From the Department of Cell Biology and Biochemistry
and ¶ Southwest Cancer Center at University Medical Center, Texas
Tech University Health Sciences Center, Lubbock, Texas 79430 and
§ Mouse Cancer Genetics Program, NCI, Frederick Cancer
Research and Development Center, Frederick, Maryland 21702
Received for publication, October 4, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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Many mRNAs in male germ cells lack the
canonical AAUAAA but are normally polyadenylated (Wallace, A. M.,
Dass, B., Ravnik, S. E., Tonk, V., Jenkins, N. A., Gilbert,
D. J., Copeland, N. G., and MacDonald, C. C. (1999)
Proc. Natl. Acad Sci. U. S. A. 96, 6763-6768).
Previously, we demonstrated the presence of two distinct forms of the
Mr 64,000 protein of the cleavage stimulation factor (CstF-64) in mouse male germ cells and in brain, a somatic Mr 64,000 form and a variant
Mr 70,000 form. The variant form was specific
to meiotic and postmeiotic germ cells. We localized the gene for the
somatic CstF-64 to the X chromosome, which would be inactivated during
male meiosis. This suggested that the variant CstF-64 was an autosomal
homolog activated during that time. We have named the variant
form " Polyadenylation is the process of eukaryotic mRNA processing
in which 3' end cleavage occurs, followed by the addition of as many as
250 adenosine residues (1, 2). Messenger RNA polyadenylation is
important for cellular processes such as transcription termination
(3-6), splicing (7, 8), mRNA transport (9), translation (10-12),
and mRNA stability (13, 14). Polyadenylation requires at least five
protein complexes, including the cleavage and polyadenylation
specificity factor (CPSF),1
the cleavage stimulation factor (CstF), two cleavage factors (CFI and
CFII), and the poly(A) polymerase (1, 2). Other factors, including the
poly(A)-binding protein II (which mediates poly(A) tail length) (15),
the U1A small nuclear ribonucleoprotein protein (which interacts with
both CPSF and the poly(A) polymerase) (16, 17), and DSEF-1 (which binds
G-rich auxiliary elements) (18), also contribute to efficient polyadenylation.
In somatic cells, the sequence AAUAAA is required for accurate and
efficient cleavage and polyadenylation, and must reside within 30 nucleotides upstream of the cleavage site in the pre-mRNA (19-23).
Ninety percent of all sequenced genes have a canonical AAUAAA sequence
(1). Recently, we noted that many mRNAs expressed in male germ
cells lack AAUAAA, yet are efficiently polyadenylated (24).2 To explain proper
polyadenylation of mRNAs lacking AAUAAA signals in germ cells, we
hypothesized the presence of a testis-specific variant of a known
polyadenylation protein. In examining the expression of the
Mr 64,000 protein of CstF (CstF-64 (25)), we
discovered a variant form of this protein in germ cells that is a
candidate for this function (24). CstF-64 interacts directly with
another subunit of the complex, CstF-77 (26), linking it to CstF-50 to
form the CstF trimer (25, 27). CstF-64 is the RNA-binding component of
CstF that binds to U- or GU-rich elements downstream of the cleavage
site during polyadenylation (28, 29). As such, CstF-64 is essential for
polyadenylation (25), for cell viability (30), and has been shown to
interact with RNA polymerase II, coupling polyadenylation and
transcription (31).
In our previous work, we showed that in mice the principal
Mr 64,000 form of CstF-64 is found in all
somatic cells, and in premeiotic and postmeiotic (but not meiotic) germ
cells (24). This form of CstF-64 is recognized by the monoclonal
antibody 3A7 (25). A variant Mr 70,000 form of
CstF-64 is found in meiotic and postmeiotic cells in the testis and to
a smaller extent in brain. We have named this form of CstF-64 " The gene for CstF-64 has recently been renamed CSTF2 in human and
Cstf2 in mouse by authorities of The Genome Data
base (32, 33), and we will adhere to that nomenclature here.
Cstf2 or CSTF2 mapped to the X chromosome in either
mouse or human (24), supporting the hypothesis that inactivation of the
X chromosome during male meiosis results in inactivation of the
X-linked Cstf2 in mouse spermatocytes. This
inactivation of the essential Cstf2 led us to
hypothesize the requirement of an alternative CstF-64 protein in
spermatocytes, which we proposed was encoded by an autosomal gene.
We describe here the cloning and characterization of m Antibody Interaction Mapping--
Full-length cDNA encoding
human CstF-64 (34) was cloned in frame into the pGEX2T vector (Amersham
Pharmacia Biotech). 3' end truncations were made by limited enzymatic
digestion of the above construct to obtain versions that terminated at
amino acids 531, 425, and 325. A fourth construct was made by
introducing a 290-bp BamHI fragment of CstF-64 encoding
amino acids 316-412 into pGEX2T. Constructs were introduced into
Escherichia coli DH5 Complementary DNA Isolation--
An adult mouse pachytene
spermatocyte cDNA library in Uni-Zap XR vector (35) was screened
using the 290-bp BamHI cDNA fragment of CstF-64 encoding
amino acids 316-412. Filters were denatured in 0.5 M NaOH,
1.5 M NaCl, neutralized in 0.5 M Tris-HCl, pH
7.4, 1.5 M NaCl, and hybridized with
[ In Vitro Transcription and Translation--
Polypeptides
corresponding to human (hCstF-64 (34)), mouse (mCstF-64 (36)), and the
mouse Peptide Mapping by Limited Proteolysis--
Radiolabeled
proteins corresponding to mCstF-64 and m
Radiolabeled Interspecific Mouse Backcross Mapping--
Interspecific
backcross progeny were generated by mating (C57BL/6J × Mus
spretus) F1 females and C57BL/6J males as described (24, 40). A
total of 205 N2 mice were used to map the Cstf2t locus. DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, and Southern blot analysis were performed as described
(41). The probe, a 514-bp DraI/XhoI fragment of
m Recombinant Protein Purification--
RNA-binding domains (RBDs)
from human (hRBD) and mouse CstF-64 (mRBD) and mouse UV Cross-linking--
0.3 µg each of hRBD, mRBD, m Epitopes for the Monoclonal Antibodies 3A7 and 6A9 Map to
Two Distinct Regions of CstF-64 cDNA CstF-64," and we describe here the cloning and
characterization of the mouse
CstF-64 cDNA, which maps to
chromosome 19. The mouse
CstF-64 protein fits the criteria of
the variant CstF-64, including antibody reactivity, size, germ cell
expression, and a common proteolytic digest pattern with
CstF-64
from testis. Features of m
CstF-64 that might allow it to promote
the germ cell pattern of polyadenylation include a Pro
Ser
substitution in the RNA-binding domain and significant changes in the
region that interacts with CstF-77.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CstF-64." In mice,
CstF-64 is recognized by the monoclonal
antibody 6A9 (24, 25).
CstF-64, a
cDNA that encodes
CstF-64 from mouse pachytene spermatocytes. The protein encoded by m
CstF-64 is similar to other isoforms of
CstF-64 from several species and retains protein motifs such as the RNA
binding, MEAR(A/G) repeats, and C-terminal domains. m
CstF-64 is
capable of encoding a protein of about Mr 70,000 that is recognized by the 6A9 monoclonal antibody. Partial protease digestion shows that peptide fragments from the protein encoded by
m
CstF-64 are identical to those of the variant CstF-64 protein obtained from mouse testis. We also show that the RNA-binding domain of
the protein encoded by m
CstF-64 is functional in vitro. Finally, Cstf2t, the gene for m
CstF-64 maps to an
autosome, chromosome 19 in mouse. These data suggest that m
CstF-64
encodes the variant form of CstF-64 seen in meiotic and postmeiotic
male germ cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells by transformation, grown to
mid-logarithmic phase, and induced for 3 h at 37 °C with 1 mM isopropyl
-D-thiogalactoside. Bacterial
extracts were prepared in SDS-PAGE loading buffer by sonication and
boiling and prepared for immunoblotting with either the 3A7 or 6A9
monoclonal antibody as described previously (24).
-32P]dCTP-labeled cDNA probe in hybridization
solution (10× SSC, 0.05 M sodium phosphate, pH 6.5, 5×
Denhardt's solution, 0.1% Na4P2O7, 0.5% SDS, 0.05 mg/ml
salmon sperm DNA) overnight at 65 °C. Filters were washed at a final
stringency of 2× SSC, 0.1% SDS at 65 °C, and exposed to film at
80 °C with an intensifying screen. Positive plaques were purified
by two additional rounds of screening. Plasmid rescue into pBluescript
SK-was according to the manufacturer's directions (Stratagene, La
Jolla, CA). Of two million plaques screened initially, 24 hybridized to
the probe, all of which represented the same mRNA transcript (see
"Results"). The longest clone (3612 bp) was designated m
CstF-64
and was sequenced by a combination of primer walking and subcloning,
using the Sequenase 2.0 kit (U. S. Biochemical Corp.). Sequences were
aligned and grouped into contigs using the SeqMan analysis program (DNAStar).
CstF-64 (m
CstF-64) cDNAs were prepared in
vitro using the T3 TNT Coupled Reticulocyte Lysate System
(Promega, Madison, WI). Products from the transcription/translation reactions were separated on a 10% SDS-PAGE and immunoblotted with either the 3A7 or 6A9 monoclonal antibody (24).
CstF-64 were made in
vitro using the T3 TNT reticulocyte lysate system (Promega) with
[35S]methionine (PerkinElmer Life Sciences). Translated
products were separated on 10% SDS-PAGE, and the band corresponding to full-length m
CstF-64 protein was excised and eluted overnight at
4 °C in Cleveland buffer (0.125 M Tris-HCl, pH 6.8, 0.5% SDS, 1% glycerol, 0.0001% bromphenol blue (34, 37)).
CstF-64 was made in vivo by incorporation of
[35S]methionine during short term culture of mouse
seminiferous tubules. Testes from 8 CD-1 mice (Charles River Breeding
Laboratories) were decapsulated and washed several times in cold PBS to
remove interstitial cells. Tubules were then washed in prewarmed DMEM lacking methionine (Cellgro, Mediatech, Inc) followed by incubation in
DMEM containing 10 mM methionine, 2 mM
glutamine, and 1.25 mCi/ml Tran35S-label (ICN) for 7 h
at 32 °C (38). Following incubation, tubules were washed in DMEM,
resuspended in RIPA (150 mM NaCl, 1% Nonidet P-40, 0.5%
deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0), and sonicated on ice. After preclearing, samples were immunoprecipitated at 4 °C
using the 6A9 monoclonal antibody and protein A-Sepharose beads as
described (29). Following immunoprecipitation, beads were washed in
RIPA, boiled in Laemmli buffer (39), and digested with 0.1 or 1.0 µg
of V8 protease (Sigma) in the presence of 5 µg of bovine IgG protein
(Bio-Rad) for 30 min at 37 °C (34, 37). Polypeptide fragments were
separated by 15% SDS-PAGE, followed by fluorography. The image was
captured on x-ray film exposed in the presence of an intensifying
screen at
80 °C for the times indicated in Fig. 3.
CstF-64 from the 3'-UTR, was labeled with
[
-32P]dCTP using a nick translation primed labeling
kit (Roche Molecular Biochemicals); washing was done to a final
stringency of 0.8× SSCP (120 mM NaCl, 5 mM sodium citrate, 20 mM sodium phosphate, pH
6.8), 0.1% SDS at 65 °C. A fragment of 0.5 kb was detected in
TaqI-digested C57BL/6J DNA, and a fragment of 1.8 kb was
detected in TaqI-digested M. spretus DNA. The
presence or absence of the 1.8-kb TaqI M. spretus-specific fragment was followed in backcross mice. A
description of the probes and RFLPs for the loci linked to
Cstf2t including Gnaq and Fas
has been reported previously (42, 43). Recombination distances were
calculated using Map Manager, version 2.6.5 (Roswell Park Cancer
Institute, Buffalo, NY). Gene order was determined by minimizing the
number of recombination events required to explain the allele
distribution patterns.
CstF-64
(m
RBD) were prepared as fusion polypeptides with N-terminal
hexahistidine tags. Construction of the plasmid hRBD (formerly known as
rHis64
247) was described previously (34). The RNA-binding domains of
mCstF-64 (nucleotides 53-897) and m
CstF-64 (nucleotides 77-598)
were cloned in frame with the hexahistidine tag of the pQE9 vector
(Qiagen, Valencia, CA) to make mRBD and m
RBD, respectively. hRBD,
mRBD, and m
RBD plasmid DNAs were transformed into UltraMAXDH5
-FT
cells (Life Technologies, Inc.), grown to mid-logarithmic phase, and
induced at 37 °C for 3 h by the addition of isopropyl
-D-thiogalactoside to 1.5 mM. His-tagged
recombinant proteins were isolated as described (34) and dialyzed
against buffer D (20 mM HEPES, pH 7.9, 0.1 M
KCl, 0.2 mM EDTA, 1.5 mM MgCl2,
10% glycerol, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride (44)) overnight at 4 °C. Recombinant
proteins were quantified by comparison to bovine serum albumin
standards after staining of 12.5% SDS-PAGE gels with Coomassie
Brilliant Blue R-250.
RBD, or
bovine IgG (Bio-Rad) was mixed with 32P-labeled SVL
substrate (3 × 104 cpm (45)) in buffer D for 30 min
at 30 °C. Reaction mixtures were exposed to 107
µJ/cm2 of ultraviolet light in a CL-1000 Ultraviolet
Cross-linker (Ultraviolet Products, Upland, CA). Control reactions were
processed without exposure to UV. Reaction mixtures were incubated with
10 units of RNaseONE (Promega, Madison, WI) at 37 °C for 15 min.
SDS-PAGE loading buffer was added, and the samples were boiled and
RNA-cross-linked polypeptides separated on 12.5% SDS-PAGE. The gel was
stained with Coomassie Blue to ensure equal loading of the recombinant proteins, destained, dried, and subjected to autoradiography at
80 °C with an intensifying screen.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The monoclonal
antibodies 3A7 and 6A9, obtained by using human CstF purified from HeLa
cells, can distinguish the somatic (3A7) and variant (6A9) forms of
CstF-64 in mice (24). In human, however, both antibodies recognize the somatic form of CstF-64 ((25) see Fig. 2). Therefore, to map the recognition sites of each antibody, polypeptides corresponding to
different regions of human CstF-64 were expressed as fusions to
glutathione S-transferase in bacteria and immunoblotted with either the 3A7 or 6A9 antibody (data not shown, summarized in Figs.
1 and 6).
View larger version (43K):
[in a new window]
Fig. 1.
Comparison of
m CstF-64 protein sequence to known CstF-64
homologs. A, protein sequence alignment of human
CstF-64 (top line) and m
CstF-64 (bottom line).
Alignment was by the ClustalV method (61). Numbering is according to
the m
CstF-64 sequence. Amino acids that differ from the human
CstF-64 are boxed. Significant features (RBD, 17-92; MEARA
repeats, 425-464; C-terminal domain, 589-630) are boxed in
gray. Inserted segments in m
CstF-64 relative to hCstF-64
are indicated by a black overline. The region thought to
interact with CstF-77 (26) is indicated by a gray overline.
Boxed segments of the human sequence represent the regions
of interaction for the 6A9 (332) and 3A7 (441) antibodies.
B, sequence identities (in percent) of known CstF-64
homologs. Shown are sequences from human (hCstF-64 (34)), mouse
(mCstF-64 (36)), mouse
CstF-64 (m
CstF-64), human brain (KIAA0689
(48)), Xenopus laevis (xCstF-64 (51)), Caenorhabditis
elegans (ceCstF-64 (52)), Drosophila melanogaster
(dCstF-64 (50)), and Saccharomyces cerevisiae (yRNA15 (27)).
Pairwise identities were determined from the alignments as above.
C, tree diagram showing the relative similarities of the
human, mouse, Xenopus, Caenorhabditis, Drosophila, and yeast
homologs of CstF-64.
By using this approach, we determined that the region where the 3A7
antibody interacted with CstF-64 lay between amino acids 426 and 531 (441-583 in Fig. 1). Similarly, the region of interaction for the 6A9 antibody lie on a BamHI fragment between amino
acids 316 and 412 (333-427 in Fig. 1). Since CstF-64 was
recognized by the 6A9 antibody but not the 3A7 antibody (24), we chose to screen a mouse pachytene spermatocyte cDNA library with the BamHI fragment of the human CstF-64 that includes the 6A9
epitope to isolate its cDNA.
Isolation of a cDNA for CstF-64 from Mouse Pachytene
Spermatocytes--
An adult mouse pachytene spermatocyte library was
the kind gift of John McCarrey (Southwest Foundation for Biomedical
Research, San Antonio, TX (35)). Our previous results suggested that
cells during this period of spermatogenesis expressed
CstF-64
exclusively of the somatic CstF-64 (24). Two million plaques were
screened using the 290-bp BamHI cDNA fragment of human
CstF-64 that was described above (see Fig. 1). Twenty four positive
clones were identified and rescued into pBluescript SK
. Each of these
plasmids represented nearly identical transcripts that differed in
length at the 5' ends. None of the cDNAs represented the mouse
somatic CstF-64 (data not shown, see Ref. 36). The plasmid containing the longest insert was designated m
CstF-64 (GenBankTM
accession number AF322194) and was chosen for further characterization.
Sequence analysis of mCstF-64 revealed a cDNA of 3596 bp size
and 16 bp of 3' poly(A). A single open reading frame of 1890 bp was
deduced that encoded a 630-amino acid protein with an estimated molecular mass of 65,893.8 Da and an isoelectric point of 7.10 (Fig.
1). The putative translation initiation codon ATG at nucleotide 77 is
in good translational consensus (46, 47). The m
CstF-64 cDNA had
76 bp of 5'-UTR and 1640 bp of 3'-UTR. There is a canonical polyadenylation sequence AATAAA at nucleotides 3576-3581.
The protein encoded by the mCstF-64 cDNA is similar to other
known forms of CstF-64 and is 69.8 and 71.6% identical, respectively, to the mouse and human somatic forms of CstF-64 (Fig. 1, B
and C). Interestingly, the protein encoded by m
CstF-64 is
more highly related (85.6% identical) to the protein encoded by
KIAA0689, a cDNA uncovered in a survey of long open reading frames
expressed in human brain (48). m
CstF-64 also has two peptide inserts (amino acids 213-231 and 498-555) relative to human, mouse, and Xenopus; these inserts are shared by KIAA0689. These data
suggest the possibility that KIAA0689 is the human ortholog of
m
CstF-64 (see "Discussion"). The inserts also probably account
for the larger apparent molecular size of the variant CstF-64 protein on SDS-PAGE (see Ref. 24 and see Fig. 2).
The downstream insert (498) contains 12 imperfect repeats of the
5-amino acid motif MQG(A/G)G; two such repeats are seen in human
CstF-64. Although it is unlikely that these repeats form a stabilized
structure such as the MEAR(A/G) repeats (Ref. 49 and see below), it is possible they perform a similar but undefined function.
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Fig. 1A shows an alignment of the mCstF-64 protein with
human CstF-64 (34). The m
CstF-64 protein shares features of known CstF-64 proteins as follows: an N-terminal RBD of the RNA recognition motif type (amino acids 17-92), proline- and glycine-rich
regions (amino acids 198-425 and 464-579), a highly conserved C
terminus (amino acids 589-630 (50)), and eight imperfect repeats of
the amino acids MEAR(A/G) (amino acids 425-464 (49)) that are repeated 12 times in human (34), mouse (36), and chicken CstF-64 ((30) Fig.
1A). The RBD of m
CstF-64 is identical to that of CstF-64 from human, mouse, and Xenopus (51) except for a serine
replacing the proline at position 41 of the protein.
Antibody Reactivity of the mCstF-64 Protein--
cDNA
clones for CstF-64, mCstF-64, and m
CstF-64 were transcribed and
translated in vitro using the rabbit reticulocyte lysate system, and proteins were tested for reactivity with the 3A7 and 6A9
monoclonal antibodies (Fig. 2). As expected, CstF-64 from human reacted
with both the 3A7 and 6A9 antibodies (Fig. 2, lanes 1).
CstF-64 from mouse reacted with 3A7 but not 6A9 (lanes 2, compare A and B). This is in agreement with our
earlier assessment that the somatic form of CstF-64 from mouse is
recognized by the 3A7 but not the 6A9 monoclonal antibody (24). In
contrast, the protein encoded by m
CstF-64 reacted with 6A9 but not
3A7 (lanes 3, compare A and B).
Furthermore, the protein recognized by 6A9 had a larger apparent
molecular weight than either the mouse or human somatic CstF-64
proteins. This suggests that m
CstF-64 has the same antibody
reactivity as the variant form of CstF-64 found in mouse testis
(24). The slowest migrating band of about 70 kDa in
the human CstF-64 sample (Fig. 2A, lane 1) may be due to posttranslational modification, possibly phosphorylation (34). The
small amount of immunoreactivity in control samples that were incubated
with vector DNA is probably due to endogenous CstF-64 protein in the
rabbit lysates (lanes 4).
Peptide Maps of the mCstF-64 Protein Are Identical to the
Pattern of the Variant CstF-64 from Testis--
To determine whether
the protein encoded by m
CstF-64 is the same as that present in mouse
testis, we compared partial protease digestion patterns of the two
proteins. Radiolabeled m
CstF-64 protein was produced in
vitro in rabbit reticulocyte lysate translation extracts in the
presence of [35S]methionine. Radiolabeled
CstF-64 was
isolated in vivo by short term culture of mouse seminiferous
tubules in medium containing [35S]methionine followed by
immunoprecipitation of the variant CstF-64 with the 6A9 antibody. Both
proteins were treated identically with either 0.1 or 1.0 µg of
Staphylococcus aureus V8 protease and analyzed by 15%
SDS-PAGE and fluorography (Fig. 3).
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By comparing the partial protease digestion profiles of the protein synthesized in vitro (Fig. 3, lanes 1-3) with the protein synthesized in vivo (lanes 4-6), the two profiles appear to share many common peptides. This suggests strongly that the two proteins share the same primary structure. In contrast, the protein encoded by the cDNA for the mouse somatic form of CstF-64, mCstF-64, has a distinctly different partial protease digestion profile, indicating that it has a different primary structure (data not shown).
Cstf2t Is on Chromosome 19 in Mouse--
The mouse
chromosomal location for the mCstF-64 gene
(Cstf2t) was determined by interspecific backcross
analysis using progeny derived from matings of ((C57BL/6J × M. spretus)F1 × C57BL/6J) mice. This
interspecific backcross mapping panel has been typed for over 2900 loci
that are well distributed among all the autosomes as well as the X
chromosome (40). C57BL/6J and M. spretus DNAs were digested
with several enzymes and analyzed by Southern blot hybridization for
informative restriction fragment length polymorphisms (RFLPs) using a
mouse cDNA probe from m
CstF-64. The 1.8-kb TaqI M. spretus RFLP (see "Experimental Procedures") was used
to follow the segregation of Cstf2t in backcross
mice. The mapping results indicated that Cstf2t is
located in the central region of mouse chromosome 19 linked to
Gnaq and Fas. Although 120 mice were analyzed for
every marker and are shown in the segregation analysis (Fig. 4), up to 167 mice were typed for some
pairs of markers. Each locus was analyzed in pairwise combinations for
recombination frequencies using the additional data. The ratios of the
total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene
order are as follows:
centromere---Gnaq---24/167---Cstf2t---1/122---Fas. The recombination frequencies (expressed as genetic distances in
centimorgans ± S.E.) are as follows:
centromere---Gnaq---14.4 ± 2.7---Cstf2t---0.8 ± 0.8---Fas.
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RNA Binding of the mCstF-64 Protein RBD--
The RBDs of all
vertebrate CstF-64s are identical except for m
CstF-64, which
contains a single amino acid difference (amino acid 41). Therefore, we
wanted to determine whether the m
CstF-64 RBD was functional in
binding RNA in a UV cross-linking assay. The RNA-binding domains of
human CstF-64 (amino acids 1-247), mouse CstF-64 (amino acids 1-247),
and m
CstF-64 (amino acids 1-174) were incubated with
32P-labeled RNA in vitro and subjected to
cross-linking with UV light (29, 34, 45) (Fig.
5). Under the conditions used, each
RBD-containing polypeptide bound covalently to RNA only in the presence
of UV light (lanes 2, 4, and 6). Minor
differences in the intensity of the RNA cross-linked protein bands were
not reproducible (data not shown). In contrast, a non-RNA
binding protein (IgG) did not bind to RNA irrespective of UV radiation (lanes 7 and 8). This suggests that all three
RNA-binding domains are functional to bind a complex RNA substrate,
although it does not address the question of RNA-binding
specificity.
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DISCUSSION |
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We recently described two forms of CstF-64, which are expressed in
distinctive patterns in mouse male germ cells (24). One form was
clearly the somatic CstF-64 that is found in nearly every cell and
tissue. The other had a more limited range and is designated CstF-64. We hypothesized that, since the gene for the somatic form
of CstF-64 (Cstf2) was located on the X chromosome
and most X-linked genes are inactivated in male meiosis,
CstF-64 was expressed from an autosomal paralog of CstF-64. In this
paper, we describe the cloning and characterization of a cDNA for
CstF-64 from adult mouse pachytene spermatocytes that has all the
properties of the gene we hypothesized (Fig.
6). Specifically, the cDNA,
designated m
CstF-64, (i) is found in a pachytene spermatocyte
cDNA library, (ii) encodes a protein with an apparent mobility on
SDS-PAGE of about Mr 70,000, (iii) protein
derived from this cDNA is recognized by the 6A9 but not the 3A7
monoclonal antibody, (iv) is encoded by the gene
Cstf2t that is on an autosome, chromosome 19, (v) encodes a protein that has a partial peptide map that is identical to
that of
CstF-64 from testis, and (vi) is most similar to KIAA0689 (48), a cDNA from brain where
CstF-64 is also found. We further showed that the RBD of m
CstF-64 functions in RNA binding despite having a Pro
Ser substitution at amino acid 41 of the RBD.
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The mCstF-64 protein is clearly the product of a different gene than
is the mouse CstF-64 protein. The cDNAs are only 69.8% identical, leading to a number of amino acid substitutions throughout the protein, rather than inclusion or exclusion of individual exons.
Furthermore, mouse backcross analysis determined that
Cstf2t is on chromosome 19 (Fig. 5) and not the X
chromosome as is Cstf2 (24). A human cDNA clone,
KIAA0689 (48), is quite similar to m
CstF-64 and is probably the
human ortholog of this gene.3
These similarities between m
CstF-64 and the human and mouse somatic
CstF-64s suggest that m
CstF-64 is the result of a duplication or
retroviral insertion of the CstF-64 gene that occurred prior to the divergence of primates and mice (Fig. 1C).
As has been noted elsewhere (50), the C termini (amino acids 589-630)
of all known CstF-64 homologs are remarkably conserved (27, 34, 36, 48,
50-52), suggesting an essential function for that region. Also
conserved is the RNA recognition motif type RNA-binding
domain at the N terminus, which is identical in all vertebrate CstF-64s
examined (human (34), mouse (36), Xenopus (51), and chicken
(53)), except mCstF-64, which has a single proline
serine change
at amino acid 41. RBDs of the RNA recognition motif type have a
well defined
motif (54-56), and serine 41 is part
of the second loop following the first
-helix in the RBD that might
alter RNA substrate specificity (57). Therefore, m
CstF-64 might have
a different RNA binding specificity than CstF-64, which would
contribute to the differences seen in germ cell polyadenylation.
Many of the amino acid substitutions in mCstF-64 relative to CstF-64
are in regions that have as yet unknown functions, including the
Gly/Pro-rich regions (Fig. 1A, amino acids
198-425 and 464-579 (34)). Interestingly, a
number of substitutions and a 19-amino acid insertion occur in the
region thought to interact with CstF-77 (Fig. 1A, amino
acids 180-260, summarized in Fig. 6), which bridges CstF
and CPSF, and symplekin, a protein whose function in the nucleus is not
known (26). This suggests the possibility that m
CstF-64 interacts
differently than CstF-64 with other proteins of the polyadenylation
complex, and the interaction might affect m
CstF-64 function in germ
cell polyadenylation.
The MEAR(A/G) repeat region of mCstF-64 is significantly different
than the 12 repeats in CstF-64 (34, 49). The region in m
CstF-64
contains only eight recognizable repeats (425), one of which is
incomplete (441), some of which have proline substitutions (430, 438), and none of which precisely match the consensus. In CstF-64, the
MEAR(A/G) repeats likely form a stable, monomeric
-helix that might
serve as a rigid structural element in polyadenylation (49). Perhaps
the degenerate MEAR(A/G) region in m
CstF-64 forms a shorter
structural variant or is dispensable, as it is in Xenopus which lacks MEAR(A/G) (51). However, the second insert in m
CstF-64 (498) includes 12 repeats of the amino acids MQG(A/G)G that might substitute for the MEAR(A/G) function.
How might mCstF-64 be involved in promoting polyadenylation of
non-AAUAAA-containing mRNAs in male germ cells? One possibility is
that the Pro
Ser substitution at amino acid 41 alters the RNA
binding affinity of the variant CstF-64 (58, 59), allowing binding to a
different downstream sequence element (29). An altered affinity of CstF
for a downstream sequence element could then influence the binding of
CPSF to an upstream element, which might or might not match the AAUAAA
consensus. Our RNA binding experiments (Fig. 5) suggest that mammalian
CstF-64 RBDs have similar affinities for nonspecific RNAs. However, the
RNA binding specificity of CstF-64 is quite different in isolation than
in complex with CstF-77, CstF-50, and CPSF (29, 60). Therefore, other
regions of m
CstF-64 and other interacting proteins might have a
strong influence on RNA substrate specificity. In light of this,
m
CstF-64 contains a number of amino acid differences in the site of
protein-protein interaction with CstF-77 and symplekin. Changes in this
region (Fig. 1A, amino acids 108-229) could
disrupt binding of CstF-64 to CstF-77 or even to symplekin, thus
dramatically altering CstF interaction with the pre-mRNA and with
CPSF.
Finally, we note the similarity of mCstF-64 to the human KIAA0689
cDNA from brain (48), suggesting the possibility that KIAA0689 is
the human ortholog of m
CstF-64. Our recent cloning of the variant
CstF-64 from a human testis cDNA library confirms this
possibility.3 This strongly suggests that the phenomenon of
non-AAUAAA polyadenylation occurs in human germ cells, as well as in
mouse. This possibility is currently under investigation.
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ACKNOWLEDGEMENTS |
---|
We thank John McCarrey for cDNA libraries; A. Michelle Wallace-Shannon, Eman Attaya, and Andreé Reuss for technical help; Susan San Francisco for sequencing; and S. Sridhara and Stuart Ravnik for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant 1 R01 HD37109-01A1, the South Plains Foundation (to C. C. M.), the NCI, Department of Health and Human Services, National Institutes of Health (to N. A. J. and N. G. C.), and a Helen Hodges Educational Charitable Trust and Raymond Green Scholarships (to B. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Cell
Biology and Biochemistry, Texas Tech University Health Sciences Center,
3601 4th St., Lubbock, TX 79430. Tel.: 806-743-2703; Fax: 806-743-2990;
E-mail: cbbccm2@ttuhsc.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M009091200
2 J.-L. Redondo and C. C. MacDonald, unpublished work.
3 B. Dass and C. C. MacDonald, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: CPSF, cleavage and polyadenylation specificity factor; CstF, cleavage stimulation factor; RBD, RNA-binding domain; RRM, RNA recognition motif; UTR, untranslated region; bp, base pair; kb, kilobase pair; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; RFLPs, restriction fragment length polymorphisms.
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