Unraveling the organization of the internal nuclear matrix: RNA-dependent anchoring of NuMA to a lamin scaffold.
Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.
Barboro
Paola
P
Istituto Nazionale per la Ricerca sul Cancro, I-16132, Genova, Italy.
D'Arrigo
Cristina
C
Diaspro
Alberto
A
Mormino
Michele
M
Alberti
Ingles
I
Parodi
Silvio
S
Patrone
Eligio
E
Balbi
Cecilia
C
D000818:Animals; D018797:Cell Cycle Proteins; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D034882:Lamins; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D015530:Nuclear Matrix / Q000737:chemistry* / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000032:analysis*; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000032:analysis; D051381:Rats; D012261:Ribonucleoproteins / Q000032:analysis; D008941:Spindle Apparatus / Q000737:chemistry* / Q000378:metabolism
D018797:Cell Cycle Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D034882:Lamins; D009687:Nuclear Proteins; C489081:Numa1 protein, rat; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D012313:RNA