Apolipoprotein II messenger RNA. Transcriptional and splicing heterogeneity yields six 5'-untranslated leader sequences.

G S Shelness,D L Williams

doi:10.1016/s0021-9258(17)42788-8

Abstract

The structure of the mRNA for apolipoprotein II (apo-II), a major avian estrogen-responsive yolk protein, has been investigated. Primer extension using a cDNA probe-primer revealed six forms of mature apo-II mRNA. S1 and primer extension analysis with intron probes showed that only three of these forms arise due to multiple sites of transcription initiation on the apo-II gene. To investigate the basis for the additional forms of apo-II mRNA, we examined their nucleotide sequence. The sequence obtained between -10 and -42, relative to translation initiation, is identical to that reported by Wieringa et al. (Wieringa, B., AB, G., and Gruber, M. (1981) Nucleic Acids Res. 9, 469-499). At position -43, however, sequence heterogeneity appears. The minor form of the sequence starting at -43 and extending in a 5' direction corresponds exactly to the published mRNA sequence. The major form of the sequence has the insertion, 5'-CAG-3', at this position. The apparent basis for this heterogeneity is an unusual intron-exon border which violates the consensus sequence found in comparable positions in most eukaryotic genes by the existence of two adjacent 5'-CAG-3' triplets after the pyrimidine (Y)-rich track. The processing ratio between intron removal at the upstream and downstream AG dinucleotide is approximately 2.5:1. This result demonstrates that the splicing mechanism allows spatial flexibility in the positioning of the highly conserved YAG triplet within the consensus sequence at the 3' splice site.

Highlights

From the Department of PharmacologicalSciences, State University ofNew York ut Stony Brook, Stony Brook, New York 11794-8651
Primer extension using a Apo-I1 is well suited for the analysis of estrogen-regulated cDNA probe-primerrevealedsix forms of mature apo-gene expression due to the compact size of the apo-I1 gene
Work to date demonstrates that apo-I1 mRNA tranapo-I1gene

Summary

RESULTS

42 "C, 135 mM KCI, 10 mM MgCl,, 0.7 mM dGTP, dATP, dTTP, and Mapping the 5' Terminus of Apo-11 mRNA by Primer dCTP, and 5 mM dithiothreitol (final volume, 40 pl). To precisely map the 5' end of the matureapo-I1 transcriptase were added to each sample and incubation was carried message, primer extension [31,32,33] wasperformed following out for 1.5 h at 42 "C. Followingthe addition of EDTA to 15 mM, the the experimental strategy shown in Fig. 1T.he 5' end-labeled reaction was made 0.2 N NaOH and incubation was continued for 1 cDNA primer used in this experiment (probeA) is derived h. The S1-protected fragments were further purified oy gene).The single stranded 5' end-labeled probe A (* indicates location treatment with 0.2 N NaOH, followed by neutralization, phenol of 32Plabel) is synthesized and annealed to 3 pgof liver RNA as extraction, and ethanol precipitation as described above for primer described under "Experimental Procedures." Following treatment extension mapping of apo-I1 precursor mRNC,. B maps on the apo-I1 mRNA to position -10 with respect to the initiationof translation [13]and is situated approximately

G GA lATl CT 2C 2

Findings

DISCUSSION