Nucleolar localization of DGCR8 and identification of eleven DGCR8-associated proteins.
We identified 11 proteins that are associated with DGCR8 by immunoprecipitation assay and mass spectrometry. These proteins included Nucleolin, ILF3 and others, most of which appeared to be involved in the RNA processing or RNA transportation. We detected at least four kinds of protein complex, such as DROSHA/DGCR8, DGCR8/Nucleolin, DGCR8/ILF3 and ILF3/XPO5, by co-immunoprecipitation. The complex formation of DGCR8 with Nucleolin was dependent on RNA. Subcellular localization analysis by the immunofluorescent microscopy and immunoelectron microscopy indicated that DGCR8 locates at the nucleolus and small foci adjacent to splicing speckles in the nucleoplasm. Furthermore, the localization of DGCR8 at the nucleolus was changed by the inhibition of RNA transcription. Thus, our studies provided additional new evidence for the involvement of various protein complexes in the molecular mechanisms of apparently complex innate RNA interference machinery.
Shiohama
Aiko
A
Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan; GSP Center, The Leading Institute of Keio University, Ibaraki, Japan.
Sasaki
Takashi
T
Noda
Setsuko
S
Minoshima
Shinsei
S
Shimizu
Nobuyoshi
N
D002466:Cell Nucleolus / Q000378:metabolism* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008957:Models, Genetic; D050779:Nuclear Factor 90 Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic; D000097796:Nucleolin
D050779:Nuclear Factor 90 Proteins; D010750:Phosphoproteins; D011506:Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000097796:Nucleolin; C474043:DGCR8 protein, human; C429643:ILF3 protein, human