Thalassaemia mutations within the 5'UTR of the human beta-globin gene disrupt transcription.
The mechanisms by which mutations within the 5' untranslated region (UTR) of the human beta-globin gene (HBB) cause thalassaemia are currently not well understood. We present here the first comprehensive comparative functional analysis of four 'silent' mutations in the human beta-globin 5'UTR, namely: +10(-T), +22(G --> A), +33(C --> G) and +(40-43)(-AAAC), which are present in patients with beta-thalassaemia intermedia. Expression of these genes under the control of the beta-globin locus control region in stable transfected murine erythroleukaemia cells showed that all four mutations decreased steady state levels of mRNA to 61.6%, 68%, 85.2% and 70.6%, respectively, compared with the wildtype gene. These mutations did not interfere with either mRNA transport from the nucleus to the cytoplasm, 3' end processing or mRNA stability. Nuclear run-on experiments demonstrated that mutations +10(-T) and +33(C --> G) reduced the rate of transcription to a degree that fully accounted for the observed lower level of mRNA accumulation, suggesting a disruption of downstream promoter sequences. Interestingly, mutation +22(G --> A) decreased the rate of transcription to a low degree, indicating the existence of a mechanism that acts post-transcriptionally. Generally, our data demonstrated the significance of functionally analysing mutants of this type in the presence of a full complement of transcriptional regulatory elements within a stably integrated chromatin context in an erythroid cell environment.
Sgourou
Argyro
A
Laboratory of Biology, Faculty of Medicine, University of Patras, Patras, Rion 261 10, Greece.
Routledge
Samantha
S
Antoniou
Michael
M
Papachatzopoulou
Adamantia
A
Psiouri
Lambrini
L
Athanassiadou
Aglaia
A
D020121:5' Untranslated Regions / Q000235:genetics*; D015870:Gene Expression; D005914:Globins / Q000235:genetics*; D006801:Humans; D009154:Mutation; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D017086:beta-Thalassemia / Q000235:genetics*
D020121:5' Untranslated Regions; D012333:RNA, Messenger; D005914:Globins