shift mutant

The EMBO Journal vol.5 no.5 pp.971 -977, 1986

Expression of a mutant vasopressin gene: differential polyadenylation and read-through of the mRNA 3' end in shift mutant

Richard Ivell, Hartwig Schmale, Brigitte Krisch1, Peter Nahke and Dietmar Richter Institut fur Zellbiochemie und klinische Neurobiologie, Universitat Hamburg, Martinistr. 52, 2 Hamburg 20, and 1Anatomisches Institut, Universitaet Kiel, 23 Kiel, FRG Communicated by D.Richter

Sequence analysis of cDNA clones derived from hypothalamic mRNA of diabetes insipidus (Brattleboro) rats shows that the vasopressin gene transcript also includes the single base deletion demonstrated in the gene. This causes a frame-shift in the C terminus of the vasopressin precursor with a reading frame open through the 3' end of the mRNA including the poly(A) sequence. Antibodies raised against a synthetic tetradecapeptide (CP-14) corresponding to the frame-shifted C terminus identified a product of mol. wt -26 000 in a reticulocyte lysate system programmed with Brattleboro hypothalamic mRNA. Immunohistochemical analysis indicated that a similar precursor is also present in vivo in neurones of the Brattleboro hypothalamus. Electrophoretic analysis of vasopressin mRNA from wild-type and mutant rat tissues revealed that (i) the hypothalamic mRNA from Brattleboro rats contains a longer stretch of poly(A) sequence than the wild-type strains; (ii) vasopressin mRNA is also present in the adrenal, ovary, testis and cerebellum, at very low levels; however, (iii) the extra-hypothalamic mRNA is considerably shorter than that in the hypothalamus because of a curtailed poly(A) sequence. Thus similar vasopressin gene transcripts are subject to a tissue-specific differential

polyadenylation. Key words: vasopressin-like precursor/oxytocin mRNA/diabetes inspidus/Brattleboro rat/gonadal neuropeptides

Introduction The nonapeptide hormone vasopressin is synthesized in the magnocellular neurones of the hypothalamus as part of a composite precursor which also includes a carrier protein neurophysin - and a glycopeptide. Hypothalamic vasopressin is released from the neurohypophysis and is responsible for regulating water resorption in the distal kidney tubules. Its absence in the syndrome of vasopressinsensitive diabetes insipidus leads to the excretion of large volumes of dilute urine and consequently to an increased fluid intake. This human disease can be studied in a model system, the Brattleboro rat, where vasopressin appears to be completely lacking from the nerve terminals of the posterior pituitary. The defect is autosomally recessive and seems only to affect the vasopressinergic neurones. Recently, the genes for both wildtpe and Brattleboro rat vasopressin precursors have been isolated and sequenced, and shown to differ by the absence of a single nucleotide in the second exon of the gene (Schmale and Richter, 1984). The consequence of this mutation is to produce a mRNA which includes a frame-shift in the 3' protein-coding region, and IRL Press Limited, Oxford, England

a

frame-

lacks a stop codon, so that, in principle, ribosomes could continue translating into the 3' end of the mRNA including the poly(A) tail. Although vasopressin mRNA is transcribed in the hypothalamus of the Brattleboro rat and in cells transfected with the isolated mutant gene, in neither system is the mRNA translated to give detectable levels of the peptide hormone (Schmale et al., 1984). This is in spite of the fact that the first two-thirds of the mRNA encoding the hormone and its proteolytic excision signals are intact. Some recent reports, however, suggest that the nonapeptide may be expressed in certain peripheral tissues in this mutant strain (Lim et al., 1984; Nussey et al., 1984). In this article we address this anomalous vasopressin expression at both transcriptional and translational levels. Results mRNA and mutated vasopressin precursor analysis The vasopressin gene sequence for the Brattleboro rat (Schmale and Richter, 1984) was obtained by the sub-cloning of a single 3.8-kb Hindu genomic DNA fragment. That the DNA sequence of the gene was reflected also in the hypothalamic transcripts was verified by cloning and sequencing a cDNA clone obtained from hypothalamic mRNA of the mutant rat (Figure 1). This mRNA encodes the predicted altered vasopressin precursor which is normal up until amino acid 63 of the neurophysin moiety; hence the precursor should contain the intact hormone as well as the signals for release and amidation of the hormone and also twothirds of the sequence of the carrier neurophysin. However, downstream of the deletion site a new reading frame starts which predicts a totally different amino acid sequence lacking recognizable proteolytic processing signals, glycosylation sites or codons for terminating protein synthesis. In theory, the mRNA should be translated through the poly(A) tail to yield a long Cterminal poly-lysine sequence. As previously described (Schmale et al., 1984) translation of the mutated mRNA in a cell-free protein synthesizing system and analysis of possible translation products by immunoprecipitation with anti-vasopressin or anti-neurophysin was ineffective in visualizing a normal-sized vasopressin precursor (mol. wt 19 000). However, longer exposures of the autoradiograms revealed that anti-vasopressin and anti-neurophysin reacted with a larger protein (mol. wt - 26 000; Schmale et al., 1984). The difference in size could be explained by read-through of the 3' non-coding region and - 150-200 adenosine residues of the poly(A) tail. To test whether the precursor synthesized in vitro did indeed correspond to that predicted from the mutant cDNA, a synthetic tetradecapeptide (CP-14) was made (Figure 1) correspond ing to part of the C terminus of the mutated precursor. This was conjugated to keyhole limpet hemocyanin and used to raise polyclonal antisera in rabbits. In the cell-free translation assay antiCP-14 antibodies precipitated a protein with a mol. wt - 26 000 (Figure 2, lane 5), which could be competed only by the synthetic peptide, CP-14, and was absent amongst the translation 971

R.Ivell et al.

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5 x 109 c.p.m./4g) as indicated in the legend to Figure 4. DNA fragments were radiolabelled using the random primer extension procedure (Feinberg and Vogelstein, 1983). To determine the length of the poly(A) tails, 10-15 ug of poly(A)+ RNA was incubated as described previously (Ivell and Richter, 1984b) with 1-2 jig of oligo(dT) and 4.5 units of RNase H for 1 h at 37°C. After ethanol precipitation the RNA was analysed as above.

60°C

Acknowledgements We would like to thank Dr. S.Morley for advice on the random primer radiolabelling method and H.Christiansen and H.Prien for skillful technical assistance and the Deutsche Forschungsgemeinschaft for financial support.

References

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