The histone-like C-terminal extension in ribosomal protein S6 in Aedes and Anopheles mosquitoes is encoded within the distal portion of exon 3

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.