Novel translational control through an iron-responsive element by interaction of multifunctional protein YB-1 and IRP2.
The eukaryotic Y-box-binding protein YB-1 functions in various biological processes, including DNA repair, cell proliferation, and transcriptional and translational controls. To gain further insight into how human YB-1 plays its role in pleiotropic functions, we here used two-hybrid screenings to identify partners of this protein; the results showed that YB-1 itself, iron-regulatory protein 2 (IRP2), and five ribosomal proteins each served as partners to YB-1. We then examined the biological effect of the interaction of YB-1 and IRP2 on translational regulation. Both in vitro binding and coimmunoprecipitation assays showed the direct interaction of YB-1 and IRP2 in the presence of a high concentration of iron. RNA gel shift assays showed that YB-1 reduced the formation of the IRP2-mRNA complex when the iron-responsive element of the ferritin mRNA 5' untranslated region (UTR) was used as a probe. By using an in vitro translation assay using luciferase mRNA ligated to the ferritin mRNA 5'UTR as a reporter construct, we showed that both YB-1 and IRP2 inhibited the translation of the mRNA. However, coadministration of YB-1 and IRP2 proteins abrogated the inhibition of protein synthesis by each protein. An In vivo coimmunoprecipitation assay showed that IRP2 bound to YB-1 in the presence of iron and a proteasome inhibitor. The direct interaction of YB-1 and IRP2 provides the first evidence of the involvement of YB-1 in the translational regulation of an iron-related protein.
Ashizuka
Megumi
M
Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan.
Fukuda
Takao
T
Nakamura
Takanori
T
Shirasuna
Kanemitsu
K
Iwai
Kazuhiro
K
Izumi
Hiroto
H
Kohno
Kimitoshi
K
Kuwano
Michihiko
M
Uchiumi
Takeshi
T
D020121:5' Untranslated Regions; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D004260:DNA Repair; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D005293:Ferritins / Q000378:metabolism; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D035942:Iron Regulatory Protein 2; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D051379:Mice; D008957:Models, Genetic; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011817:Rabbits; D012270:Ribosomes / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D051840:Y-Box-Binding Protein 1
D020121:5' Untranslated Regions; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D005293:Ferritins; D007501:Iron; D005982:Glutathione Transferase; D035942:Iron Regulatory Protein 2