mRNAs in the erythroid tissues of transgenic mice - NCBI

The EMBO Journal vol.8 no.9 pp.2613 - 2619, 1989

Novel metabolism of several j0 -thalassemic 3-globin mRNAs in the erythroid tissues of transgenic mice

SaiKiang Lim, John J.Mullins1'2, Chuan-Min Chen, Kenneth W.Gross' and Lynne E.Maquat Departments of Human Genetics and 'Molecular and Cellular Biology, Roswell Park Memorial Institute, Buffalo, NY 14263, USA 2Present address: Pharmakologisches Institut der Universitat Heidelberg, Im Neuenheimer Feld 366, Heidelberg, FRG

Communicated by F.Grosveld

Mice that are transgenic for human f0-thalassemic ,B-globin alleles were generated in order to study how ,0-thalassemic mutations affect (-globin RNA metabolism in erythroid tissues. Three thalassemic alleles were studied, each of which harbors either a frameshift or a nonsense mutation. These mutations result in the premature termination of f-globin mRNA translation and an abnormally low level of 3-globin mRNA in the peripheral blood of thalassemic patients. Comparative studies of mice that express any of the f0-thalassemic transgenes with mice that express a normal human ,Bglobin transgene demonstrated that all three thalassemic mRNAs are metabolized in erythroid tissues abnormally. RNA blotting and Si nuclease transcript mapping revealed for each thalassemic transgene that (i) the full-length mRNA is abnormally short-lived and (ii) in addition to full-length mRNA, three more stable yet smaller RNAs are present. These smaller RNAs are polyadenylated and lack the mRNA 5' end. Key words: f3-globin/premature translation termination/ thalassemia/transgenic mice/truncated RNAs

level) of 3-globin mRNA in peripheral blood (Ramirez et al., 1978) since blood is comprised of cells that are derived from the bone marrow and that are, for the most part, transcriptionally inactive. To date, the degradative mechanism in bone marrow remains unknown. Interestingly, HeLa cells that have been transiently transfected with the Kurdish Jewish allele do not degrade the product mRNA at an abnormally fast rate (Maquat and Kinniburgh, 1985). This finding suggests that, like j-globin gene transcription, the rapid turnover of the Kurdish Jewish 3-globin mRNA may be particular to erythroid cells. To obtain an experimental system in which to study further how the premature termination of ,B-globin mRNA translation affects 3-globin RNA metabolism, three WBthalassemic alleles, including the Kurdish Jewish allele, were introduced into the germline of mice in linkage to all of the cis-acting sequences that are required for qualitatively appropriate temporal and cell-type expression. Here we report that murine erythroid cells, like human erythroid cells, metabolize thalassemic RNA differently than RNA from a normal human 3-globin gene. In the case of each of the three thalassemic transgenes, murine cells produce properly processed albeit abnormally short-lived mRNA as well as three smaller RNAs. These smaller RNAs are not detected in the erythroid cells of mice that express a normal human f-globin transgene. Since the common feature of these thalassemic alleles is the premature termination of human ,3-globin mRNA translation, a mechanism that recognizes this translational defect is functional in both human and murine erythroid cells.

Results Introduction The ,B0-thalassemias are a heterogeneous group of inherited human anemias that are attributable to mutations within or upstream of the (-globin gene (Orkin and Kazazian, 1984). The majority of these mutations are frameshift or nonsense mutations within an exon that have no effect on gene transcription or RNA splicing but result in the premature termination of (3-globin mRNA translation. Those mutations that have been investigated are invariably associated with an abnormally low level of 3-globin mRNA in the peripheral blood of affected individuals. As such, they most likely accelerate the rate of 1-globin mRNA decay. In fact, the best characterized mutation, a G C base-pair deletion within codon 44 that is prevalent in the Kurdish Jewish population (Kinniburgh et al., 1982), has been proven to reduce the half-life of fully processed (3-globin mRNA in thalassemic bone marrow cells (Maquat et al., 1981). Human ,B-globin mRNA normally has a half-life of at least 16.5 h (Ross and Pizarro, 1983), whereas the Kurdish Jewish mRNA has a half-life of 30 min (Maquat et al., 1981). This very short half-life explains the virtual absence ( < 2% of the normal -

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Transgene structure The three 30°-thalassemic alleles that were chosen for study consisted of (i) a Kurdish Jewish allele that harbors a single base pair deletion in codon 44 and produces an mRNA that terminates translation between codons 60 and 61; (ii) a Turkish allele that harbors a 2 bp deletion in codon 8 and produces an mRNA that terminates translation at codons 21-22; and (iii) an Italian allele that harbors a nonsense mutation in codon 39 (Table I). Transgenic animals (reviewed in Gordon and Ruddle, 1985; Wagner and Stewart, 1985; Palmiter and Brinster, 1986) were generated using the purified 7.7 kb HindIm fragment of either p(Kurd) l0°-thal or p(Ital) l0°-thal DNA, or the purified 6.0 kb HpaI-HindIII fragment of p(Turk) O°-thal DNA (Figure 1). These fragments consist of the thalassemic allele plus all flanking promoter and enhancer sequences that are required for qualitatively proper temporal and tissue-specific gene regulation (Townes et al., 1985; Magram et al., 1985; Kollias et al., 1986; Behringer et al., 1987; Trudel et al., 1987). A total of 14 independently derived transgenic (TG) lines were analyzed, each of which carried 5-250 copies of the intact allele (data not shown). 2613

S.Lim et al. Table I. 30-Thalassemic ,B-globin transgenes

Type of allele

Kurdish Jewish

Type of

Sequence change

mutation

in mRNA

Site of mRNA translation terminationa

Steady-state

frameshift

AC, codon 44

codons 60-61