Lymphocyte p56L32 is a RNA/DNA-binding protein which interacts with conserved elements of the murine L32 ribosomal protein mRNA.
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.
Severson
W E
WE
Veterinary Molecular Biology, Montana State University, Bozeman 59717, USA.
Mascolo
P L
PL
White
M W
MW
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002417:Cattle; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D008214:Lymphocytes / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics*; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000737:chemistry
D017931:DNA Primers; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C041806:ribosomal protein L32; D004247:DNA