Analysis of a noncanonical poly(A) site
reveals a tripartite mechanism
for vertebrate poly(A) site recognition
Krishnan Venkataraman, Kirk M. Brown,1and Gregory M. Gilmartin2
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA
At least half of all human pre-mRNAs are subject to alternative 3? processing that may modulate both the
coding capacity of the message and the array of post-transcriptional regulatory elements embedded within the
3? UTR. Vertebrate poly(A) site selection appears to rely primarily on the binding of CPSF to an A(A/U)UAAA
hexamer upstream of the cleavage site and CstF to a downstream GU-rich element. At least one-quarter of all
human poly(A) sites, however, lack the A(A/U)UAAA motif. We report that sequence-specific RNA binding of
the human 3? processing factor CFImcan function as a primary determinant of poly(A) site recognition in the
absence of the A(A/U)UAAA motif. CFImis sufficient to direct sequence-specific, A(A/U)UAAA-independent
poly(A) addition in vitro through the recruitment of the CPSF subunit hFip1 and poly(A) polymerase to the
RNA substrate. ChIP analysis indicates that CFImis recruited to the transcription unit, along with CPSF and
CstF, during the initial stages of transcription, supporting a direct role for CFImin poly(A) site recognition.
The recognition of three distinct sequence elements by CFIm, CPSF, and CstF suggests that vertebrate poly(A)
site definition is mechanistically more similar to that of yeast and plants than anticipated.
[Keywords: mRNA 3? processing; polyadenylation; poly(A) site recognition]
Received January 14, 2005; revised version accepted April 21, 2005.
The process of mRNA 3? end formation is not simply a
perfunctory step in eukaryotic gene expression. At least
one-half of all human genes are subject to alternative 3?
processing (Iseli et al. 2002), the consequences of which
may impact the protein coding capacity of the message,
as well as its localization, translation efficiency, and sta-
bility (Edwalds-Gilbert et al. 1997). Moreover, poly(A)
site selection may be modulated in a developmental and
tissue-specific manner. In addition, pre-mRNA 3? pro-
cessing contributes directly to transcription termination
(Zorio and Bentley 2004), pre-mRNA splicing (Proudfoot
et al. 2002), and mRNA export (Hammell et al. 2002; Lei
and Silver 2002). While the processing of constitutive
poly(A) sites has been examined in considerable detail,
the fundamental mechanisms responsible for the regula-
tion of alternative poly(A) site selection have yet to be
fully elucidated (Barabino and Keller 1999).
The processing of the majority of human poly(A) sites
involves the recognition of an AAUAAA or AUUAAA
hexamer by CPSF, coupled with the binding of CstF to a
GU-rich downstream element (DSE) (Zhao et al. 1999).
The binding of CPSF and CstF appears to be sufficient, at
least in vitro, to direct the assembly of a 3? processing
complex composed of at least 14 different proteins. In
vivo, however, the hexamer and DSE alone are unlikely
to suffice for poly(A) site definition. The recognition of
an authentic poly(A) site within a nascent RNA in vivo
appears to rely on the “biosynthetic context” provided
by the transcription elongation complex (Proudfoot
2004). At least nine 3? processing proteins are recruited
to the transcription complex, at least in part through
interactions with the C-terminal domain (CTD) of the
largest subunit of RNA polymerase II (RNAPII) (Calvo
and Manley 2003). The colocalization of 3? processing
factors, along with capping enzymes and spliceosome
components, to the transcription elongation complex, al-
lows for the cooperative interaction of these processing
machineries within an “mRNA factory” (Zorio and
Cotranscriptional recognition of a poly(A) site pro-
vides an elegant mechanism for the identification of a
processing site demarcated by a limited set of sequence
motifs. Yet the mechanisms that regulate the selection
of alternative poly(A) sites within a pre-mRNA, or allow
for the recognition of poly(A) sites that lack the canoni-
cal A(A/U)UAAA motif, are poorly understood. Se-
quences that function to enhance the efficiency of 3?
processing have been identified upstream of several ca-
nonical poly(A) sites (Zhao et al. 1999). Such elements
might contribute to the regulation of poly(A) site selec-
1Present address: Department of Biochemistry and Molecular Pharma-
cology, University of Massachusetts, Worcester, MA 01605, USA.
E-MAIL firstname.lastname@example.org; FAX (802) 656-8749.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/
GENES & DEVELOPMENT 19:1315–1327 © 2005 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/05; www.genesdev.org 1315
tion, although the molecular mechanisms by which they
function are largely unclear. In this work, we have iden-
tified a mechanism by which the 3? processing factor
CFImcontributes to poly(A) site recognition and, specifi-
cally, to the recognition of a human poly(A) site that
lacks the canonical A(A/U)UAAA hexamer.
CFImis an essential, heterodimeric pre-mRNA 3? pro-
cessing factor, unique to metazoans, composed of a small
subunit of 25 kDa and a large subunit of 59, 68, or 72 kDa
(Ruegsegger et al. 1996). The human CFIm59- and 68-
kDa subunits are encoded by paralogous genes, while the
nature of the 72-kDa protein remains unclear. The CFIm
59- and 68-kDa proteins possess an N-terminal RNP-
type RNA-binding domain (RBD), a central proline-rich
domain, and a C-terminal RS-like alternating charge
domain—a structure similar to that of the SR family of
proteins that function in basal and regulated pre-mRNA
splicing (Graveley 2000). The structure of the CFImlarge
subunit, along with the identification of both the 25- and
68-kDa subunits as components of human spliceosomes
(Rappsilber et al. 2002; Zhou et al. 2002), suggests a po-
tential role for CFImin the coordination of 3? processing
and pre-mRNA splicing. Such a role is supported by
the specific interaction of the 25-kDa subunit with the
70-kDa protein of U1 snRNP (Awasthi and Alwine 2003)
and the interaction of the 68-kDa subunit with the
SR proteins Srp20, 9G8, and hTra-2? (Dettwiler et al.
2004). The CFIm68/25-kDa heterodimer has been shown
to be sufficient to reconstitute CFImfunction in vitro
(Ruegsegger et al. 1998), and SELEX analysis has indi-
cated that the 68/25-kDa heterodimer preferentially
binds the sequence UGUAN (N = A > U ? C/G) (Brown
and Gilmartin 2003). Ruegsegger et al. (1998) demon-
strated that CFImcan function to facilitate pre-mRNA
3? processing complex assembly and to enhance the rate
and overall efficiency of poly(A) site cleavage in vitro.
In vitro experiments have also shown that CFImcan
function to autoregulate the 3? processing of the pre-
mRNA encoding the CFIm68-kDa subunit through
the binding of a set of UGUAA elements that flank and
overlap the AAUAAA hexamer (Brown and Gilmartin
In this report, we demonstrate that CFImhas a funda-
mental role in poly(A) site recognition. Sequence-spe-
cific RNA binding of CFImwas found to function as the
primary determinant for the recognition of a human
poly(A) site lacking the A(A/U)UAAA hexamer. Further-
more, we show that CFImcan function to direct se-
quence-specific, A(A/U)UAAA-independent poly(A) ad-
dition in vitro, through its ability to recruit the CPSF
subunit hFip1 and poly(A) polymerase to the RNA sub-
strate. A direct role for CFImin poly(A) site recognition
is supported by chromatin immunoprecipitation (ChIP)
analysis indicating that CFImis recruited to the tran-
scription unit, along with CPSF and CstF, during the
initial stages of transcription. Taken together, the data
indicate that three sequence-specific RNA-binding fac-
tors participate in vertebrate poly(A) site recognition,
CFIm, CPSF, and CstF, and suggest that the mechanisms
of poly(A) site recognition in yeast, plants, and verte-
brates are more similar than previously appreciated
(Graber et al. 1999; Proudfoot and O’Sullivan 2002).
PAPOLA and PAPOLG gene paralogs as a model
system for the analysis of poly(A) site recognition
EST database analysis has indicated that over a quarter of
all human transcripts are processed at noncanonical
poly(A) sites that deviate in one or more positions from
the A(A/U)AAA consensus (Beaudoing et al. 2000). Mu-
tagenesis studies have indicated that such noncanonical
sites are processed poorly, if at all, in vitro (Sheets et al.
1990). To begin to understand the mechanisms that al-
low for the recognition of noncanonical poly(A) sites, we
chose to examine the noncanonical poly(A) site of the
human PAPOLG gene (also referred to as neo-poly(A)
polymerase [Topalian et al. 2001]) in parallel with the
canonical poly(A) site of its paralog, PAPOLA. The pri-
mary poly(A) site of PAPOLG (as determined by EST
analysis) does not possess a sequence with more than a
4-nt match to the A(A/U)UAAA motif. The PAPOLA
and PAPOLG genes, which encode poly(A) polymerase ?
(PAP?) and poly(A) polymerase ? (PAP?), respectively,
derive from gene duplication, as evidenced by a common
intron/exon structure. PAP? and PAP? have an overall
amino acid sequence similarity of 71%.
As illustrated in Figure 1, the 3? ends of PAPOLA and
PAPOLG are strikingly distinct and highly conserved
among vertebrates. The conserved sequences upstream
of each poly(A) site encompass multiple copies of po-
tential CFIm-binding sites of the form UGUAN (N = A >
U ? C/G). As denoted by the boxed sequences, the hu-
man PAPOLA poly(A) site contains six UGUAN ele-
ments within 131 nucleotides (nt) upstream of the cleav-
age site. The PAPOLG poly(A) site contains seven
UGUAN elements within 119 nt upstream of the cleav-
age site in a pattern that is clearly distinct from that of
the PAPOLA poly(A) site. The PAPOLG gene contains a
single UGUAN element within 130 nt downstream of
the cleavage site, while the PAPOLA gene has none. The
following experiments were undertaken to test the hy-
pothesis that sequence-specific binding of CFImto the
UGUAN elements of PAPOLA and PAPOLG contrib-
utes to poly(A) site recognition and processing.
CFIm-binding sites enhance 3? processing at the
canonical PAPOLA poly(A) site in vitro
We first addressed the contribution of the four conserved
UGUAN elements immediately upstream of the PAPOLA
AAUAAA hexamer to poly(A) site function. Single G-
to-C point mutations were introduced into the two
UGUAN elements distal to the hexamer (PAP??1), the
two UGUAN elements proximal to the hexamer
(PAP??2), or the combination of all four elements
(PAP??1/2) (Fig. 2A). In addition, a single U-to-G point
mutation was introduced into the AAUAAA hexamer
(PAP??hex). As illustrated in Figure 2B, lane 1, the wild-
type PAPOLA poly(A) site was efficiently cleaved in
Venkataraman et al.
1316 GENES & DEVELOPMENT
were resolved on a 10% polyacrylamide–SDS gel and subjected
to Western analysis with either the His-probe antibody or the
CPSF160K anti-peptide antibody. As a positive control for each
Western, a 5% aliquot of the starting material was also run.
HRP-conjugated secondary antibodies were detected by ECL
ChIPs were performed according to the Farnham lab protocol
chips.html). Chromatin was prepared from four confluent T75
flasks of HeLa cells. The antibodies used were as follows:
RNAPII CTD monoclonal antibodies H5, H14, and 8WG16
were obtained form BAbCO; RuvBL1 [NMP 238 (N-15)],
RbAp46 (N-19), Mta1L1 (C-20), TFIIH 62 (Q-19), and CPSF 160
[CPSF1 (E-20)] were obtained from Santa Cruz Biotechnology.
The CFIm25 antibody was an affinity-purified rabbit anti-pep-
tide antibody raised against the sequence YTFGTKEPLYEK
DSS. Anti-CstF 64 (3A7) was a kind gift of C. MacDonald (De-
partment of Cell Biology and Biochemistry, Texas Tech Univer-
sity, Health Sciences Center, Lubbock, TX). Oligonucleotide
primer sequences for PCR amplification are available upon re-
We thank the Vermont Cancer Center DNA Analysis Facility
for support in DNA sequencing and molecular imaging.
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Poly(A) site recognition by CFIm
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