Analysis of U1 small nuclear RNA interaction with cyclin H.
TFIIH is a general transcription and repair factor implicated in RNA polymerase II transcription, nucleotide excision repair, and transcription-coupled repair. Genetic defects in TFIIH lead to three distinct inheritable diseases: xeroderma pigmentosa, Cockayne syndrome, and trichothiodystrophy, with xeroderma pigmentosa patients being highly susceptible to skin cancer. Earlier data revealed that the cyclin H subunit of TFIIH associates with U1 small nuclear RNA, a core-splicing component. In addition to its role in RNA processing U1 small nuclear RNA also regulates diverse stages of transcription by RNA polymerase II both in vivo and in vitro, including abortive initiation and re-initiation. Here we identify structural components of U1 and cyclin H implicated in the direct interaction and show how they affect function. Because of unique features of cyclin H we have developed a new methodology for mapping RNA interaction with the full-length cyclin H polypeptide based on electrospray ionization tandem mass spectrometry. We also demonstrate the importance of U1 stem-loops 1 and 2 for the interaction with cyclin H. Functional assays implicate the identified interaction with U1 in regulation of the activity of the cyclin H associated kinase CDK7.
O'Gorman
William
W
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.
Thomas
Benjamin
B
Kwek
Kon Yew
KY
Furger
Andre
A
Akoulitchev
Alexandre
A
D003432:Cross-Linking Reagents / Q000378:metabolism; D056748:Cyclin H; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D018983:DNA Footprinting; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D014158:Transcription, Genetic; D000095043:Cyclin-Dependent Kinase-Activating Kinase
C534751:CCNH protein, human; D003432:Cross-Linking Reagents; D056748:Cyclin H; D016213:Cyclins; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human