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120s Biochemical Society Transactions ( I99 I) I 9 The indentification of protein-RNA interactions within the 5'untranslated region of human preproinsulin mRNA. STUART W . KNIGHT and KEVIN DOCHERTY of Department of Medicine, University Birmingham, Queen Elizabeth Hospital, Birmingham, B15 2TH, U.K. The biosynthesis of insulin is controlled at both transcriptional [l] and translational levels [2]. A rise in the plasma level of glucose triggers an increase in both de novo insulin synthesis and secretion of stored insulin. In the short term the increase in biosynthesis is actinomycin D resistant implying the usage of pre-existing mRNA [3]. There is a redistribution of alreadyinitiated preproinsulin mRNA from the cytoplasmic ribosome fraction to the membrane-bound polysome fraction mediated by the signal recognition particle [4] in response to increasing glucose levels. The specificity of the glucose-induced increase in insulin biosynthesis is probably mediated via interactions with the preproinsulin mRNA. The 5'untranslated region (5'UTR) of the human preproinsulin mRNA consists of 59 nucleotides upstream of the AUG translation start codon [5]. There is a high degree of sequence homology between species in this region: the rat I and I1 5'UTRs have respectively 59.4% and 62.5% homology with human mRNA whereas monkey has 88.1% homology [6]. The region does not have any palindromic sequences which favour the creation of a hairpin loop secondary structure [7] or an upstream ORF 181. Both of these features have been implicated in the regulation of translation. We were interested in evaluating the role of the human preproinsulin 5'UTR mRNA sequences in the glucose-mediated control of translation. To investigate protein binding to the 5'UTR of the human preproinsulin mRNA a set of RNA oligonucleotides were synthesised corresponding to this region. The RNAprotein interactions were then analysed using gel retardation. Five overlapping RNA oligonucleotides corresponding to the 5'UTR were synthesised from DNA oligo templates: A(-1 to -2O), B(-11 to -30), C(-21 to -40), D(31 to - 5 0 ) , and E(-41 to -59). These DNA templates contained the 17 nucleotide T7 RNA polymerase primer recognition sequence at their 3' end facillitating the transcription of the RNA oligos using TI [9]. The transcription RNA polymerase reaction gave a mixture of RNA sequences of varying length; the full length RNA was purified using a denaturing acrylamide gel. The RNA-protein interactions were then assessed by gel retardation using a native 6% polyacrylamide gel. For the preparation of protein extracts cells were lysed in ice cold 1% Triton X-100, 25mM Tris-HCL (pH 7.5), 40mM KC1 in the presence of protease inhibitors, and then centrifuged at 100,000 g for 60 minutes at 4" C to remove cell debris and membranes. Protein extracts from both non-insulin secreting cell lines (monkey kidney COS 7 and human liver HEP G2), and insulin secreting cell lines (rat pancreatic pcell RIN m5f and the Syrian hamster HIT T15 cell line) were utilised. The COS I and HEP G2 extracts gave banding patterns of greater than two retarded bands for each RNA sequence, RIN m5F and HIT T15 extracts gave much less well defined bands, but this may be due to the presence of nuclear proteins or lower abundance of the particular proteins in these extracts. The sequence specificities of these protein-RNA interactions were determined using excess cold RNA sequence. The interaction of protein(s) with RNA sequences A (-1 to -20) and E (-41 to -59) were abolished by prior incubation with excess concentrations of all other unlabelled RNA sequences. RNA sequence B (-11 to -30) and C (-21 to -40) were specifically competed by excess A (-1 to20), B (-11 to -30) and C (-21 to -40). The interaction of protein(s) with RNA sequence C (-21 to -40) were competed with excess C (-21 to -40) and D (-31 to -50), while the proteins binding to RNA sequence D (-31 to -50) were competed with excess C (-21 to -40), D (-31 to -50) and E (-41 to -59). These data indicate two sites of specific protein interactions within the human preproinsulin mRNA 5' UTR localised in the regions (-11 to -30) and (-31 to50). These preliminary studies have identified sequence-specific protein-RNA interactions within the human preproinsulin mRNA 5'UTR. The identity of the proteins and the significance of their interaction remains to be elucidated. The cell-type distribution of these RNAbinding proteins is not restricted to insulin secreting cells. This lack of cell sepecific expression does not exclude these proteins from a role in the cellular mechanism of preproinsulin translational control. Further information concerning the molecular weight of the RNA-binding proteins involved can be obtained by UVcrosslinking and SDS-PAGE analysis. S.W.Knight is supported by a Medical Research Council research studentship 1. Boam, D.S.W., Clark, A.R. & Docherty, K. (1990) J. Biol. Chem. 265, 8285-8296. 2. Itoh, N. & Okamoto, H. (1980) Nature 283, 100-102. 3. Permutt, A. & Kipnis, D. (1972) J. Biol. Chem. 247, 1194-1199. 4. Welsh, M., Scherberg, N., Gilmore, R., & Steiner, D.F. (1983) Biochem. J. 235, 459-467. 5. Bell, G.I., Pictet, R. L . , Rutter, W. J., Cordell, B., Tischer, E. & Goodman, H. M. (1980) Nature 284, 26-32. 6. Bell, G.I. & Sanchez-Pescador, R. (1984) Diabetes 33, 297-300. 7. Aziz, N. & Munro, H. N. (1987) Proc. Natl. Acad. Sci. USA 84, 8478-8482. 8. Hinnebush, A. G. (1984) Proc. Natl. Acad. Sci. USA 81, 6442-6446. 9. Milligan, J., Groebe, N.R., Witherell, G. W. & Uhlenbeck, 0. C. (1987) Nucleic Acids Res. 15, 8783-8798.