THE NUCLEAR MATRIX PROTEIN P255 IS A HIGHLY PHOSPHORYLATED FORM OF RNA-POLYMERASE-II LARGEST SUBUNIT WHICH ASSOCIATES WITH SPLICEOSOMES

The monoclonal antibody CC-3 recognizes a phosphodependent epitope on a 255 kDa nuclear matrix protein (p255) recently shown to associate wi th splicing complexes as part of the [U4/U6.U5] tri-snRNP particle [Ch abot et al, (1995) Nucleic Acids Res. 23, 3206-3213], In mouse and Dro sophila cultured cells the electrophoretic mobility of p255, faster in the latter species, was identical to that of the hyperphosphorylated form of RNA polymerase II largest subunit (Ilo), The CC-3 immunoreacti vity of p255 was abolished by 5,6-dichloro-1-beta-o-ribofuranosylbenzi midazole, which is known to cause the dephosphorylation of the C-termi nal domain of subunit Ilo by inhibiting the TFIIH-associated kinase. T he identity of p255 was confirmed by showing that CC-3-immunoprecipita ted p255 was recognized by POL3/3 and 8WG16, two antibodies specific t o RNA polymerase II largest subunit. Lastly, the recovery of RNA polym erase II largest subunit from HeLa splicing mixtures was compromised b y EDTA, which prevents the interaction of p255 with splicing complexes and inhibits splicing. Our results indicate that p255 represents a hi ghly phosphorylated form of RNA polymerase II largest subunit physical ly associated with spliceosomes and possibly involved in coupling tran scription to RNA processing.