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Gene
2000 Oct 03;2561-2:293-302. doi: 10.1016/s0378-1119(00)00375-9.
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4SR, a novel zinc-finger protein with SR-repeats, is expressed during early development of Xenopus.
Ladomery M
,
Marshall R
,
Arif L
,
Sommerville J
.
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The protein C4SR contains two cysteine(4) (C(4)) zinc-finger motifs at its amino terminus, a stretch of acidic residues in the middle and a series of serine-arginine (SR) repeats at its carboxyl terminus. A cDNA clone encoding the zinc-finger domain was first selected from a Xenopus laevis oocyte expression library on the basis of the ability of the fusion protein to stably bind an RNA probe. The mRNA encoding C4SR is expressed during oogenesis, and the protein is present at a constant level in oocytes and early embryos. The C4SR protein is expressed in transcriptionally active erythroblasts but not in transcriptionally inert mature erythrocytes. An epitope-tagged C4SR protein, expressed in oocytes, associates with nascent transcripts at many loci in lampbrush chromosomes and is absent from storage particles (snurposomes) containing the normally recognized complement of RNA splicing components. It is likely that C4SR is involved in pre-mRNA transcription/packaging rather than in exon splicing. The zinc-finger motif, present as two copies in C4SR, is also present in a range of transcription-associated proteins. We suggest the descriptor (DW)C(4), in which DW refers to the invariant aspartic acid (D)/tryptophan (W) dipeptide that precedes the first cysteine residue, for this distinctive zinc-finger structure.
Fig. 1. Cloning of Xenopus C4SR, primary structure of the protein and composition of recombinant proteins expressed. (A) Selection of RNAbindingproteinsfromthe lgt11expression library.Theplaque-liftmembranewasincubatedinthepresenceof32P-labelledriboprobe.Themembrane was rinsed in binding buï¬er and quartered, and each quarter was washed extensively in buï¬er containing either 0.05, 0.5, 1.0 or 2.0 NaCl before setting up for autoradiography. In general, plaques for subcloning and sequencing were selected as those giving positive signals after the 0.5 M NaCl washes. (B) Amino acid sequence of C4SR and box diagram of motifs and domains. The two zinc-ï¬nger motifs (letters in shaded boxes labelled Znf1 and Znf2); a region rich in aspartic and glutamic acid residues (italic letters D and E and cross-hatched box labelled acidic); a region containing many repeats of the sequence serineâarginine (boxed and vertical stripes labelled SR-repeats); a putative nucleotide triphosphate-binding site (underlined and horizontal striped box labelled ATP); a glycine-rich âhingeâ (double-underlined and horizontally striped box labelled G); three regions rich in arginine and lysine residues (bold letters and black boxes labelled B), which could be used for nuclear import. The total length of the cDNA sequence is 1186 bp, and the 5â and 3â UTRs are shown as single lines. (C) Composition of the two fusion proteins used in this study: GST-G2 is expressed in bacteria from the bacteriophage T7 promoter (T7p) and consists of glutathione-S-transferase (broken box labelled GST) fused to the zinc-ï¬nger region encoded by cDNA clone G2; T7-C4SR is expressed from the cytomegalovirus promoter (CMVp) and consists of the complete coding sequence of C4SR with a bacteriophage T7-epitope (broken box labelled T7) inserted at the amino terminus.
Fig. 2. Expression of C4SR at the RNA and protein levels in Xenopus oocytes, embryos and erythroid tissues. (A) Relative abundance of transcripts hybridized by the G2 antisense riboprobe on slot blots of total ovarian RNA recovered from oligo(dT) chromatography as polyadenylated (A+) and non-polyadenylated (A−) fractions and of RNA extracted from six oocytes at stage II (late previtellogenesis) to stage VI (full grown; Dumont, 1972). (B) Riboprobe G2 characterization and hybridization to Northern blots of polyadenylated RNA isolated from stage II oocytes. Riboprobe N5 and its hybridization proï¬le to a blot from the same gel are included for comparison. Clone N5 encodes the mRNA-masking protein FRGY1 (Tafuri and Wolffe, 1990). Numbers on the left indicate the probe sizes, numbers on the right, the hybridized transcript sizes, both in kilobases (kb). (C) Immunoblots, using anti-G2, of protein extracted from oocytes (O); from eight-cell cleavage (C), blastula (B), gastrula (G), neurula (N) and tailbud (TB) embryos; and from mature erythrocytes (E), red blood cells from anaemic blood (A) and bone marrow (M). Each well on SDS-PAGE contains protein equivalent to two oocytes or two embryos or 105 erythroid cells. GST-G2 (G2) is included as a positive reaction marker. (D) Immunostaining, usinganti-G2, of a smearof anaemicXenopusblood. Positive reaction(Anti-G2) is seen in only thelarger erythroblast nuclei, and not in smaller erythrocyte nuclei, as determined by staining with 4,6-diamidino-2-phenylindole (DAPI).
Fig. 3. Expression of recombinant C4SR in Xenopus oocytes. (A) Nuclei and cytoplasms were separated under oil at 18 h after microinjection of plasmid DNA into oocyte nuclei. The recombinant protein, tagged with T7-epitope, was detected after SDS-PAGE by immunoblotting with anti-T7 monoclonalantibody.AsinglebandwithapparentMWof56 kDawasdetected onlyinthenuclearextract(equivalent to10nuclei).(B)Immunoblot of extract of 40 nuclei from microinjected oocytes, fractionated by centrifugation through a 5 ml linear gradient of 10â30% glycerol at 30000 rpm for 3 h in a SW55 rotor (Beckman). (C) Immunostaining of lampbrush chromosomes isolated from the nuclei of microinjected oocytes. The chromosome preparations were immunostained using mouse anti-T7, to detect recombinant C4SR, and rabbit anti-p116, to detect endogenous snRNA U5-associated protein p116. C4SR was visualized with ï¬uorescein-conjugated anti-mouse IgG (FITC), and p116 was visualized with rhodamine-conjugated anti-rabbit IgG (TRITC). The preparations were counterstained with 4,6-diamidino-2-phenylindole to detect DNA (DAPI) and were also viewed by phase contrast (PC).
Fig. 4. Deï¬nition of a novel C2âC2 zinc-ï¬nger motif. Amino-acid sequences were aligned using the program ClustalW. Protein names were taken from database entries (Accession Nos are given). The label âF1â4â after the protein name denotes the zinc-ï¬nger number within a given protein, which ranges from one to four. Invariant residues are denoted by an asterisk, and highly conserved residues by a semi-colon. The consensus is: [DWxCx1â4CxxxNaxxRxxCx2â4Cxxx+] where â+â denotes basic and âaâ aromatic. Note that in two yeast examples, the conserved arginine is positioned at two, rather than at three, residues after the conserved aromatic. Furthermore, the basic residue, four positions after the fourth cysteine is present in all but three cases. In addition to the highly conserved residues, which are highlighted, other shared features are noted; for example, the conserved aspartic acid is often preceded by glycine. We suggest the name â(DW)C4 zinc-ï¬nger motifâ, as the aspartic acid (D) and tryptophan (W) residues always precede the ï¬rst cysteine. References, where available, are given in the text.
Fig. 5. Location of the (DW)C4 zinc-ï¬nger motif with respect to other motifs in a range of proteins. In the Arabidopsis protein T13L16.1, the zinc ï¬nger is the only identiï¬able motif throughout the protein and is repeated four times. Xenopus C4SR, rat Zis, mouse Zfp265 and human ZIS are very similar in structure. In human EWS (in addition to FUS/TLS, not shown), Drosophila CAZ (Cabeza) and human HTAFII68, the zinc ï¬nger is present in combination with the RNA-binding domains: RRM and arginineâglycineâglycine (RGG) repeats. The sequences of the zinc-ï¬nger motifs are as listed in Fig. 4.
