Lymphocyte p56L32 is A RNA/DNA-Binding Protein which Interacts with Conserved Elements of the Murine L32 Ribosomal Protein mRNA

Abstract

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...

AXenopus laevisHomologue of the La Autoantigen Binds the Pyrimidine Tract of the 5′ UTR of Ribosomal Protein mRNAsin Vitro: Implication of a Protein Factor in Complex Formation

“…Two of them, named A and B (57 kDa and 47 kDa), were found to interact with the pyrimidine tract, while the others, C and D (31 kDa and 24 kDa), were shown to bind the downstream region of the 5' UTR. Similarly, a protein of 57 kDa has been described to bind the pyrimidine tract of mouse rp-L32 mRNA (Kaspar et al, 1992;Severson et al, 1995). Here, we report the purification and characterization of the Xenopus 57 kDa protein that binds the L4-mRNA pyrimidine tract, and its identification as La, the Xenopus homologue (Scherly et al, 1993) of human La autoantigen (reviewed by Pruijn et al, 1990).…”

“…It appears that other proteins, which have been shown to interact with the 5' UTR of r-protein mRNA downstream of the pyrimidine tract (Cardinali et al, 1993), might be involved in the regulation. It has been found that this downstream region, together with the pyrimidine tract, is relevant in translational regulation in mammalian cells (Avni et al, 1994) and that mutations in this sequence alter protein binding (Severson et al, 1995). However, other elements that may be implicated in a more complex mechanism have to be kept in mind.…”

Feedback Inhibition of Poly(A)-binding Protein mRNA Translation

“…This protein may also be involved in providing the observed cell specificity toward the effect of TOP on Pabp1 mRNA translation. Although TOP binding proteins have been shown for some TOP mRNAs (55)(56)(57)(58)(59)(60)(61), their role in mRNA translation is uncertain, because the level of TOP binding activity does not change under conditions that inhibit translation of TOP mRNAs (37). It is not clear whether all TOP elements behave similarly.…”

Cellular nucleic acid binding protein binds a conserved region of the 5′ UTR of Xenopus laevis ribosomal protein mRNAs

“…In our previous studies some proteins were identi®ed to bind speci®cally in vitro to the pyrimidine tract and to the region downstream of the 5 H UTR of Xenopus L4 mRNA (Cardinali et al, 1993). Similarly, a protein has been described to bind the pyrimidine tract of mouse rp-L32 mRNA (Kaspar et al, 1992;Severson et al, 1995). In a recent study the rp-mRNA pyrimidine tract binding protein was identi®ed as the Xenopus homologue of human La autoantigen (Scherly et al, 1993) and its binding was dependent on the interaction with at least one protein factor.…”