Abstract
Analyses of apolipoprotein II mRNA with chemical and enzymatic probes showed that double- and single-stranded regions were distributed uniformly along the mRNA except for a large (72 nucleotides) single-stranded region containing the translation stop codon. Secondary structure models constrained by the experimental data were made by varying the distance (along the mRNA) over which base pairing was allowed. Four prominent secondary structures were seen with restrictions of 165, 330, or 659 nucleotides suggesting that such structures from via local interactions over distances of 50-120 nucleotides. Predicted long range interactions involve only 2-3 base pairs while local interactions involve helices of 4-10 base pairs. Predicted helices of greater than or equal to 4 base pairs occur primarily within exons, raising the possibility that prominent secondary structures in mRNAs may be largely due to intraexonic base pairing. Tests of single- and double-stranded domains by oligonucleotide-directed RNase H cleavage and primer extension were in accord with the structure model and with nuclease and chemical modification data. The model predicting base pairing between the coding and the 3' noncoding regions was tested by RNase H cleavage followed by oligo(dT)-cellulose chromatography to separate 5' and 3' mRNA fragments. Most (82%) of the 5' fragment remained associated with the 3' noncoding region in a structure with a tm = 50 degrees C in 0.2 M Na+ suggesting that this stem could be stable in vivo. This stem may be stable in the isolated mRNA, but would likely occur transiently in polyribosomal apolipoprotein II mRNA due to ribosome transit through the 5' side of the stem. Alternate structures may occur in this region during ribosome transit and play a role in translation termination or in determining the susceptibility of the mRNA to degradation.
Highlights
Predicted Structures of Apolipoprotein I1 mRNA Constrained by Nuclease and DimethylSulfate Reactivity: Stable Secondary Structures Occur Predominantly in Local Domainsvia Intraexonic Base Pairing*
Four prominentsecondary structures were seen with restrictions of 165, 33o0r,659 nucleotides suggesting that such structures form via local interactions over avian liver in which apoII’ mRNA and VTG I1 mRNA are expressed at highlevels in laying hensorin roosters or immature birds after the administration of exogenous estrogen (Bergink et al, 1974; Chan et al, 1976)
Recent studies have shown that the degradation rates of apoI1 and VTG I1 mRNAs during hormone withdrawal are dramatically altered by the duration of prior estrogen treatment
Summary
Cleavagesproduced by S1 RNase were largely confirmed with one or more of the mRNA Structure Analyses other probes suggesting that single-stranded domains are not dramatically different at pH 5 as compared to pH 7.5. The. The specific enzymatic and chemical probes, RNases T1, double- andsingle-stranded regions aredistributed moder-. Cobra venom (CV) RNase a rather long single-stranded region bound by nucleotides wasused to map double-stranded regions. Several other regions showed no reactivity using apoII mRNA-specific DNA primers and reverse tran- with any of the probes suggesting that such regions may be scriptase was used to locate cleavage and chemical modifica- inaccessible in the three-dimensional structure of the mRNA. Five end-labeled One such region shown in Fig. 3 containsthe AUG translation probe-primers (I, 11,111,E, and G, Table I) spaced at approx- start codon. Modeling of ApoZI mRNA Secondary Structure throughout the 659-nucleotide apoII mRNA
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