In previous studies of the ribosomal protein L32 mRNA, we demonstrated that a conserved polypyrimidine tract found in the 5'-untranslated region (5'-UTR) was required for translational regulation in vivo and that a 56-kDa protein (~5 6~~~) from T-lymphocytes specifically interacts with this sequence [Kaspar, R. L., Kakegawa, T., Cranston, H., Morris, D. R. & White, M. W. (1992) J. Biol. Chem. 267, Here we show that ~5 6~~~ binding to the L32 5'-UTR is complex and requires other 5'-UTR RNA sequences in conjunction with the polypyrimidine tract. Deletion and site-directed mutagenesis studies revealed that binding of ~5 6~~~ to the L32 5'-UTR requires a second RNA element, GGUGGCUGCC, 15 nucleotides downstream from the polypyrimidine tract. In contrast, L32 RNA transcripts altered in this downstream element were good substrates for binding of the polypyrimidine binding proteins from HeLa nuclear extracts, indicating that these proteins have RNA-binding specificities distinct from ~5 6~~' .Competition analysis demonstrated that ~5 6 "~~ will bind to DNA as well as RNA with identical sequence specificity and similar affinity. Single or double-stranded DNAs composed of the L32 5'-UTR sequences were found to specifically compete with L32 RNA transcripts for p56'd3'binding. The L32 5'-UTR downstream element, GGUGGCUGCC, which is required for ~5 6~~~ binding, has previously been implicated as a transcriptional element of the L32 gene. The ability of p56"" to bind this sequence as DNA or FWA suggests ~5 6~~~ may have a dual role in the regulation of ribosomal protein mRNA accumulation and translation.Keywords. Polypyrimidine; translation ; ribosome, RNA-binding protein; transcription.Ribosome biogenesis requires the synchronized production of roughly 70-80 RNA and protein components since, with rare exceptions, cells do not accumulate unassembled components. In mammalian cells, the balance of ribosomal proteins (r-proteins) is maintained by the rapid degradation of unassembled rproteins, while changes in r-protein synthesis are coordinated through mechanisms regulating r-protein gene expression (reviewed in [I]). Depending on the cell type, r-protein synthesis may be principally regulated at the level of r-protein mRNA accumulation or mRNA translation, although regulation by a combination of these levels is known to occur in cells such as mitogen-activated lymphocytes [2].The mechanism regulating the translational utilization of rprotein mRNA in mammalian cells is characterized by changes in the partitioning of the total cellular r-protein mRNA between two compartments, mRNP particles and polysomes [1]. In contrast to mRNAs which are mRNP-bound and heterogeneously loaded on polysomes [ 3 ] , the r-protein mRNAs show a distinct bimodal distribution between these compartments, with individual mRNAs found on polysomes whose size is consistent with the full loading of the respective mRNAs coding capacity [2, 41. Correspondence to M. W. White, Veterinary Molecular Biology, Fax: + I 406 994 4303. Abbreviations. r-proteins, r...
In previous studies of the ribosomal protein L32 mRNA, we demonstrated that a conserved polypyrimidine tract found in the 5'-untranslated region (5'-UTR) was required for translational regulation in vivo and that a 56-kDa protein (p56L32) from T-lymphocytes specifically interacts with this sequence [Kaspar, R. L., Kakegawa, T., Cranston, H., Morris, D. R. & White, M. W. (1992) J. Biol. Chem. 267, 508-514]. Here we show that p56L32 binding to the L32 5'-UTR is complex and requires other 5'-UTR RNA sequences in conjunction with the polypyrimidine tract. Deletion and site-directed mutagenesis studies revealed that binding of p56L32 to the L32 5'-UTR requires a second RNA element, GGUGGCUGCC, 15 nucleotides downstream from the polypyrimidine tract. In contrast, L32 RNA transcripts altered in this downstream element were good substrates for binding of the polypyrimidine binding proteins from HeLa nuclear extracts, indicating that these proteins have RNA-binding specificities distinct from p56L32. Competition analysis demonstrated that p56L32 will bind to DNA as well as RNA with identical sequence specificity and similar affinity. Single or double-stranded DNAs composed of the L32 5'-UTR sequences were found to specifically compete with L32 RNA transcripts for p56L32 binding. The L32 5'-UTR downstream element, GGUGGCUGCC, which is required for p56L32 binding, has previously been implicated as a transcriptional element of the L32 gene. The ability of p56L32 to bind this sequence as DNA or RNA suggests p56L32 may have a dual role in the regulation of ribosomal protein mRNA accumulation and translation.
The NADPH oxidase plays an important role in immune and nonimmune cell functions. Because rabbits represent an established model for studying a number of important disease processes that involve NADPH oxidase activity, we carried out studies to clone and sequence all five rabbit leukocyte NADPH oxidase genes. Comparison of the rabbit sequences with those of other species showed that, with the exception of p67phox, the rabbit phox proteins were highly conserved. In contrast, rabbit p67phox had a very divergent C-terminus and was 17 amino acids longer than any other known p67phox homolog. This was surprising, given the high degree of conservation among all of the phox proteins sequenced previously. To evaluate the functional consequences of this difference, wild-type rabbit p67phox and a mutated rabbit p67phox missing the C-terminal 17 amino acids were expressed and analyzed in a cell-free assay. Our results show that the full-length and truncated rabbit p67phox proteins were able to support oxidase activity, although the truncated form reproducibly supported a higher level of activity than full-length p67phox. These studies contribute to our understanding of the nature of the leukocyte NADPH oxidase in different species and will be valuable in future research using the rabbit model.
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