Posttranscriptional Defects in p-Globin Messenger RNA Metabolism in ,f-Thalassemia ABNORMAL ACCUMULATION OF p-MESSENGER RNA PRECURSOR SEQUENCES

A B S T R A C T The production of ,B-globin messenger RNA (mRNA) in /3-thalassemic erythroblasts was studied during pulse-chase incubations with [3H]uridine. Globin [3H]mRNA was quantitated by molecular hybridization to recombinant DNA probes complementary to globin mRNA and mRNA precursor sequences. Each of six patients with ,8+-thalassemia produced normal amounts of globin a and ,8 [3H]mRNA during a 20-min pulse incubation, but the 1/3a [3H]mRNA ratio declined to steady-state levels during a chase incubation, suggesting posttranscriptional defects in ,3-globin mRNA metabolism. ,/-globin mRNA precursor production was estimated by measurement of [3H]RNA sequences hybridizing to a pure DNA probe containing only the large intervening sequence (intron) of the /8-mRNA precursor. Four of the patients exhibited abnormal accumulation of 3H-,8-intron sequences (2-10 times normal), indicating abnormal posttranscriptional processing. In the remaining two patients, one ofwhom is known to carry a mutation in the small intron of the ,3-globin gene, accumulation of large 3H p3_ntron RNA and ,3-globin [3H]mRNA was normal in nuclei, but the ratio of,//a [3H]mRNA in cytoplasm was reduced, suggesting a different posttranscriptional defect in /3-mRNA processing. These findings imply the existence of heterogeneous posttranscriptional abnormalities in /8-globin mRNA metabolism in different patients with ,8-thalassemia. The initial rates of y- and 8-mRNA synthesis were low in all patients, suggesting that the low Preliminary results of these studies were presented at the National Meeting of the American Society for Clinical In-vestigation, 25-27 April 1981, San Francisco, Calif. (Clin. Res. 29: 512a [Abstr.]). sub-tracting control per washed the individual probes. Measurements of globin synthesis and steady-state mRNA levels. Globin biosynthesis was measured by incubating intact bone marrow cells and/or reticulocytes with [3H]leucine, as described (21, 22). Measurements of the steady-state amounts of globin mRNA present in the bone marrow aspirates at the time the sample was taken were accomplished by saturation hybridization techniques, using [32P]deoxy cytidine triphosphate (dCTP) purified a- and 83-cDNA (specific activity 8 x 107 cpm/,ug cDNA) under stringent hybridization conditions (78°C, 0.2 M sodium phosphate buffer, pH 6.8, 0.5% SDS); the methods used were those described by us earlier (22, 27). The steady-state S/a-mRNA ratios in total erythroblast RNA were measured by comparing the slopes of the saturation hybridization curves. The values presented in Results were determined using the same pulse-labeled RNA preparations used to detect newly synthesized

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