CIRP2, a major cytoplasmic RNA-binding protein in Xenopus oocytes

Abstract

In an attempt to isolate mRNA-binding proteins we fractionated Xenopus oocyte lysate by oligo(dT)-cellulose chromatography. A 20 kDa protein was the major component of the eluate. cDNA cloning revealed that this protein is a Xenopus homolog of the cold-inducible RNA-binding protein (CIRP) which was originally identified in mammalian cells as a protein that is overexpressed upon a temperature downshift. This Xenopus protein, termed here xCIRP2, is highly expressed in ovary, testis and brain in adult Xenopus tissues. In oocytes it is predominantly localized in the cytoplasm. By biochemical fractionation we provide evidence that xCIRP2 is associated with ribosomes, suggesting that it participates in translational regulation in oocytes. Microinjection of labeled mRNA into oocytes followed by UV cross-linking of the oocyte lysate led to identification of two major RNA-binding activities. Immunoprecipitation of the RNA-binding proteins demonstrated that one is xCIRP2 and that the other contains FRGY2. FRGY2, which is one of the principal constituents of mRNA storage particles involved in translational masking of maternal mRNA, has an RNA-binding domain conserved to those of bacterial cold shock proteins. Possible implications of the highly abundant expression in oocytes of cold shock RNA-binding proteins of both eukaryotic and prokaryotic types are discussed.

Figure 1

Oligo(dT)–cellulose chromatography. Xenopus oocyte lysate was incubated with oligo(dT)–cellulose. Then after extensive washing with binding buffer, the bound materials were eluted with binding buffer containing 25% formamide. Aliquots of total lysate (lane 1), unbound materials (lane 2) and the eluate (lane 3) were electrophoresed by 12% SDS–PAGE and stained with Coomassie brilliant blue. FRGY2 and the abundant 20 kDa protein (p20) are indicated by arrows and the sizes of molecular weight markers (lane M) are shown on the left.

Figure 2

cDNA cloning of xCIRP2. The deduced amino acid sequence of cloned xCIRP2 cDNA (Xenopus 2) is aligned with previously reported amino acid sequences, XCIRP (Xenopus 1, accession no. AB007597), human CIRP (D78134), mouse CIRP (D78135) and Mexican axolotl RBP (U71299). ClustalW with minor visual modifications was used to produce the alignments. Conserved amino acids are indicated by asterisks. RNP1 and RNP2 sequences in the RNA recognition motif and RGG sequences are boxed. The sequences of three peptides (K1, K2 and K3) that were microsequenced from gel-purified p20 are indicated. C1 indicates the synthesized peptide sequence used to prepare the polyclonal antibodies.

Figure 3

Expression of xCIRP2. (A) Recombinant xCIRP2 with a 6× histidine tag was prepared. One microgram of the recombinant protein was analyzed by 12% SDS–PAGE and stained with Coomassie brilliant blue along with molecular weight markers (lane 1). Oocyte lysate (2 µg protein) was subjected to immunoblotting with rabbit antisera against recombinant xCIRP2 (lane 2) or against the C-terminal peptide C1 (lane 3; see Fig. 2 for sequence of the peptide). (B and C) The levels of xCIRP2 proteins and mRNAs in Xenopus tissues and oocytes were examined by immunoblotting (top) and northern blotting (bottom), respectively. Five micrograms of protein from tissues (B) or a lysate equivalent to 0.25 oocytes (C) were used for immunoblotting with the antiserum raised against the C-terminal peptide. Lysate from ovaries was extracted with 1,1,2-trichlorotrifluoroethane (Freon) to remove yolk proteins (B, top, lane 4). xCIRP2 mRNA in 10 µg total tissue RNA (B) or in RNA equivalent to one oocyte (C) was examined by northern blotting. Note that the 2.8 kb mRNA was barely detected in the ovaries as well as in the oocytes. (D) Stage VI oocytes were fractionated into nuclei and cytoplasm. The expression of xCIRP2 in total (T), cytoplasmic (C) and nuclear (N) fractions was examined by immunoblotting with anti-xCIRP2 C-terminal peptide antibodies. To confirm the fractionation process, localization of FRGY2 and TAF-I in the same fractions was also examined.

Figure 4

FRGY2 and xCIRP2 are the major mRNA-binding proteins in Xenopus oocytes. (A) ³²P-labeled Xenopus histone H1 mRNA was injected into the cytoplasm of Xenopus oocytes. The oocytes were harvested 1 h later and the oocyte lysate then stored on ice without (lane 1) or with (lanes 2–4) irradiation with UV light and digested with RNase A. Aliquots of UV-irradiated samples were immunoprecipitated with antibodies against the recombinant xCIRP2 protein (lane 3) or anti-FRGY2 antibodies (lane 4). The samples were analyzed by 12% SDS–PAGE and visualized by autoradiography. (B) A similar experiment to that in (A) was performed with CAT mRNA. The UV-irradiated sample (lane 1) was immunoprecipitated with anti-xCIRP2 antibodies (lane 2) or anti-FRGY2 antibodies (lane 3).

Figure 5

Gel retardation assay with recombinant xCIRP2 and FRGY2. (A) A gel retardation assay using ³²P-labeled histone H1 mRNA (lane 1) was performed with xCIRP2 (18 kDa) and FRGY2 (35 kDa) proteins. RNA was first mixed on ice with 0.07 (lanes 5–8) or 0.7 µg (lanes 9–12) FRGY2 protein and then 0.18 (lanes 2, 6 and 10), 0.9 (lanes 3, 7 and 11) or 3.6 µg (lanes 4, 8 and 12) xCIRP2 protein were added. The mixture was incubated at 30°C for 20 min and electrophoresed in a 1% agarose gel in 0.5× TBE buffer at room temperature. (B) Radiolabeled histone H1 mRNA, 0.36 µg (lanes 5–8) xCIRP2 protein and 0.07 (lanes 3–6) or 0.7 µg (lanes 7 and 8) FRGY2 protein were incubated as in (A). Anti-xCIRP2 antibody (0.6 µg, lanes 2, 4, 6 and 8) was then added and the mixture further incubated at 30°C for 10 min. The reactions were electrophoresed in a 1% agarose gel in 0.5× TBE buffer at room temperature.

Figure 6

xCIRP2 protein is associated with heavily sedimenting structures. Oocytes were fractionated as described in Materials and Methods. (Top) Total lysate (T) and the S100 (S) and P100 (P) fractions were analyzed by immunoblotting using anti-xCIRP2 antibodies. Oocytes were homogenized in a buffer containing 100 (lanes 1–3) or 500 mM NaCl (lanes 4–6). For lanes 7–9, total lysate (100 mM NaCl) was digested with RNase A prior to ultracentrifugation. (Bottom) RNA was purified from S100 (lanes 2, 5 and 8) and P100 (lanes 3, 6 and 9), electrophoresed in a 1% agarose gel containing formaldehyde and visualized by staining with ethidium bromide. 28S and 18S indicate rRNAs.

Figure 7

xCIRP2 co-sediments with ribosomes in density gradients. (A) Oocyte lysate was irradiated with UV light and fractionated through a 15–40% sucrose gradient. The distribution of xCIRP2 and FRGY2 was examined by immunoblotting (western). RNA prepared from every other fraction was electrophoresed in an agarose gel containing formaldehyde and transferred to a nylon membrane. The membrane was stained with methylene blue (Total RNA; 28S and 18S indicate rRNAs) and histone H1 mRNA was detected by northern blotting. The positions of stored mRNP and ribosomes are indicated. (B) Oocyte lysate was fixed either by irradiation with UV light or with formaldehyde. The samples were fractionated through 20–60% Nicodenz gradients. Proteins and RNA were detected as in (A). Positions of free proteins, mRNP and ribosomes are indicated.