Hernandez Vida P, Higgins LeeAnn, Schwientek Melinda Sue, Fallon Ann M
Department of Entomology, University of Minnesota, 1980 Folwell Ave, St Paul, MN 55108, USA.
Insect Biochem Mol Biol. 2003 Sep;33(9):901-10. doi: 10.1016/s0965-1748(03)00095-x.
In eukaryotic cells, ribosomal protein S6 (RPS6) is the major phosphorylated protein on the small ribosomal subunit. In the mosquitoes Aedes aegypti and Aedes albopictus, the cDNA encoding RPS6 contains 300 additional nucleotides, relative to the Drosophila homolog. The additional sequence encodes a 100-amino acid, lysine-rich C-terminal extension of the RPS6 protein with 42-49% identity to histone H1 proteins from the chicken and other multicellular organisms. Using mass spectrometry we now show that the C-terminal extension predicted by the cDNA is present on RPS6 protein isolated from ribosomal subunits purified from Ae. albopictus cells. To expand our analysis beyond the genus Aedes, we cloned the rpS6 cDNA from an Anopheles stephensi mosquito cell line. The cDNA also encoded a lysine-rich C-terminal extension. However, in An. stephensi rpS6 the extension was approximately 70 amino acids longer than that in Ae. albopictus, and at the nucleotide level, it most closely resembled histone H1 proteins from the unicellular eukaryotes Leishmania and Chlamydomonas, and the bacterium Bordetella pertussis. To examine how the histone-like C-terminal extension is encoded in the genome, we used PCR-based approaches to obtain the genomic DNA sequence encoding Ae. aegypti and Ae. albopictus rpS6. The sequence encoding the histone-like C-terminal extension was contiguous with upstream coding sequence within a single open reading frame in Exon 3, indicating that the lysine-rich extension in mosquito RPS6 is not the result of an aberrant splicing event. An in silico investigation of the Anopheles gambiae genome based on the cDNA sequence from An. stephensi allowed us to map the An. gambiae gene to chromosome 2R, to deduce its exon-intron organization, and to confirm that Exon 3 encodes a C-terminal histone-like extension. Because the C-terminal extension is absent from Drosophila melanogaster, we examined a partial cDNA clone from a Psychodid fly, which shares a relatively recent common ancestor with the mosquitoes. The absence of the C-terminal extension in the Psychodid rpS6 cDNA suggests that the unusual RPS6 structure is restricted to a relatively small group of flies in the Nematocera.
在真核细胞中，核糖体蛋白S6（RPS6）是小核糖体亚基上主要的磷酸化蛋白。在埃及伊蚊和白纹伊蚊中，相对于果蝇的同源物，编码RPS6的cDNA含有300个额外的核苷酸。该额外序列编码一个100个氨基酸的、富含赖氨酸的RPS6蛋白C末端延伸，与鸡和其他多细胞生物的组蛋白H1蛋白具有42 - 49%的同一性。利用质谱分析，我们现在表明，cDNA预测的C末端延伸存在于从白纹伊蚊细胞纯化的核糖体亚基中分离出的RPS6蛋白上。为了将我们的分析扩展到伊蚊属之外，我们从斯氏按蚊的一个细胞系中克隆了rpS6 cDNA。该cDNA也编码一个富含赖氨酸的C末端延伸。然而，在斯氏按蚊的rpS6中，该延伸比白纹伊蚊中的大约长70个氨基酸，并且在核苷酸水平上，它与单细胞真核生物利什曼原虫和衣藻以及百日咳博德特氏菌的组蛋白H1蛋白最为相似。为了研究基因组中如何编码这种组蛋白样C末端延伸，我们使用基于PCR的方法获得了编码埃及伊蚊和白纹伊蚊rpS6的基因组DNA序列。编码组蛋白样C末端延伸的序列与外显子3中单个开放阅读框内的上游编码序列相邻，这表明蚊子RPS6中富含赖氨酸的延伸不是异常剪接事件的结果。基于斯氏按蚊的cDNA序列对冈比亚按蚊基因组进行的电子研究，使我们能够将冈比亚按蚊基因定位到2R染色体上，推断其外显子 - 内含子组织，并确认外显子3编码一个C末端组蛋白样延伸。由于黑腹果蝇不存在C末端延伸，我们检查了一种毛蠓的部分cDNA克隆，毛蠓与蚊子有相对较近的共同祖先。毛蠓rpS6 cDNA中不存在C末端延伸表明，这种不寻常的RPS6结构仅限于长角亚目相对较小的一组果蝇。
