Stable repression of ribosomal protein Li synthesis in Xenopus oocytes by microinjection of antisense RNA ( oogenesis / ribosome biosynthesis / hybrid - arrested

The synthesis of an endogenous ribosomal protein, L1, is selectively and efficiently inhibited by microinjection of antisense Li RNAs into Xenopus oocytes. Repression of Li synthesis is achieved within 12 hr and is maintained for 48 hr. RNase-protection assays reveal the formation of RNARNA duplexes in vivo between the endogenous Li mRNA and injected antisense transcripts. Partial-length antisense RNAs, complementary to only the 3'-terminal region of Li mRNA, repress translation as effectively as a full-length antisense RNA, indicating that complementarity to the 5' region of Li mRNA is not required for efficient inhibition. The use of antisense RNA to repress synthesis of an endogenous ribosomal protein provides a functional basis for determining mechanisms that integrate ribosomal protein synthesis with ribosome assembly during oogenesis. Defining the mechanisms that coordinate the synthesis of ribosomal proteins is an integral aspect of understanding eukaryotic ribosome biosynthesis. The expression of Xenopus ribosomal protein genes is developmentally regulated. During oogenesis, synthesis of ribosomal proteins and rRNAs results in accumulation of sufficient ribosomes to support protein synthesis through development of the swimming tadpole, which consists of =106 cells. This elevated synthesis of ribosomal proteins during oogenesis and the requirement to impose somatic regulation of ribosome production during embryogenesis renders Xenopus a model system for the analysis of ribosomal protein gene expression during vertebrate development. Previous studies (1-3) have indicated that translational control is important for the coordinate regulation of these genes during Xenopus oogenesis and embryogenesis. Additional control ofribosomal protein Li accumulation is mediated at the level of splicing Li mRNA and by posttranslational proteolysis ofLi protein, which is potentially linked to 60S subunit assembly (4, 5). The mechanisms that balance the accumulation of ribosomal proteins and rRNAs with subunit assembly are ascertained most directly by analysis ofmutants in which synthesis of individual ribosomal components is eliminated. Such a genetic approach to the study of ribosome biosynthesis in Xenopus is restricted to homozygous anucleolate embryos, which do not synthesize 18S and 28S rRNAs (3). Mutants of Xenopus ribosomal protein genes do not exist, in contrast to minute mutants which have been mapped to several loci encoding ribosomal proteins in Drosophila (6-8). A series of recent studies have demonstrated the efficacy of using antisense RNA to repress the expression of specific genes in Xenopus oocytes (9, 10), mammalian cells (11-13), Drosophila (14, 15), and Dictyostelium (16). A common mechanism for repression involves duplex formation between an antisense RNA and its corresponding mRNA. These hybrids induce several pleiotropic effects including "hybrid arrest" of translation in vivo, decreased transport of transcripts from the nucleus to the cytoplasm, and increased turnover of mRNAs. The use of antisense RNA provides a means to complement analyses ofXenopus ribosomal protein gene regulation achieved by both the use of anucleolate embryos and the introduction ofexogenous genes and transcripts into oocytes. I have utilized microinjection of complementary RNAs (cRNAs) to Li mRNA in order to inhibit the expression of this ribosomal protein in fully grown oocytes. Efficient and selective repression of Li synthesis is achieved within 12 hr after microinjection of antisense RNA and is maintained for 48 hr. The formation ofRNA-RNA duplexes in vivo has been ascertained by RNase-protection assays. Formation of these hybrids appears to enhance the degradation of endogenous Li mRNA. These results indicate that repression of endogenous Li synthesis by antisense RNA provides a basis to resolve the levels at which production of individual ribosomal proteins is coupled to ribosome assembly during oogenesis. MATERIALS AND METHODS Plasmids and Synthesis of Antisense Transcripts with SP6 RNA Polymerase. A 1.3-kilobase (kb) cDNA corresponding to Li mRNA (5, 17) was isolated from an oocyte cDNA library constructed in XgtlO (18), using pXomi02 (19) as an Li-specific probe. The nucleotide sequence of this cDNA indicates that it contains the complete Li coding region and the entire 3' untranslated sequence but is lacking the first 10 residues of the 5' untranslated leader (17). Four antisense RNAs, aL1-1.3, aLl-1.16, aLi-0.41, and aLi-0.27, (with transcript lengths of 1.3, 1.16, 0.41, and 0.27 kb, respectively) were transcribed from the Li cDNA inserted in the "antisense" orientation of pSP65 and linearized with BamHI, HincII, Bgl II, and Nde I, respectively (Fig. 1). An additional antisense RNA, aL1-0.14, was synthesized by subcloning the 140-bp HincII-EcoRI fragment into pSP64 and linearizing with EcoRI (Fig. 1). In vitro synthesis of capped transcripts with SP6RNA polymerase was performed as described (10). RNAs were stored under ethanol at -20°C. Oocyte Microinjection, Protein Extraction, and Two-Dimensional Gel Electrophoresis. Li antisense RNAs were redissolved in sterile 88 mM NaCl for microinjection into stage VI oocytes isolated by manual dissection of mature Xenopus laevis ovaries. Oocytes were maintained in modified Barth's saline (MBS) at 18°C (21). Oocyte proteins were metabolically labeled by incubation of control and injected oocytes in MBS containing [35S]methionine (1 mCi/ml; 1 Ci = 37 GBq). Newly synthesized proteins were isolated from oocytes and 35S-labeled ribosomal proteins were analyzed by two-dimensional gel electrophoresis as described (1). Polyacrylamide gels were treated with Amplify (Amersham) and dried prior Abbreviations: bp, base pair(s); kb, kilobase(s); cRNA, complementary RNA; MBS, modified Barth's saline. 8639 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8640 Developmental Biology: Wormington