
(500ng); time points were 5, 10, 15, 20 and 30min. Mutant CPSF-73
(500ng) did not exhibit RNase activity (lanes 7 and 14).
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affinity chromatography and gel-filtration chromatography. Yeast CPSF-100
(Ydh1, residues 1–720) was expressed and purified by nickel–agarose affinity
chromatography, anion exchange and gel-filtration chromatography.
CrystalsofhumanCPSF-73wereobtainedatroomtemperaturebythesitting-
drop vapour diffusion method. In an attempt to increase the occupancy of zinc
ions, some crystals were grown from a reservoir solution that also contained
0.5mM ZnCl2. Crystals of yeast CPSF-100 were obtained at 4uC by the same
protocol. A solution that had been infected by a fungus was crucial for the
crystallization of this protein16. The crystals were frozen in liquid propane for
data collection at 100K.
Data collection, structure determination and refinement. X-ray diffraction
data were collected on an ADSC CCD at the X4A beamline of Brookhaven
National Laboratory. The diffraction data were collected at the zinc absorption
peak for crystals of CPSF-73, and at the gold absorption peak for a KAu(CN)2
derivativeof CPSF-100.Thestructureof CPSF-73 wasdeterminedby thesingle-
wavelengthanomalousdiffractionmethod29,usingtheanomaloussignalofzinc.
The structure of CPSF-100 was determined by the single-isomorphous replace-
ment method, supplemented with anomalous diffraction. The crystallographic
information are summarized in Supplemental Table 2. Figs 1–3 were produced
with Ribbons30.
Pre-mRNA 39-end cleavage assay. CPSF-73 was pre-incubated with 5mM
CaCl2at 37uC for 30min. Cleavage assays were carried out in reaction mixture
(10ml)containing,1nglabelledRNAsubstrates,10mMHEPES(pH7.9),10%
(v/v) glycerol, 50mM KCl, 0.25mM DTT, 0.25mM PMSF, 0.125mM EDTA,
50mM CaCl2, RNase inhibitor (2 units), 500ng BSA and indicated amounts of
recombinantCPSF-73.CleavedRNAswereisolatedandfractionatedon6%urea
PAGE. The data were analysed by Phospho-Imager.
Received 18 July; accepted 20 October 2006.
Published online 26 November 2006.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank K. Ryan for discussions; B. Tweel for characterizing
the fungus; R. Abramowitz, J. Schwanof and X. Yang for setting up the X4A
beamline at the NSLS; and J. Khan and Y. Shen for help with data collection at the
synchrotron. This research is supported in part by grants from the National
Institutes of Health.
Author Information The atomic coordinates have been deposited at the Protein
Data Bank (accession numbers 2I7T, 2I7V and 2I7X). Reprints and permissions
information is available at www.nature.com/reprints. The authors declare no
competing financial interests. Correspondence and requests for materials should
be addressed to L.T. (ltong@columbia.edu).
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