Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction.
Coding region determinant-binding protein (CRD-BP) binds to the 3'-UTR of microphthalmia-associated transcription factor (MITF) mRNA to prevent its targeted degradation by miR-340. Here, we aim to further understand the molecular interaction between CRD-BP and MITF RNA. Using point mutation in the GXXG motif of each KH domains, we showed that all four KH domains of CRD-BP are important for their physical association with MITF RNA. We mapped the CRD-BP-binding site in the 3'-UTR of MITF RNA from nts 1330-1740 and showed that the 49-nt fragment 1621-1669 is the minimal size MITF RNA for binding. Upon deletion of nts 1621-1669 within the nts1550-1740 of MITF RNA, there was a 3-fold increase in dissociation constant Kd, which further confirms the critical role sequences within nts 1621-1669 in binding to CRD-BP. Amongst the eight antisense oligonucleotides designed against MITF RNA 1550-1740, we found MHO-1 and MHO-7 as potent inhibitors of the CRD-BP-MITF RNA interaction. Using RNase protection and fluorescence polarization assays, we showed that both MHO-1 and MHO-7 have affinity for the MITF RNA, suggesting that both antisense oligonucleotides inhibited CRD-BP-MITF RNA interaction by directly binding to MITF RNA. The new molecular insights provided in this study have important implications for understanding the oncogenic function of CRD-BP and development of specific inhibitors against CRD-BP-MITF RNA interaction.
Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
Gerrit van
GV
Rensburg
Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
Sebastian
S
Mackedenski
Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
Chow H
http://orcid.org/0000-0001-6136-7058
CH
Lee
D020413:3' Untranslated Regions / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D006801:Humans; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017423:Sequence Analysis, RNA
D020413:3' Untranslated Regions; C406144:IGF2BP1 protein, human; C497234:MITF protein, human; D051739:Microphthalmia-Associated Transcription Factor; D012333:RNA, Messenger; D016601:RNA-Binding Proteins