(Received for publication, July 7, 1995; and in revised form, September 5, 1995)
From the
The Xenopus Y-box protein FRGY2 has a role in the translational silencing of masked maternal mRNA. Here, we determine that FRGY2 will recognize specific RNA sequences. The evolutionarily conserved nucleic acid-binding cold shock domain is required for sequence-specific interactions with RNA. However, RNA binding by FRGY2 is facilitated by N- and C-terminal regions flanking the cold shock domain. The hydrophilic C-terminal tail domain of FRGY2 interacts with RNA independent of the cold shock domain but does not determine sequence specificity. Thus, both sequence-specific and nonspecific RNA recognition domains are contained within the FRGY2 protein.
The Xenopus laevis Y-box protein FRGY2 is a major component of ribonucleoprotein storage particles containing maternal mRNA within Xenopus oocytes (Murray et al., 1992; Deschamps et al., 1992; Tafuri and Wolffe, 1993a, 1993b). FRGY2 has an active role in facilitating the translational silencing or masking of maternal mRNA (Richter and Smith, 1984; Ranjan et al., 1993; Bouvet and Wolffe, 1994). How FRGY2 interacts with mRNA and how mRNA packaged by the FRGY2 protein is rendered accessible to the translational machinery is not understood.
The Y-box proteins
contain a nucleic acid-binding domain conserved between prokaryotic and
eukaryotic organisms (reviewed by Wolffe (1994a, 1994b)). The
prokaryotic Y-box proteins regulate the cold shock response (Goldstein et al., 1990; La Teana et al., 1991; Jones et
al., 1992). These proteins contain a single nucleic acid-binding
structure known as the cold shock domain (CSD) ()(Wistow,
1990). The cold shock domain is a five-stranded
-barrel containing
a well characterized RNA binding motif RNP-1 (Schindelin et
al., 1993; Schnuckel et al., 1993; Landsman, 1992; Burd
and Dreyfuss, 1994a, 1994b). The cold shock domain will bind
selectively to duplex (La Teana et al., 1991; Jones et
al., 1992) and single-stranded DNA (Schindelin et al.,
1993; Schnuckel et al., 1993). However, an exact DNA binding
specificity is yet to be determined.
The eukaryotic Y-box proteins have been reported to bind with some selectivity to a wide variety of duplex and single-stranded nucleic acids including the Y-box (CTGATTGGCCAA) (Marello et al., 1992; Wolffe et al., 1992; Murray, 1994) (reviewed by Wolffe (1994a)); however, the exact nucleic acid binding specificity was not determined. The eukaryotic Y-box proteins recognize DNA through the conserved CSD (Kolluri et al., 1992; Tafuri and Wolffe, 1992). The recognition of RNA has been proposed to be either predominantly via the CSD (Ladomery and Sommerville, 1994) or through interactions with islands of basic/aromatic amino acids in the C-terminal domain of those Y-box proteins (Murray, 1994).
In this work we establish that both the oocyte-specific Y-box protein FRGY2 and a somatic homolog FRGY1 can have highly specific interactions with RNA. We determine that the cold shock domain is required for specific recognition of RNA. The C-terminal domain of FRGY2 has nonspecific interactions with RNA that may account for the more efficient association of wild type FRGY2 with RNA in comparison with the cold shock domain alone. We discuss the significance of these observations for the potential roles of FRGY2 in transcriptional and translational control.
Two steps of PCR amplification were performed to generate the constructs for point mutants of FRGY2 protein. Primers K12 (5`-GTTGATAGCTCCGGCGCCGTTGCGAACGTTAAACC-3`; mutations corresponding to aa 55 and 57 of FRGY2 are underlined) and K13 (5`-AACGGCGCCGGAGCTATCAACAGAAATGACACCAAA-3`) were synthesized to obtain constructs for PM1 and CP1 proteins, and K14 (5`-GTTGATAA(G/A)TCCCAAGCCGTTGCG AACGTTAAACC-3`) and K15 (5`-AACGGCTTGGGA(T/C)TTATCAACAGAAATGACACCAAA-3`) were for PM2, CP2, and CP3 proteins. FRGY2 cDNA was amplified by PCR with primer sets of K9/K12 and K13/K8 for PM1 or K9/K14 and K15/K8 for PM2. PCR products were isolated and used for secondary PCR amplification with the primer set of K9/K8. The products from secondary PCR were used as the templates of PCR to generate constructs for CP1, CP2, and CP3 with the primer set of K20/K19. The final products were cloned into pGEX-4T-3 vector as described above. All constructs were completely sequenced to exclude the possible introduction of unintended fortuitous mutations during amplification and cloning.
To obtain GST-fusion FRGY2 protein
and its derivatives, BL21(DE3) pLysS was transformed with each pGEX
construct. A 300-500-ml culture was induced by
isopropyl-1-thio--D-galactopyranoside to synthesize
GST-fusion proteins. C-terminal deletion mutants D4, CSD, CP1, CP2, and
CP3 were purified from bacterial lysate using glutathione-Sepharose 4B
according to the method suggested by the manufacturer (Pharmacia).
Since wild type, D5, D6, PM1, and PM2 proteins did not bind to
glutathione-Sepharose efficiently, they were purified from the pelleted
inclusion bodies of Escherichia coli lysate through
SP-Sepharose chromatography as described (Tafuri and Wolffe, 1992),
except that the step of precipitation with ammonium sulfate was omitted
and the proteins were eluted from SP-Sepharose with phosphate buffer
containing 0.75 M NaCl after washing the column with the
buffer containing 0.5 M NaCl.
The full-length FRGY1 and FRGY2 proteins with T7 gene 10 leader peptides (T7-FRGY2) were expressed in E. coli BL21(DE3) transformed with the pET constructs (Tafuri and Wolffe, 1992). The Y-box proteins were purified from the pelleted inclusion bodies of E. coli lysate through an SP-Sepharose column as described above.
RNA was
selected from this random pool by coimmunoprecipitation with the FRGY1
and FRGY2 fusion proteins (Tsai et al., 1991). 1-5
µg of full-length T7 gene 10 fusion proteins was bound to 4 mg of
protein A beads using the antibody against the gene 10 epitope
(Novagen). After three washes with NT2 buffer (50 mM Tris, pH
7.4, 150 mM NaCl, 0.05% Nonidet P-40, 1 mM
MgCl), the protein and 200-500 ng of RNA were
incubated for 7 min in 100 µl of reaction mixture (20 mM KCl, 150 mM NaCl, 50 mM Tris, pH 7.5, 0.05%
Nonidet P-40, 2.5% polyvinyl alcohol, 1 mM MgCl
, 1
mM EGTA, 50 µg/ml poly(A), 2 µg/ml vanadyl
ribonucleoside complex, 0.5 mg/ml tRNA, 125 µg/ml bovine serum
albumin, 1 mM dithiothreitol, 80 units/ml RNasin (Promega).
Following this incubation, the RNA was washed 5 times with NT2 buffer.
Ten µg of carrier tRNA was then added to the immunoprecipitated
RNA. It was then subjected to phenol extraction and ethanol
precipitation. Rounds two to five had identical binding conditions
except that 0.5 M urea was added to the final NT2 buffer wash
to reduce nonspecific binding as described previously.
Reverse transcription and reconstitution of the transcription template were as described previously (Tsai et al., 1991). RNA was reverse transcribed using 0.1 µg of primer RevUniv with avian myeloblastosis virus reverse transcriptase (1 h at 42 °C using conditions recommended by the supplier). Following reverse transcription, the cDNA was resuspended in 10 µl of double distilled water. Three µl of the cDNA was subjected to 35 cycles of PCR under the conditions already noted. The new transcription template was then used to repeat the above transcription, coimmunoprecipitation, and reverse transcription steps for four additional rounds. After the last PCR the cDNA was digested with BamHI and then subcloned in pGem/BamHI. Then the clones were sequenced; 13 sequences for Y1 and 10 for Y2 contained a 6-nucleotide (nt) sequence that is very similar in all these clones (see Fig. 1).
Figure 1: FRGY1 and FRGY2 bind to RNA with sequence selectivity. A, DNA sequences obtained by Selex (see ``Materials and Methods'') for FRGY1 and FRGY2. The DNA sequence corresponding to the RNA initially selected is shown. B, RNA footprinting of FRGY1 and FRGY2 bound to RNA containing a binding consensus sequence. Methylation protection (see ``Materials and Methods'') is shown for FRGY1 (lanes 1-4) and FRGY2 (lanes 9-12). Sequencing markers are shown (lanes 5-8). The sequence containing the Y-box consensus sequence (YRS) is indicated by the vertical bar. Dimethyl sulfate (DMS) alkylates accessible A and C nucleotides; sites of protection within the YRS are indicated by asterisks.
To introduce mutations into the FRGY2-binding site, three double-stranded oligonucleotides containing the sequences shown (5`-AATTCGACATT(A/C)ACA(T/G)(C/G)AGACAAATAAAAATCATCCGTTGATATAAATAGAGCT-3`, 5`-AATTCGACATT(A/G)(A/G)CATC(A/T)AGACAAATAAAAATCATCCGTTGATATAAATAGAGCT-3`, or 5`-AATTCGACATTA(A/C)(A/A)(A/C)TCAGACAAATAAAAATCATCCGTTGATATAAATAGAGCT-3`; EcoRI and SacI sites are underlined) were cloned in EcoRI- and SacI-digested pGEM vector (Promega). Plasmid DNA was sequenced, and one of the mutants, M3/M4-1, was used to generate the nonspecific RNA for competition used in this study.
To obtain shorter RNA probes, double-stranded oligonucleotides K22/K23 (5`-AATTCTAATACGACTCACTATAGGGGCCGACATTAACATCAGACAAAGGTAC-3`) or K5/K6 (5`-GATCCTAATACGACTCACTATAGGGCGTTAACATCAGACAAG-3`) were used as the templates for in vitro transcription with T7 RNA polymerase. Transcription with K22/K23 and K5/K6 gave transcripts of 30 and 15 nt, respectively, and these were isolated from a polyacrylamide gel. Non-labeled RNA for competitors were synthesized by transcribing the EcoRI and HindIII-digested pGEM construct containing a 2.14 wt site (see Table 2) or the HindIII-digested pGEM construct containing a M3/M4-1 mutated site with T7 RNA polymerase.
Figure 2:
RNA binding of GST-fusion FRGY2 proteins. A and B, diagrams of FRGY2 protein and its
derivatives. The cold shock domain (striped box), RNP-1 motif (filled box), and the basic (+) and acidic(-) amino
acid clusters are indicated on the wild type FRGY2 protein. Deletion
and point mutants are shown by open boxes. C,
purification of FRGY2 proteins and GST-fusion derivatives used in this
study. Approximately 0.4 µg of each protein was electrophoresed in
4-20% gradient polyacrylamide gel containing SDS. The gel was
stained with Coomassie Brilliant Blue. D, GST-fusion FRGY2
proteins were tested for RNA binding activity (see ``Materials
and Methods''). The P-labeled 2.14 wt RNA probe (95
nt) (lanes 2 and 12) was incubated with 0.1 µg (lanes 3, 5, 7, 9, 13, 15, and 17) or 0.25 µg (lanes 4, 6, 8, 10, 14, 16, and 18) of GST-fusion
FRGY2 proteins and subjected to gel retardation assay. The 2.14 wt RNA
probe, which was heat-treated at 95 °C for 5 min and chilled on ice
immediately, was electrophoresed in parallel (lanes 1 and 11). The positions of two RNA-protein complexes formed between GST-FRGY2 (WT) and the probe are indicated by dots.
Full-length FRGY2 was purified either as a GST-fusion protein or as a T7 gene 10 fusion protein (Fig. 2C) (see ``Materials and Methods''). The mutant proteins were purified as GST fusions (Fig. 2C). The C-terminal tail of FRGY2 is an extended hydrophilic structure extremely sensitive to proteolysis (Deschamps et al., 1992). Thus, immunoblotting using polyclonal antibodies against FRGY2 reveals that the other proteins of smaller size than the full-length expressed protein are degradation products (data not shown). Gel retardation experiments (Fig. 2D) using a 95-nucleotide RNA probe containing the specific YRS show the GST-FRGY2 protein and the deletion mutants retaining the CSD together with either the N-terminal (D4) or C-terminal domain (D5) bound efficiently to the probe (Fig. 2D, lanes 3 and 4, lanes 15 and 16, and lanes 9 and 10, respectively). The CSD or the C-terminal tail domain alone (D6) bound less well to the probe (Fig. 2D, lanes 17 and 18 and lanes 13 and 14, respectively). Point mutations of both aromatic amino acids in the RNP-1 motif (PM1) reduced RNA binding (Fig. 2D, lanes 5 and 6), whereas a single point mutation (PM2) had relatively little effect on RNA binding within the context of the full-length protein (Fig. 2D, lanes 7 and 8). Control experiments (not shown) indicated that the GST moiety has no RNA binding activity in a gel retardation (or filter binding) assay. We conclude that both the CSD and the C-terminal tail can contribute to the association of FRGY2 with RNA. Within the context of the full-length protein, the RNP-1 motif within the CSD has an important role. Both the N- and C-terminal domains of FRGY2 that flank the CSD can stabilize interaction with RNA.
We wished to establish a
more quantitative assessment of the relative binding affinity of FRGY2
compared with the various deletion and point mutations. In order to
minimize the possibility that differential binding is influenced by
differential recovery of active protein in the various recombinant
protein preparations rather than true differences in binding affinity,
we prepared all the proteins in parallel and examined their interaction
with the YRS consensus (the 2.14 probe) and a mutant RNA sequence (the
M3/4.1 probe, see Table 2and Fig. 5) in parallel. The
filter binding analysis of protein binding to the YRS consensus (Fig. 3A) reveals that GST-FRGY2 recognizes the 2.14 wt
RNA probe with highest affinity (K = 0.2
10
M). The deletion mutants
containing the CSD or tail domain alone (D6), the N-terminal domain
including the CSD (D4), or point mutations in the RNP-1 motif all have
reduced affinity (K
= 0.8-1
10
M). The use of a nonspecific RNA
sequence (M3/4.1) reduced the binding of GST-FRGY2 (to a K
of 1.0
10
M) (Fig. 3B). We conclude that the filter binding
experiments confirm the general indications of affinity derived from
gel shift analysis (Fig. 2).
Figure 5: Effect of point mutations in the conserved YRS consensus RNA sequence on FRGY2 binding. The filter binding of T7 FRGY2 (see ``Materials and Methods'') to various radiolabeled RNAs containing the Y-box consensus sequence (A) or various mutant RNAs (B-D) is shown. The percentage of RNA bound to the input is plotted. The legends to the data points are shown in each graph. The sequences of each mutant are indicated in Table 2.
Figure 3:
RNA binding curves for FRGY2 and mutants.
A filter binding assay (see ``Materials and Methods'') was
performed with GST-FRYG2 (), PM1 (
), D4 (
), D6
(
), and the CSD (
). A,
P-labeled RNA
containing the consensus sequence (2.14 wt, AACAUC) was used as the
probe. B, one of the point mutants in the consensus sequence
(M3/4-1, AACAUG, see Table 2) was used as the probe. The
data show the fraction of RNA bound as a
percentage.
We extended this analysis to the
properties of the CSD in isolation. Using the gel retardation assay, we
found that point mutations of the RNP-1 motif (Fig. 4A)
dramatically reduced stable interaction with RNA probes (Fig. 4B). Quantitative measurements revealed a
reduction in K from
10
M for CSD to less than 10
M in the mutant proteins (not shown). A single point mutation of the
RNP-1 motif has a more severe consequence for the CSD in isolation (Fig. 4B, lanes 10-12) than for the
full-length FRGY2 protein (Fig. 2D, lanes
7-8). These differences might be due to the stabilizing
influence of the C-terminal tail domain on FRGY2 interaction with RNA.
Comparable effects are obtained when FRGY2 binds to DNA (Tafuri and
Wolffe, 1992).
Figure 4: Requirements for RNA binding of FRGY2. A, diagram of GST-fusion FRGY2 deletion mutants containing the CSD. B, gel retardation assay using the 2.14 wt RNA probe (lane 1) was performed with 0.1 µg (lanes 2, 4, 7, 10, and 13), 0.25 µg (lanes 3, 5, 8, 11, and 14), and 0.625 µg (lanes 6, 9, 12, and 15) of GST-fusion FRGY2 proteins. C, binding of FRGY2 protein to RNA probes of various lengths. Gel retardation assays were performed with 10 fmol of 95 nt (lanes 2-5), 50 fmol of 30 nt (lanes 6-9), or 50 fmol of 15 nt (lanes 10-13) RNA probe containing a 2.14 wt binding site. The amount of T7-FRGY2 was 0 µg (lanes 2, 6, and 10), 0.04 µg (lanes 3, 7, and 11), 0.1 µg (lanes 4, 8, and 12), and 0.25 µg (lanes 5, 9, and 13). The positions of free probes and RNA-protein complexes are shown by asterisks and dots, respectively. MspI-digested pBR322 was electrophoresed in parallel (lane 1).
The association of FRGY2 with DNA is dependent on the length of the DNA probe used to assess binding (Tafuri and Wolffe, 1992). In our experiments we use RNA probes of 95 nt in length. Since earlier studies on selective RNA binding have generally made use of short 47-nt probes (Murray, 1994) or homopolymers of ill defined length (Ladomery and Sommerville, 1994), we explored whether RNA binding depends on the length of RNA probe. We find that little or no stable association of FRGY2 occurs with an RNA probe of 30 or 15 nt in length even though the YRS is present, whereas efficient binding occurs with a probe of 95 nt (Fig. 4C). Thus some of the reported discrepancies in nucleic acid binding selectivity may depend on the length of the probe and/or competitor in nucleic acid binding studies.
Our gel retardation experiments with T7-FRGY2 reveal that two complexes assemble on the 95-nt YRS RNA as the excess of protein over probe is increased (Fig. 6A, lanes 1-5). Since identical bands are seen with nonspecific RNA that does not contain a YRS (Tafuri and Wolffe, 1993a; Tafuri et al., 1993), this may reflect both specific and nonspecific protein-RNA interactions. In vivo the FRGY2 protein interacts with mRNA such that one protein binds every 40 nucleotides (Darnbrough and Ford, 1981). We chose an excess of FRGY2 over the RNA probe that led to the accumulation of a single complex (Fig. 6A, lane 6) and competed this complex with an increasing excess of RNA competitors (Fig. 6A, lanes 7-12). Competition with the 95-nt RNA containing the YRS was much more effective than when this sequence was altered.
Figure 6:
FRGY2 protein binds to a 2.14 wt site in a
sequence-dependent manner. A, gel retardation assay was
performed with P-labeled 2.14 wt RNA as the probe and
titration of T7-FRGY2 (lanes 1-5). The amount of FRGY2
was 0 µg (lane 1), 0.04 µg (lane 2), 0.1
µg (lane 3), 0.25 µg (lane 4), and 0.625
µg (lane 5). Sequence specificity of RNA binding was
examined in lanes 6-12. Ten fmol of RNA probe were
incubated with 0.1 µg of T7-FRGY2 in the absence (lane 6)
or presence of an increasing amount of non-radiolabeled 2.14 wt RNA (lanes 7-9) or M3/M4-1 RNA (lanes
10-12). The amount of competitor RNA was 0.6 pmol (lanes
7 and 10), 2 pmol (lanes 8 and 11), and
6 pmol (lanes 9 and 12). B, competition gel
retardation assay was performed using the 2.14 wt RNA probe with 0.5
µg of GST-Y2 CSD (lanes 1-3), 0.25 µg of GST-Y2
D4 (lanes 4-6), and GST-Y2 D6 (lanes
7-9). Fifteen pmol (lanes 2 and 3) or 10
pmol (lanes 5, 6, 8, and 9) of non-radiolabeled 2.14
wt RNA (lanes 2, 5, and 8) or M3/M4-1 RNA (lanes
3, 6, and 9) were added to the binding reaction as
competitors. S is with specific competitor. NS is
with nonspecific competitor.
We next examined whether deletion mutants of FRGY2 containing either the CSD alone, the N terminus including the CSD, or the C-terminal portions of the protein would have specific interactions with RNA. The C-terminal tail domain of FRGY2 binds to the specific RNA probe, but this interaction is competed equivalently by both specific and nonspecific RNA competitors (Fig. 6B, lanes 7-9). In contrast, the CSD alone (Fig. 6B, lanes 1-3) and the N terminus of FRGY2, which includes the CSD (Fig. 6B, lanes 4-6), bind to the specific RNA probe such that binding is selectively competed by the YRS sequence. This suggests that the CSD contributes to the specific recognition of RNA but requires the flanking N- and C-terminal domains of FRGY2 for a high affinity interaction (see Fig. 3). The CSD is the only conserved amino acid sequence in the N-terminal portion of FRGY1 and FRGY2 (Tafuri and Wolffe, 1990) leading to recognition of the same YRS consensus. Since RNA binding is weakened by mutation of the RNP-1 motif (Fig. 2D, lanes 5 and 6, Fig. 3), it appears that the RNP-1 motif may have a role in maintaining the structural integrity of the CSD necessary for discrimination between various RNA sequences.
In the oocyte, FRGY2 and a highly related protein mRNP3 package mRNA (Deschamps et al., 1992; Murray et al., 1992; Tafuri and Wolffe, 1993a, 1993b). These proteins interact with mRNA with a stoichiometry such that one protein binds approximately every 40 nucleotides (Darnbrough and Ford, 1981; Marello et al., 1992). This repetitious binding requires that the FRGY2 protein binds to a wide variety of RNA sequences, and thus FRGY2 must also retain the capacity to package mRNA with relatively little sequence specificity. By analogy with the packaging of the vast majority of nuclear DNA with the histone proteins (Wolffe, 1992), the Y-box proteins might package the vast majority of cytoplasmic mRNA (Tafuri and Wolffe, 1993a, 1993b; Wolffe, 1994a). Just as the histone proteins provide a default mechanism that directs the repression of basal transcription (Grunstein, 1990; Wolffe, 1992) so might the Y-box proteins provide a default mechanism for the repression of translation (Tafuri and Wolffe, 1993a, 1993b; Ranjan et al., 1993; Bouvet and Wolffe, 1994). However, just as the histones can have highly selective interactions with DNA in spite of their general packaging function and thereby influence the events of transcription (Wolffe, 1994b), we have found that the Y-box proteins can also bind to RNA with sequence selectivity in spite of their general packaging function (Fig. 1, Fig. 3, Fig. 5, and Fig. 6). Thus the exact sequence of mRNA will influence the context and affinity with which the Y-box proteins bind and thus the association of other proteins necessary for the translational activation or silencing of maternal mRNA. Moreover, the selective association of Y-box proteins with particular RNA sequences might nucleate the assembly of masking proteins on mRNA. Future experiments will explore these possibilities.
FRGY1 is found in the nuclei of somatic cells (Tafuri and Wolffe, 1990; Ranjan et al., 1993). The function of the somatic Y-box proteins is unknown. They have been proposed to also influence the translation of mRNA (Evdokimova et al., 1995); however, other experiments suggest a role in transcriptional control (Tafuri and Wolffe, 1990, 1992; Ting et al., 1994; Kashanchi et al., 1994; Kerr et al., 1994; MacDonald et al., 1995). Our definition of the Y-box proteins as capable of sequence-selective RNA recognition provides a potential explanation for their selective influence on the transcription process independent of specific association with DNA. It is possible that the Y-box proteins might interact with specific nascent RNAs and communicate with the basal transcriptional machinery in a manner analogous with that of TAT-TAR (Berkhout et al., 1989). Although RNA binding by the prokaryotic Y-box proteins has not yet been examined, it is possible that sequence-selective RNA binding might contribute to regulation of the cold shock response (Goldstein et al., 1990). A requirement to maintain the capacity to interact with a variety of specific nucleic acids might contribute to the striking evolutionary conservation of the cold shock domain itself.