Download Price, DK, Zhang, F, Ashley, CT and Warren, ST: The chicken FMR1 gene is highly conserved containing a CTT 5\' untranslated repeat and encodes an RNA-binding protein. Genomics 31:3-12 (1996).

GENOMICS 31, 3–12 (1996)
Article No. 0002
The Chicken FMR1 Gene Is Highly Conserved with a CCT 5*Untranslated Repeat and Encodes an RNA-Binding Protein
DOUGLAS K. PRICE,1 FUPING ZHANG, CLAUDE T. ASHLEY, JR.,
AND
STEPHEN T. WARREN2
Howard Hughes Medical Institute and Departments of Biochemistry and Pediatrics,
Emory University School of Medicine, Atlanta, Georgia 30322
Received August 22, 1995; accepted October 17, 1995
Willems, 1994). The first reported disease containing
this type of mutation, fragile X syndrome, is the most
frequent inherited form of mental retardation. Fragile
X syndrome segregates as an X-linked dominant disorder with reduced penetrance and has been mapped to
the fragile X mental retardation (FMR1) gene located
at human Xq27.3 (Warren and Ashley, 1995; Warren
and Nelson, 1994). FMR1 contains a (CGG)n trinucleotide repeat in the 5*-untranslated region (UTR) in both
human (Verkerk et al., 1991) and mouse (Ashley et al.,
1993a), and expansion of this repeat is the mutation
responsible for nearly all of the fragile X cases (Fu et
al., 1991; Verkerk et al., 1991). Once the trinucleotide
repeat expands over approximately 230 copies, the
FMR1 gene becomes hypermethylated and transcriptionally inactive, resulting in the absence of the fragile
X mental retardation protein (FMRP) (Pieretti et al.,
1991; Sutcliffe et al., 1992). Due to this lack of transcription of FMR1, fragile X syndrome is classified as
a loss-of-function type of mutation. As expected, fragile
X syndrome patients without repeat expansion are occasionally found and have FMR1 deletions (deVries et
al., 1994; Gedeon et al., 1992; Gu et al., 1994; Hirst et
al., 1995; Meijer et al., 1994; Quan et al., 1995; Tarleton
et al., 1993; Wöhrle et al., 1992) or, in the case of a
single patient, a missense FMR1 mutation (De Boulle
et al., 1993).
Protein domains within mammalian FMRP that are
common to ribonucleotide particle (RNP) families have
been identified (Ashley et al., 1993b; Siomi et al.,
1993b). The first of these domains, the K homology
(KH) domain, originally described in the hnRNP K protein (Siomi et al., 1993a), consists of a highly conserved
I/L/V-I-G-X2-G-X2-I sequence and a series of regularly
spaced hydrophobic residues. Two of these KH domains
are highly conserved in FMRP of human and mouse
(Ashley et al., 1993a). Importantly, the single missense
mutation resulting in fragile X syndrome is due to a
change in a highly conserved residue of one of the KH
domains (De Boulle et al., 1993). Also present in the
FMRP sequence of mouse and human are two copies
of an Arg-Gly-Gly (RGG) box motif (Ashley et al., 1993b;
Siomi et al., 1993b). RGG boxes have previously been
The transcriptional silencing of the human gene,
fragile X mental retardation 1 (FMR1), is due to abnormal methylation in response to an expanded 5*-untranslated CGG trinucleotide repeat and accounts for
most cases of fragile X syndrome, a frequent inherited
form of mental retardation. Although the encoded
fragile X mental retardation protein (FMRP) is known
to have properties of a RNA-binding protein, the precise function of FMRP remains to be elucidated. We
report the cloning of the chicken homolog of FMR1 and
show strong evolutionary conservation, with nucleotide and amino acid identities of 85 and 92%, respectively, between chicken and human. In place of the
mammalian CGG trinucleotide repeat, a 99-nt tripartite repetitive element containing a CCT trinucleotide
repeat flanked on both sides by dinucleotide repeats
was identified. Blocks of highly conserved 3*-untranslated sequence were also found. Within the coding region, two copies each of the highly conserved K homology motif and the Arg-Gly-Gly (RGG) box motif, both
ribonucleotide particle family domains implicated in
RNA binding, were identified. Chicken FMRP was
found to bind RNA in vitro, and this activity correlated
with the presence of the carboxy-terminal portion of
the protein that includes the RGG motifs. q 1996 Academic
Press, Inc.
INTRODUCTION
In the last 4 years, 11 genetic loci displaying trinucleotide repeat expansion have been identified, with
8 of these loci being linked to heritable human diseases
(Burke et al., 1994; Fu et al., 1992; Group, 1993; Kawaguchi et al., 1994; Knight et al., 1993; Koide et al., 1994;
La Spada et al., 1991; Nancarrow et al., 1994; Orr et
al., 1993; Parrish et al., 1994; Verkerk et al., 1991;
1
Present address: Department of OB/GYN, Carolinas Medical
Center, P.O. Box 32861 Room 4035, Charlotte, NC 28232.
2
To whom correspondence should be addressed at Howard Hughes
Medical Institute, Emory University School of Medicine, Rollins Research Building, 1510 Clifton Road, Atlanta, GA 30322. Telephone:
(404) 727-5979. Fax: (404) 727-5408. E-mail: [email protected]
y.edu.
3
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0888-7543/96 $12.00
Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
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PRICE ET AL.
identified in many RNA-binding proteins and have
been implicated in RNA–protein interactions (Dreyfuss et al., 1993; Kiledjian and Dreyfuss, 1992).
FMRP has recently been shown to have RNA-binding
capabilities in vitro toward RNA homopolymers, its
own FMR1 message, and a sizable fraction of human
fetal brain RNAs (Ashley et al., 1993b; Siomi et al.,
1993b). It has been suggested that the absence of the
normal FMRP in fragile X patients could alter posttranscriptional regulation of specific cellular RNAs,
thus accounting for the pleiotropic phenotype associated with the syndrome (Ashley et al., 1993b; Siomi et
al., 1994). In favor of the loss-of-function hypothesis,
the lone I304N missense mutation in human FMR1
has been reported to abolish RNA-binding activity in
vitro (Siomi et al., 1994).
To delineate further the important functional domains of the FMR1 gene and protein, we have cloned
and sequenced the FMR1 gene from chicken. Alignments of amino acid and nucleotide sequence from
chicken, human, and mouse show that the gene is
highly conserved with a 5*-untranslated, trimodal repetitive element containing, within its core, a CCT trinucleotide repeat rather than the mammalian CGG repeat. It is also demonstrated that chicken FMRP, like
its mammalian homologs, is capable of specifically
binding both chicken and mouse FMR1 message in in
vitro RNA-binding assays.
MATERIALS AND METHODS
cDNA library screening. An adult female chicken brain cDNA
library (Clontech) in lgt10 was plated with host strain C600hfl.
Plates containing approximately 50,000 plaques were lifted with 15cm Biodyne nylon filters (Pall) and UV cross-linked. After a 2- to
3-h prehybridization at 657C in 12 ml of 250 mM Na3PO4 , 1 mM
EDTA, 250 mM NaCl, 7% (w/v) SDS, 10% (w/v) PEG (8000 MW), 1%
BSA, and 200 mg/ml denatured salmon sperm DNA, the filters were
hybridized for 12–14 h in the presence of a random-primed (Amersham megaprime kit), radiolabeled DNA probe and 12 ml of fresh
hybridization solution. The filters were washed at 657C in 21 SSC,
0.1% SDS 1 4 for 30 min each, 11 SSC, 0.1% SDS 1 1 for 20 min,
and 0.51 SSC, 0.1% SDS 1 1 for 15 min. Filters were then exposed
to Kodak XAR film and intensifying screens for 12–18 h at 0807C.
Secondary screening was as above with plaque densities of 300–600
per plate. The DNA from positive plaques was prepared (Silhavy et
al., 1984) and digested with EcoRI to free the cDNA insert from
the l arms, and the inserts were subcloned into pBluescript KS(0)
(Stratagene).
DNA sequencing and analysis. The plasmid subclones were sequenced using double-stranded templates on an ABI 373A automated
DNA sequencer using the Taq DyeDeoxy Terminator Cycle Sequencing Kit (ABI) as described by the manufacturer. Initial primers were
the commercially available M13-20 and reverse pBluescript vector
primers, and subsequent primer-walking primers were synthesized
on an ABI 392 DNA/RNA synthesizer and purified over Nensorb Prep
nucleic acid purification columns (DuPont NEN). DNA and protein
sequences were analyzed with GeneWorks Version 2.1 software (IntelliGenetics, Inc.). Secondary structure predictions were generated
by DNAsis 2.0 (Hitachi).
Reverse transcription PCR. Reverse transcription–PCR (RTPCR) was performed using the GeneAmp RNA PCR kit (Perkin–
Elmer). Each RT-PCR reaction contained 50–500 ng of chicken brain
poly(A)/ RNA (Clontech) as template, with random hexamers sup-
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plied in the kit used for first-strand synthesis. Subclone RT1 was
created by amplification using 15 pmol of primers F1 (5*-AGGTGGACCAACTACGTTTGGAAAG-3*), nts 1301–1325 of chicken FMR1,
and R1 (5*-GCCTACTATTTGTCTTTGGCCTAAC-3*), nts 1992–
2016 of chicken FMR1. Full-length cDNA representing the entire
coding region of chicken FMR1 was generated by RT-PCR using
primers F4 (5*-AGAAAGAGGTGCTGAGTCTGGG-3*), nts 173–194,
and R1, as described above. All PCR amplifications were performed
in a Perkin–Elmer 480 thermal cycler for 35 cycles (denaturation,
1 min, 957C; annealing, 1 min, 667C; extension, 1 min, 727C). Both
of the above PCR products were first cloned into PCR II vector (Invitrogen) as described by the manufacturer, and clone R1 was sequenced directly from the PCR II vector using vector primers supplied by the manufacturer (Invitrogen). The full-length chicken
cDNA product was subcloned into pBluescript (Stratagene) for subsequent in vitro transcription/translation, and the integrity of this
clone, designated pcFMR1, was confirmed by DNA sequencing as
described. To confirm the absence of the chicken equivalent of the
human FMR1 exons 11 and 12, RT-PCR was performed under the
exact same conditions as above using primers 1112F (5*-CTTGATGAAGATACCTGCACAT-3*), nts 985–1006 of chicken, and 1112R
(5*-AGCTCCAATCTGTCGAAGCTGCTC-3*), nts 1339–1362 of
chicken. The control reaction contained 50 ng pcFMR1 cDNA as template, using the same primers and conditions as above.
In vitro transcription, in vitro translation, and RNA-binding
assays. The pcFMR1 construct was linearized for transcription with
XhoI or AccI. In vitro transcription was performed using the T3/T7 in
vitro transcription kit (Stratagene) according to the manufacturer’s
instructions. Protein translation was carried out in rabbit reticulocyte lysate from the in vitro express kit (Stratagene). Binding experiments using biotinylated RNA were as described by Ashley et al.
(1993b). Briefly, 2 ml in vitro translated, 35S-labeled protein and 80
ng biotinylated chicken FMR1 RNA were mixed in a 10-ml reaction
volume containing 1 ml 101 binding buffer (200 mM Hepes, pH 7.9;
20 mM MgCl2 ; 100 mM ZnCl2 ; 0.2% Nonidet P-40, 700 mM NH4Cl),
1 ml 50 mg/ml yeast tRNA, and DEPC H2O. Binding reactions were
allowed to proceed at room temperature for 30 min, followed by capture and washing of the biotinylated FMR1 RNA and any bound
protein using streptavidin-conjugated magnetic beads (Dynal) as
previously described (Ashley et al., 1993b). Captured protein was
eluted by boiling in standard SDS sample buffer for 5 min and visualized by SDS–polyacrylamide gel (12%) electrophoresis followed by
fluorography.
RESULTS
Isolation of cDNA clones. A chicken brain cDNA library from an adult female chicken was screened using
the mouse FMR1 cDNA Mc 2.17 (Ashley et al., 1993a)
as a probe. Two positive clones were isolated, and the
cDNA sequence was obtained. The first clone, Cc2-1,
contained 900 bp of sequence homologous to the mouse
and human FMR1 genes, including approximately 500
bp of coding sequence (Fig. 1). The other clone, Cc2-5,
displayed homology to mammalian FMR1 beginning
400 bp upstream of the translational stop codon and
ending 600 bp into the 3* UTR. Additional clones containing more 5* sequence, designated Cc4-2, Cc4-3, and
Cc4-5, were isolated by rescreening of the chicken
cDNA library using clone Cc2-1 as probe, and a single
gap in the coding region was filled by RT-PCR from
chicken brain RNA (Fig. 1).
Nucleotide sequence of chicken FMR1. Overlapping
cDNA clones were aligned, producing a chicken FMR1
cDNA contig containing 222 nt of 5* UTR, a single large
open reading frame encoding a putative polypeptide of
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ALIGNMENTS OF THE CHICKEN FMR1 GENE
5
FIG. 1. Map of the chicken FMR1 subclones and contig. Unique cDNA clones constituting the chicken FMR1 contig (thick line) are
depicted (thin lines). The relative position of the putative intiation codon (ATG), translational stop codon (TAA), and repetitive motif (R)
are indicated.
566 amino acids (using initiator methionine defined in
mammalian species), and 619 nt of 3* UTR (Fig. 2).
Compared to human FMR1, the chicken nucleotide sequence was 85% identical within the coding region. Notably, identity within the 3* UTR was 81%, with highest levels of identity (ú94%) localized to two distinct
blocks of 3* sequence located approximately 60 and 600
bp downstream of the translational stop codon, respectively. Comparison of the 5* UTR of chicken and human
revealed 61% identity overall. Although the CGG trinucleotide repeat of mammalian species was not observed, a novel cryptic CCT trinucleotide repeat with
the sequence (CCT)5GCT(CCT)2 was observed. This
CCT repeat segment was flanked upstream by a cryptic
CA dinucleotide repeat displaying the sequence
(CA)2CC(CA)2CCA(CA)7CC and downstream by a cryptic GA dinucleotide repeat with the sequence (GA)6AA(GA)2AA. Altogether, these repetitive sequences constitute a 99-base tripartite repetitive element situated 55
nt upstream of the presumptive initiator methionine,
in relatively close agreement with the known positions
of the mouse and human CGG repeats, which are located 66 and 69 nt, respectively, upstream of the initiator methionine (Ashley et al., 1993a). A single large gap
in the alignment of the chicken FMR1 sequence with
those of human and mouse was found to correspond to
exons 11 and 12 of the human gene (Eichler et al.,
1993).
To confirm the absence of the exons 11 and 12 equivalent in the chicken FMR1 message from brain, RT-PCR
using chicken brain poly(A)/ RNA was performed with
primers flanking the deletion. Analysis of the PCR
product by agarose gel electrophoresis revealed a single
377-base product corresponding to the predicted distance between the primers in the absence of exons 11
and 12 and of the same mobility as a PCR product
generated from the full-length chicken FMR1 cDNA
(Fig. 3). No additional bands indicative of alternative
splicing were observed, indicating that messages in-
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cluding material representative of exons 11 and 12
were not present in detectable amounts in chicken
brain FMR1 RNA. Indeed, while alternative splicing
of exon 12 of the mammalian FMR1 gene has been
documented in multiple tissues (Ashley et al., 1993a;
Verkerk et al., 1993), alternative splicing events involving exon 11 have never been observed. Consistent with
these observations, the absence of the equivalent of
human exons 11 and 12 has also been reported recently
in Xenopus laevis FMR1 message (Siomi et al., 1995),
suggesting that these exons could represent late additions to the FMR1 gene after the divergence of mammals and fowl.
Deduced amino acid sequence. While the reading
frame of the chicken FMR1 contig remained open at the
5* end, the fact that strong nucleotide identity began
precisely at a conserved ATG previously shown to be
the translational initiation codon of the mouse FMR1
gene (Ashley et al., 1993a; Verkerk et al., 1993) suggested that the coding region of the chicken FMR1 gene
also began here (see Fig. 2). Based on this prediction,
an alignment of the putative chicken FMR1 gene product with that of human and mouse was generated (Fig.
4). The predicted 63.9-kDa cFMRP polypeptide was
found to be 92% identical and 95% similar to both the
human and the mouse FMR1 gene products. Included
in this conserved sequence were the KH domains (positions 222–251 and 284–314 in chicken, respectively),
thought to be important in either RNA binding (Siomi
et al., 1994) or RNA–protein interactions (Siomi et al.,
1993a), which were identical to those of human with
the exception of a single amino acid residue (Fig. 4).
Also conserved in cFMRP were the two copies of the
RGG motif (Arg-Gly-Gly; amino acid positions 468 and
479) previously observed in FMRP from mammals,
which represent yet another RNP family motif implicated in RNA binding (Dreyfuss et al., 1993; Kiledjian
and Dreyfuss, 1992). Thus, given the previously docu-
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PRICE ET AL.
FIG. 2. Alignment of the murine, human, and chicken FMR1 contigs. Nucleotides identical in all three contigs are boxed. The translational initiation and termination codons are shaded. The 5*-repetitive element of chicken is underlined. The gap in the chicken sequence
is due to the absence of exons 11 and 12 in the chicken genome.
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AP-Genomics
ALIGNMENTS OF THE CHICKEN FMR1 GENE
7
FIG. 2—Continued
mented interaction of mammalian FMRP with RNA
(Ashley et al., 1993a; Siomi et al., 1993a), it appeared
likely that cFMRP might also display such an activity.
RNA-binding assays. RNA-binding assays using
cFMRP were carried out to establish whether the minute differences in the primary structure of chicken
FMR1 would affect the ability of cFMRP to bind its
FIG. 3. RT-PCR of chicken brain RNA to verify the absence of
mammalian exons 11 and 12. Ethidium bromide-stained agarose gel
of the RT-PCR product obtained from amplification of chicken brain
poly(A)/ RNA in the absence (lane 1) and presence (lane 2) of reverse
transcriptase. As a control, the PCR product obtained from amplification of the pcFMR1 cDNA with the same primer pair is shown
(lane 3). Molecular weight markers are shown as well (Stds).
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own mRNA. To generate a full-length chicken FMR1
sequence, RT-PCR of chicken poly(A)/ RNA was performed using primers specific for 5* and 3* UTR sequences. The cloned PCR product, designated pcFMR1,
was shown via DNA sequencing to be identical to the
previously generated cDNA contig, with the exception
of the absence of the equivalent of the first 51 nt of
human FMR1 exon 17 (data not shown). Given that
the human exon 17 is known to contain an alternate
splice acceptor site at precisely the same location in
the sequence (Eichler et al., 1993; Verkerk et al., 1993),
the absence of these nucleotides, and thus the 17 amino
acids that they encode, was attributed to alternative
splicing of the chicken FMR1 gene. The pcFMR1 plasmid was then linearized and used to generate a runoff
transcript in vitro that consisted of 60 bp of 5* UTR,
the entire chicken FMR1 coding region, and the first
83 bp of 3* UTR. In vitro translation of this message in
the presence of L-[35S]methionine was then performed,
producing a single major product of Ç65 kDa, in
agreement with the predicted size of 63.9 kDa calculated from the predicted amino acid sequence (Fig. 5A).
Through the use of an alternate restriction site for linearization of the pcFMR1 plasmid, an additional Ç55kDa polypeptide that retained both KH domains intact
but lacked the RGG boxes near the carboxy terminus
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PRICE ET AL.
FIG. 4. Amino acid alignment of chicken, human, and mouse FMRP. Identical amino acids are boxed. The 66-amino-acid gap in the
chicken sequence corresponds to exons 11 and 12 of mammalian species. The RNP family motifs, the KH motifs, and the RGG boxes are
underlined.
was generated (Fig. 5A). A negative control, parathyroid precursor protein, was also translated. Each of
these polypeptides was then assayed for its ability to
interact with biotinylated chicken FMR1 RNA as previously described (Ashley et al., 1993b), and captured
protein was visualized by denaturing gel electrophoresis followed by fluorography. As depicted, full-length
cFMRP, like its murine counterpart (Ashley et al.,
1993b), was capable of interacting with its own message in vitro (Fig. 5B). In addition, cFMRP was shown
to interact with full-length FMR1 message from mouse
(data not shown). Importantly, truncated cFMRP was
not captured in this assay, suggesting that the carboxyterminal portion of FMRP that includes the RGG box
FIG. 5. In vitro translations and RNA-binding assays of chicken
FMRP and other polypeptides. (A) In vitro translated, 35S-labeled
chicken FMRP (lane 1), truncated FMRP lacking the RGG motifs
(lane 2), and negative control parathyroid precursor protein (lane 3).
Apparent molecular weights of cFMRP (65 kDa) and parathyroid
precursor protein (15 kDa) are indicated. (B) Labeled protein captured by biotinylated chicken FMR1 RNA. (Lane 1) cFMRP; (lane 2)
truncated cFMRP; (lane 3) parathyroid hormone precursor protein.
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motifs could represent the site of RNA interaction (Fig.
5B). Similar results have been obtained with analogous
human (Siomi et al., 1993a) and murine FMRP polypeptides (C. T. Ashley, unpublished results). Parathyroid precursor protein, which has been used in previously documented experiments as a negative control
(Ashley et al., 1993b), was also not captured, attesting
to the specificity of the assay.
DISCUSSION
We report the characterization of the chicken homolog of the gene FMR1, the gene involved in fragile X
syndrome. In humans, it is known that expansion of a
CGG trinucleotide repeat in the 5* UTR of the FMR1
gene represents the mutation responsible for the majority of cases of fragile X syndrome (Warren and Ashley, 1995; Warren and Nelson, 1994). While this trinucleotide repeat is known to be conserved in mouse
(Ashley et al., 1993a) and a wide variety of other mammals (Eichler, 1995), no such CGG repeat was identified in chicken. Instead, a novel repetitive element comprised of a cryptic CCT trinucleotide repeat flanked on
either side by distinct cryptic dinucleotide repeats was
found. Although the nucleotide content of the upstream
repetitive elements of the mammalian and fowl FMR1
homologs has apparently evolved, it would appear that
certain aspects of the various repeats, such as their
repetitive nature or their position relative to the translational start site, have been maintained. Also noted
AP-Genomics
ALIGNMENTS OF THE CHICKEN FMR1 GENE
was the fact that the 5* UTR of chicken FMR1, like
that of all mammals (Ashley et al., 1993a; Eichler,
1995), was highly GC-rich (65%). Certain residues, especially guanine and cytosine, appear highly conserved, as there are 12 cytosine and 11 guanine residues conserved between the three species within the
region designated the repetitive element in chicken.
More specifically, in the alignment of the trinucleotide
repeats of the three species, there are 9 conserved cytosines in a row, each separated by two nucleotides, resulting in a general consensus sequence (CXX)9 with X
representing any nucleotide. Although the function of
these conserved residues or repetitive sequences is unknown, one hypothesis is that the conservation of these
residues is indicative of functional significance, such
as a recognition site for protein binding. Proteins binding in this region of the FMR1 gene could be involved
in either transcriptional or translational regulation, as
has been suggested (Richards et al., 1993). Indeed, with
regard to the latter, GC-rich 5* UTR sequences have
been linked to posttranscriptional control (Godeau et
al., 1986; Muller and Witte, 1989).
Another difference between the human and the
chicken FMR1 genes is the apparent absence in chicken
of the equivalent of mammalian exons 11 and 12 (Ashley et al., 1993a; Eichler et al., 1993; Verkerk et al.,
1991), implying that these exons were introduced into
the mammalian gene after the divergence of chicken
approximately 290 million years ago, but prior to the
separation of mouse 90 million years ago. In support of
this supposition, human exons 11 and 12 were recently
shown to be absent from FMR1 cDNAs cloned from
Xenopus laevis (Siomi et al., 1995). One plausible explanation for the addition of these exons is that they provide diversity or enhancement-of-function necessary in
the higher evolved species. In agreement with this hypothesis, it has been documented previously that exon
12, which represents the largest hydrophobic peak on
a hydrophobicity profile of murine FMRP, is alternatively spliced in all mammalian tissues analyzed thus
far (Ashley et al., 1993a; Verkerk et al., 1993). The
apparent absence of these exons in the chicken FMR1
cDNA could also represent alternative splicing of
FMR1 in chicken brain, although we detect no additional bands by RT-PCR in this tissue. In addition, in
previous analyses of alternative splicing of the human
and mouse FMR1 gene in various tissues (Ashley et al.,
1993a; Verkerk et al., 1993), no evidence for alternative
splicing of exon 11 was found. However, regardless of
whether these exons were late-acquired or are alternatively spliced, it is clear that they are not necessary
for RNA-binding activity in vitro, since RNA-binding
activity of cFMRP is preserved in their absence. It will
be of interest to deduce the genomic structure of the
chicken FMR1 gene to clarify these issues.
The FMR1 coding region has been markedly conserved over time, with 92% identity between human
and chicken. It has been noted previously that silent
positions of all X-linked genes are more strongly con-
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9
FIG. 6. Comparison of rates of evolution of cFMRP with various
other X-linked and autosomal genes. The relation between estimated
proportions of differences per amino acid site (»K…) between pairs of
species against time, since each pair diverged from a common ancestor for FMR1 (Fu et al., 1991), glucose-6-phosphate dehydrogenase
(Fouts et al., 1988; Kaslow et al., 1987; Kimura, 1983) dystrophin
(Ho et al., 1988; Hoffman et al., 1987), and a-globin (Jackson, 1993).
served than those of autosome-linked genes and that
the mutation frequency of X-linked genes may be limited due to the existence of many vital genes on this
chromosome (Miyata et al., 1987). However, in a comparison of the rate of evolution of FMR1 with that of
the X-linked dystrophin (Hoffman et al., 1987; Lemaire
et al., 1988) and glucose-6-phosphate-dehydrogenase
(G6PD) genes (Fouts et al., 1988; Ho et al., 1988; Kaslow et al., 1987), as well as with autosomal a-globin
(Kimura, 1983), FMR1 evolution appears to be slower
than that of other X-linked genes (Fig. 6). Therefore,
FMR1 appears relatively intolerant of mutations, and
this may have functional significance.
Notably, evolutionary conservation of FMR1 is
shown to extend into the 3* UTR of the gene, with
80.6% identity between chicken and human over the
nucleotides identified thus far. Two large blocks of 3*
sequence display much higher identity, 98% over 54 nt
(positions 1989–2043, Fig. 2) and 94% over 185 nt
(2358–2542, Fig. 2). A high degree of sequence conservation in this region of the FMR1 gene has also been
observed between Xenopus and human (Siomi et al.,
1995). Conserved blocks such as these could represent
sites for direct protein interaction and/or regions involved in stem loop formation. In favor of the latter,
secondary structure analysis of the 3* region of chicken
FMR1 predicts the formation of a stable stem loop
structure, with segments of the two conserved blocks
forming the double-stranded stem (Fig. 7). The identification of these conserved regions along with the prediction of their involvement in stem loop structures is
again suggestive of functional significance, although no
specific role has been ascribed to these conserved blocks
thus far.
Like the mammalian FMRP, cFMRP retains the ability to bind its own mRNA in vitro. The cFMRP has also
been shown in our laboratory to interact with mouse
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PRICE ET AL.
FIG. 7. Predicted secondary structure of the 3*-untranslated region of chicken FMR1. (A) Computer-generated secondary structure
prediction of nucleotides 2017–2452 of chicken FMR1. (B) Magnification of boxed region in (A) showing base pairing of conserved 3* UTR
sequences. Numbers correspond to the chicken FMR1 sequence.
FMR1 RNAs, and the mouse FMRP been shown to bind
both chicken and mouse FMR1 RNA (data not shown).
These findings strongly suggest that a common conserved binding site exists in the FMR1 messenger RNA
molecules from various species, perhaps represented
by one or more of the highly conserved regions of the
FMR1 gene, in particular, the 3* UTR.
The demonstration of RNA-binding activity of
cFMRP in the absence of the equivalent of mammalian
exons 11 and 12 indicates that these exons are not
essential for RNA interaction. Additionally, it would
appear that the amino acids corresponding to the first
17 amino acids of human exon 17 (Eichler et al., 1993)
are not required either, since the cFMRP polypeptide
shown to interact with FMR1 messenger RNAs in this
work had these amino acids excluded, presumably secondary to alternative splicing. Importantly, a truncated FMRP polypeptide lacking the RGG box motif,
but with the KH domains present, is shown to be inactive in this RNA-binding assay, suggesting that either
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the RGG box motif or nearby residues mediate the protein–RNA interaction. These data are consistent with
those of Siomi et al. (1993a), who also demonstrated
the dependency of the carboxy terminus of human
FMRP upon the interaction with RNA homopolymers.
The data reported above demonstrate the highly conserved nature of the FMR1 gene. Nucleic acid similarities were widespread and unexpectedly pronounced in
the 3* UTR, suggesting a functional importance for this
portion of the gene consistent with emerging data on
the 3* UTR of other loci (Jackson, 1993). It is particularly notable that the 5* CGG trinucleotide repeat
found in the mammalian FMR1 genes, the site of the
unusual mutational event of unstable trinucleotide repeat expansion in fragile X syndrome, is absent. However, the finding of a highly repetitive sequence in this
location of the chicken gene, including a CCT trinucleotide repeat, is highly interesting, as no function has
yet been found for these repeats despite their relatively
common occurrence in 5* UTRs of mammalian genes
AP-Genomics
ALIGNMENTS OF THE CHICKEN FMR1 GENE
(Riggins et al., 1992). The protein identity also is remarkable in its conservation between human and
chicken. As one might expect, the highly conserved
chicken protein binds RNA in a manner analogous to
its mammalian counterparts. Although the comparison
of mammalian FMRP to lower eukaryotes might be
more informative with regard to functionally important
amino acid residues, it is curious that despite repeated
attempts by a number of laboratories, FMR1 homologues have not yet been found in any invertebrate
organism.
ACKNOWLEDGMENTS
D.K.P. is an Associate and S.T.W. an Investigator of the Howard
Hughes Medical Institute.
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