Cleavage of the Ewing tumour-specific EWSR1-FLI1 mRNA by hammerhead ribozymes.
BACKGROUND
Ewing family tumours (EFT) are the second most common bone tumours in children and adolescents. In the majority of EFT, EWSR1-FLI1 (Ewing sarcoma breakpoint region 1-Friend leukaemia virus integration 1) fusion proteins can be detected and EWSR1-FLI1 substantially contributes to the malignant phenotype of EFT. Therefore, inactivation of EWSR1-FLI1 is an interesting strategy for EFT therapy.


METHODS
A ribozyme with specificity for EWSR1-FLI1 was developed and the activity in vitro was investigated. Synthetic RNAs corresponding to EWSR1-FLI1 were used as substrates. In addition, the total RNA from EFT cells was used as substrate and the rapid amplification of cDNA ends method for the detection of the cleavage products was used.


RESULTS
The ribozyme cleaved the synthetic RNA in a sequence specific manner with high efficiency in vitro. Furthermore, the expected cleavage products were detected after digestion of the total cellular RNA with this ribozyme. A point mutation in the catalytic centre of the ribozyme abolished enzymatic activity.


CONCLUSIONS
The RNA corresponding to EWSR1-FLI1 is accessible for ribozyme mediated inactivation and ribozymes are able to cleave EWSR1-FLI1 specific RNA in the presence of a high background of normal cellular RNAs.
Hühn
Regina
R
Department of Paediatrics, Children's Cancer Research Centre, Martin-Luther-University Halle-Wittenberg, D-06097 Halle, Germany.
Staege
Martin S
MS
Hesse
Manuela
M
Liebig
Beate
B
Burdach
Stefan E G
SE
D001483:Base Sequence; D001859:Bone Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics* / Q000378:metabolism; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics* / Q000378:metabolism; D034802:RNA-Binding Protein EWS; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012512:Sarcoma, Ewing / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
D002148:Calmodulin-Binding Proteins; C500986:EWSR1 protein, human; C078433:FLI1 protein, human; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D034802:RNA-Binding Protein EWS; D016601:RNA-Binding Proteins; C427204:hammerhead ribozyme