Splicing of albuminmRNA precursors in Nagase analbuminemic rats

Abstract

The Nagase analbuminemic rat (NAR) is a mutant strain of Sprague Dawley rat defective in serum albumin synthesis. This strain is characterized by a markedly reduced level of albumin mRNA in the liver cytoplasm and elevated levels of albumin RNA sequences in liver nuclei. These characteristics led to the suggestion that a post-transcriptional defect in albumin mRNA synthesis is the reason for analbuminemia in these animals.;In the present work, the primary structure of albumin mRNA in liver cytoplasm of NAR was examined by RNAase protection mapping, Northern blot analysis and PCR amplification, cloning and sequencing of cDNA covering the G, H and I exon region. The results of these experiments indicated absence of the H exon sequence from albumin mRNA in NAR liver.;Northern blot analysis of nuclear poly A{dollar}\sp{lcub}+{rcub}{dollar} and poly A{dollar}\sp{lcub}-{rcub}{dollar} RNA fractions of NAR liver compared to normal rat demonstrated that the H exon is skipped during splicing of albumin mRNA precursors. This is consistent with a model for splice site selection that involves scanning of intron sequences in a 3{dollar}\sp\prime{dollar} to 5{dollar}\sp\prime{dollar} direction. It can also be concluded that binding of splicesome components at one end of the H exon may be required for binding of other components at the other end of the same exon.;Southern blot analysis of cDNA synthesized from nuclear RNA of NAR and normal rat liver provided evidence suggesting that the H-I intron is spliced out from the primary transcript before the G-H intron. A strong branch site was mapped in the H-I intron but not in the G-H intron. This suggested that the strength of the branch site within a given intron may play an important role in determining the order of excision of this intron relative to adjacent introns, probably by determining the affinity for U{dollar}\sb{lcub}2{rcub}{dollar}SnRNP binding and initiation of splicesome assembly. On the other hand, a unidirectional, 5{dollar}\sp\prime{dollar} to 3{dollar}\sp\prime{dollar} or 3{dollar}\sp\prime{dollar} to 5{dollar}\sp\prime{dollar} order of excision of the complete intron set from the rat albumin gene primary transcript is argued against by Northern blot analysis of nuclear RNA of NAR and normal rat liver using probes from the Z-A, H-I and M-N introns. (Abstract shortened with permission of author.).