Alu-DNA repeat-binding protein p68 is a part of Alu-RNA containing alpha-RNP

An Alu-DNA repeat-binding protein with a molecular mass of 68 kDa (p68) is identified in the somatic human cell nucleoplasm. Gel mobility shift assay (GMSA), South-western blotting and affinity purification on DNA attached to the carrier were used in the identification. GMSA revealed multiple comple xes with the exponential dependence of their relative mobility. A narrow bi nding site of the p68 was revealed using synthetic oligonucleotides. It is located between the A-box and B-box of the RNA polymerase III promoter and is identical to that reported for the Alu-binding protein from human sperma tozoids. The same narrow binding site, the similarity of the isolation proc edure from germ and somatic cells, and similar binding properties and molec ular masses suggest homology of the two proteins. Antibodies raised against Alu-protein complexes led to hypershift of the complexes in GMSA and stain ed p68 in active fractions in human spermatozoids and in Alu-RNA-containing alpha-RNP particles. Immunofluorescence of a HeLa cell monolayer revealed an intranuclear dot pattern with the dots corresponding to euchromatin area s and some dots located at the cell periphery in the cytoplasm. alpha-RNP p articles bound Alu-DNA in vitro and contained p68 as shown using the immuno gold procedure. Alu-DNA binding activity was revealed in cytoplasm as well as in nucleoplasm. The possible nature of the main Alu-DNA binding protein and its involvement in the particle structure are discussed.