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 with splicing complexes as part of the [U4/U6.U5] tri-snRNP particle [Chabot et al. (1995) Nucleic Acids Res. 23, 3206-3213]. In mouse and Drosophila 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 (IIo). The CC-3 immunoreactivity of p255 was abolished by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, which is known to cause the dephosphorylation of the C-terminal domain of subunit IIo by inhibiting the TFIIH-associated kinase. The identity of p255 was confirmed by showing that CC-3-immunoprecipitated p255 was recognized by POL3/3 and 8WG16, two antibodies specific to RNA polymerase II largest subunit. Lastly, the recovery of RNA polymerase II largest subunit from HeLa splicing mixtures was compromised by EDTA, which prevents the interaction of p255 with splicing complexes and inhibits splicing. Our results indicate that p255 represents a highly phosphorylated form of RNA polymerase II largest subunit physically associated with spliceosomes and possibly involved in coupling transcription to RNA processing.
Vincent
M
M
Département de Médecine, Recherche en Sciences de la Vie et de la Santé, Université Laval, Ste-Foy, Québec, Canada. mvincent@rsvs.ulaval.ca
Lauriault
P
P
Dubois
M F
MF
Lavoie
S
S
Bensaude
O
O
Chabot
B
B
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002460:Cell Line; D004330:Drosophila; D029721:Drosophila Proteins; D004492:Edetic Acid / Q000494:pharmacology; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D051379:Mice; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism*; D012326:RNA Splicing; D017381:Spliceosomes / Q000201:enzymology*; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D051758:Transcription Factor TFIIH; D014157:Transcription Factors; D019473:Transcription Factors, TFII
D000911:Antibodies, Monoclonal; D029721:Drosophila Proteins; D046911:Macromolecular Substances; D047428:Protein Kinase Inhibitors; D035381:TATA-Binding Protein Associated Factors; C084976:Taf6 protein, Drosophila; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D004492:Edetic Acid; D011494:Protein Kinases; D012319:RNA Polymerase II