Linnen JM et al. (1993), Two related localized mRNAs from Xenopus laevis...

XB-ART-22480

Gene 1993 Jun 30;1282:181-8.

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Two related localized mRNAs from Xenopus laevis encode ubiquitin-like fusion proteins.

Linnen JM , Bailey CP , Weeks DL .

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The uneven distribution of maternal mRNAs in unfertilized eggs and the unequal inheritance of these molecules by dividing blastomeres may be one mechanism for determining cell fate during embryogenesis. Complementary DNA (cDNA) clones corresponding to maternal mRNAs localized to specific regions of the Xenopus laevis egg have been previously identified and cloned [Rebagliati et al., Cell 42(1985) 769-777]. The maternal mRNA, An1, was originally identified as being localized to the animal hemisphere of X. laevis eggs and early embryos. We describe here the two proteins encoded by two An1 mRNA isoforms which we designate An1a and An1b. These mRNAs are both approximately 3.0 kb long and are concentrated in the animal hemisphere of unfertilized eggs. The predicted amino acid (aa) sequences encoded by An1a and An1b correspond to 76.9 and 78.6 kDa, respectively, and are 88% identical. Both proteins contain a single N-terminal ubiquitin (Ub)-like domain (50% identical to X. laevis Ub) and a putative Zn(2+)-binding region near the C terminus. Unlike Ub polyproteins and most Ub fusion proteins, the N-terminal Ub-like domain found in the An1 proteins does not undergo proteolytic processing. In contrast to earlier studies showing that the An1 mRNA represents a strictly maternal transcript, we report that both related An1 transcripts are found in later embryonic stages and in all adult tissues tested.

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Species referenced: Xenopus laevis
Genes referenced: ada.2 nos1 tbx2 zfand4

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	Fig. I. Restriction maps of Anfa and Anlb cDNA clones and alignment of the two deduced proteins. (A) Restriction maps of Ada and An/b are shown. The nearly full-length Ada and Anlb cDNAs are approximately 2.9 kb and 2.8 kb and encode proteins predicted to be 76.9 kDa and 78.5 kDa, respectively. (B) An alignment of the two proteins encoded by the Ada and Anfb mRNAs according to the algorithm of Needleman and Wunsch (1970) is shown. Solid vertical lines indicate exact aa matches; single and double dots indicate conservative replacements. The shaded box (aa 28-103) shows the region of Ub homology found in both proteins. The open box (aa 625-693 in An la; aa 633-701 in Anl b) indicates homology to the PvPR3 protein from Phaseolus vulgaris which includes the putative Zn 2’-finger-metal-binding domain common to both proteins. Underlined aa are possible PEST sequences identified using the PEST-FIND program (Rogers et al., 1986). Ada and Anlb have GenBank accession Nos. LO8474 and L08475. Methods: cDNA clones were isolated from a phage hgt IO oocyte cDNA library by screening with the original 650-bp An1 isolate (Rebagliati et al., 1985). Additional clones were obtained from the same oocyte library by PCR (Friedman et al., 1990) and RT-coupled PCR amplification of oocyte RNA (Kawasaki, 1990). DNA sequences were obtained for both strands by the dideoxynucleotide chain-termination procedure (Sanger et al., 1977) using [a-“S]dATP and the Sequenase” Version 2.0 kit (Tabor and Richardson, 1987). All nt and aa sequences were analyzed using the sequence analysis software package of the Genetics Computer Group, Madison, WI (Devereux et al., 1984).
	Fig. 2. Comparison of Anla (a) and Anlb (b) Ubil sequences to Xenopus Ub (u) and Anla and Anlb C-terminal sequences to the C-terminal sequences of PvPR3 (p) from Phaseolus vulgaris. (A) The aa sequences of Anla and An1 b (aa 28-104) compared to the 76 aa of Xenopus Ub. Solid vertical lines indicate exact aa matches; single and double dots indicate conservative replacements according to the method of Needleman and Wunsch (1970). Both Anl isoforms have about 50% identity to Xenopus Ub. Considering conservative substitutions, Anla is 65% similar, and Anlb is 67% similar. (B) The aa sequences of Anla (aa 625-693) and Anlb (633-701) compared to the PvPR3 protein from Phaseolus vulgaris (aa 70-137). Both An I proteins have about 50% identity to this novel protein. This C-terminal region in all three proteins encodes a possible metal-binding domain.

	Fig. 4. The Ubil domains of An la and An I b do not undergo proteolytic processing in rabbit reticulocyte lysates. Separation of in vitro rabbitreticulocyte- synthesized proteins by SDS-PAGE is shown in panel A. Translation of sequences encoding the full-length Anla protein are in lane I. A mutant form of the Anla protein that lacks the N-terminal Ubil domain is in lane 2; this translation product serves as a size standard for the remaining C-terminal polypeptide of a proteolytically processed protein. Panel B (lane I) shows the separation of translation products from a mRNA that contains two contiguous Ub-encoding sequences (Agell et al., 1988) on a 0.1 M phosphate:6 M urea/O.l% SDS/l 5% polyacrylamide gel system (Ley, 1984). The results indicate that the diubiquitin polypeptide was proteolytically processed to form a Ub monomer (about 8.5 kDa). No unprocessed Ub dimers. expected to be about I7 kDa, were detected. Lanes 2 and 3 demonstrate that small molecular weight polypeptides from the translation of pSP64TAnla (lane 2) and pSP64T-AnlaAUbil (lane 3) were not detected. The asterisk at about I4 kDa indicates globin protein. Methods: The An/ sequences translated in vitro were cloned into the translation vector pSP64T (Krieg and Melton, 1984). The plasmids pSP64T-Anla and pSP64T-AnlaAUbil were used in a Promega (Madison, WI) TNTTM SP6 transcription-translation reticulocyte The insert in pSP64T-AnlaAUbil the first nt of An/a coding and therefore not encode N-terminal Ubil This insert pSP64T was by PCR of the codingsequencesusingtheSprimer(S-CCCCAG ATCTACCATGG CAATTAATACACGCAGAGTGCCG)andthe3’primer(S’-CCCAG AGATCTAGATTTTTGGAAGTTTG).TheS’oligoprimerencodesa Pro’“4+Ala replacement and also encodes a consensus eukaryotic translation initiation site just prior to this new Ala’@‘(Kozak, 1984a.b). Both of these primers contain an engineered BgllI site which facilitated cloning of the PCR-generated fragments into an unique Bg/ll site in pSP64T. Approximately I pg of each circular plasmid was added directly to the reticulocyte lysates (25 pl reaction volume) containing 45 pCi of [“SJmethionine with a specific activity of II20 Ciimmol (present in Tran3’S-label), After incubation for 2 h at 30 C, the translation products were separated by 0.1% SDS- 10% PAGE (Laemmli. 1970) or by a 0.1 M phosphate/6 M urea/O.l% SDS/15% polyacrylamide gel system (Ley, 1984). The gel was fixed in 50% methanol/IO% acetic acid for 30 min. soaked in distilled water for 30 min, Fluoro- HanceTM for 30 min. dried, and exposed to Kodak XAR-5 film at - 70’ c.