Cell, Vol. 27, 543-553, December 1981 (Part 2). Copyright 0 1981 by MIT Unstable P-Globin mRNA in mRNA-Deficient p” Thalassemia Lynne E. Maquat, Alan J. Kinniburgh, Eliezer A. Rachmilewitz* and Jeffrey Ross McArdle Laboratory for Cancer Research University of Wisconsin 450 N. Randall Avenue Madison, Wisconsin 53706 * Department of Hematology Hadassah University Hospital, Mount-&opus Jerusalem, Israel Summary The molecular defect in four Kurdish Jews with homozygous, mRNA-deficient $ thalassemia was investigated. Electrophoretic profiles of pulse- labeled (Y- and &globin RNAs are similar to those of non-thalassemics; therefore, at least one of the thalassemic P-globin alleles is transcribed. During a 30 min actinomycin D chase, most of the (Y- and fl-globin mRNA precursors and processing inter- mediates are converted to mRNA-sized RNA. Thal- assemic and non-thalassemic fi-globin RNAs are indistinguishable, as determined by Sl nuclease mapping and RNA blotting. Non-thalassemic /I-glo- bin mRNA is stable during a 30 min actinomycin chase, but 30%-75% of the thalassemic mRNA- sized molecules is degraded during that period. We conclude that the absence of P-globin mRNA in this disease results from rapid turnover of /?-globin mRNA-sized molecules. Introduction The p thalassemias are a heterogeneous group of inherited anemias that affect /I-globin chain produc- tion in man (for review see Weatherall and Clegg, 1979). In ,B’ thalassemia, erythrocytes contain re- duced levels of structurally normal ,&globin mRNA and protein. This phenotype results from inefficient processing of the /?-globin mRNA precursor to mRNA (Kantor et al., 1980; Maquat et al., 1980; Benz et al., 1981). Perhaps the partial splicing reactions that re- move intervening sequences (IVS) from primary tran- scripts (Kinniburgh et al., 1978; Kinniburgh and Ross, 1979 and manuscript in preparation; Avvedimento et al., 1980; Maquat et al., 1980; Grosveld et al., 1981; Kinniburgh, unpublished data) occur more slowly in ,&’ thalassemic cells than in normal cells (A. J. Kinni- burgh and L. E. Maquat, unpublished data; Spritz et al., 1981; Westaway and Williamson, 1981). /3’ thalassemia is a severe anemia characterized by the absence of ,&globin protein synthesis in reticulo- cytes (Conconi et al., 1972; Dow et al., 1973; Prit- chard et al., 1976; Ramirez et al., 1976; Benz et al., 1978). There are two classes of p” thalassemia, based on mRNA levels: those in which no P-globin mRNA is detected by molecular hybridization analysis (Forget et al., 1974; Ottolenghi et al., 1975; Comi et al., 1977; Godet et al., 1977; Benz et al., 1978; Old et al., 1978; Ramirez et al., 1978); and those in which P-globin mRNA nucleotide sequences are detected (Kan et al., 1975; Forget et al., 1976; Ramirez et al., 1976; Ot- tolenghi et al., 1977; Benz et al., 1978; Old et al., 1978; Comi et al., 1977). In some cases the ,&globin mRNA is translated in vitro in the presence of trans- lation factors from normal cells (Conconi et al., 1972; Dreyfus et al., 1972; Rowley and Kosciolek, 1972). In other cases the /3-globin mRNA cannot be translated and may contain nonsense mutations (Conconi et al., 1972; Temple et al., 1977; Chang and Kan, 1979; Chang et al., 1980). There are no large deletions or rearrangements of the /3-globin genes in most (Otto- lenghi et al., 1975; Ramirez et al., 1976; Tolstoshev et al., 1976; Godet et al., 1977; Ramirez et al., 1978), but not all (Flavell et al., 1979; Orkin et al., 1979, 1980) cases of ,@’ thalassemia. It seemed likely that the phenotype of mRNA-defi- cient j3” thalassemia resulted from a mutation (or mutations) that affects transcription, RNA processing, RNA transport (nucleus to cytoplasm), mRNA stability or a combination thereof. We have investigated p- globin RNA processing and mRNA stability in Jewish patients with homozygous p” thalassemia (Cividalli et al., 1977). These patients emigrated to Israel from Kurdistan Province. Their P-globin genes do not have large deletions (Ramirez et al., 1975). Their reticulo- cytes, like those of patients from southern Italy, Asia and Algeria (Forget et al., 1974; Forget et al., 1976; Tolstoshev et al., 1976; Godet et al., 1977), contain little (less than 3% of normal), if any, ,&globin mRNA (Ramirez et al., 1978). The results indicate that at least one of the @globin alleles is transcribed in each patient. The P-globin mRNA precursor is polyadenyl- ated and is indistinguishable from non-thalassemic precursor, as determined by Sl nuclease mapping and RNA blotting techniques. The precursor is proc- essed to mRNA-sized RNA, but the half-life of this RNA is only 30 min. The instability of mRNA-sized molecules in the nucleated bone marrow erythroid cell accounts for the essential absence of ,&globin mRNA sequences in the peripheral blood reticulocyte. Results Hematologic Studies of Patients All four of the homozygous ,f3” thalassemic patients are severely anemic (Table 1) and are dependent upon transfusions to maintain a peripheral blood he- moglobin concentration of 9 g/dl (normal is 12-l 4 g/ dl). The absence of @globin chain synthesis in periph- eral blood reticulocytes without compensating pro- duction of y-globin chains is diagnostic of p” thalas- semia (see Cividalli et al., 1977 for more detailed clinical analyses of M. B. and S. R.). The P/a globin mRNA ratio is approximately one in normal individuals