THE SURF-6 PROTEIN IS A COMPONENT OF THE NUCLEOLAR MATRIX AND HAS A HIGH BINDING-CAPACITY FOR NUCLEIC-ACIDS IN-VITRO

The recently identified novel protein SURF-6 is shown to be a componen t of the nucleolar matrix. Immunofluorescence analysis demonstrated th at SURF-6 was localized in residual nucleoli of in situ nuclear matrix preparations of mouse fibroblast cells (NIH 3T3), which were depleted of soluble and chromatin related proteins. Immunoblot analysis of bio chemical nucleolar subfractions confirmed that SURF-6 was present in t he nucleolar matrix fraction, and was absent from the fractions of sol uble proteins released by DNase or RNase. The capacity of SURF-6 to bi nd nucleic acids was investigated in vitro. Both endogenous SURF-6 fro m nuclear extracts and recombinant SURF-6 exhibited a strong binding c apacity for nucleic acids. It was shown that SURF-6 bound to both DNA and RNA, however, it showed stronger binding to RNA. The presence and nuclear distribution of SURF-6 during the cell cycle was explored by i mmunofluorescence analysis. It was shown that SURF-6 was always found in the nucleolus regardless of the phase of the tell cycle suggesting that it is a structural protein constitutively present in nucleolar su bstructures. The colocalization of SURF-6 with the major nucleolar pro teins B23 and fibrillarin, which are known to be involved in the proce ssing of ribosomal RNA (rRNA), was examined both in interphase and mit osis by double immunolabeling of cells. SURF-6 was found to be largely coincident with both proteins in interphase and it was distributed in the same cellular locations, namely the perichromosomal layer, the cy toplasm and prenucleolar bodies, in mitosis. However, colocalization o f SURF-6 with fibrillarin and B23 was only partial in interphase, and the dynamics of its localization was not completely the same as those of either fibrillarin or B23 during mitosis. Taken together, these res ults indicate that SURF-6 is a novel nucleolar matrix component and im ply that SURF-6 might support nucleolar matrix structure and function( s) via its association with nucleic acids. We propose that SURF-6 may be involved in processing of rRNA, based on its cytological characteri stics, but at stages in ribosomal biogenesis which are different from those for fibrillarin and B23.