A human splicing factor, SKIP, associates with P-TEFb and enhances transcription elongation by HIV-1 Tat.

HIV-1 Tat binds human CyclinT1 and recruits the CDK9/P-TEFb complex to the viral TAR RNA in a step that links RNA polymerase II (RNAPII) C-terminal domain (CTD) Ser 2 phosphorylation with transcription elongation. Previous studies have suggested a connection between Tat and pre-mRNA splicing factors. Here we show that the splicing-associated c-Ski-interacting protein, SKIP, is required for Tat transactivation in vivo and stimulates HIV-1 transcription elongation, but not initiation, in vitro. SKIP associates with CycT1:CDK9/P-TEFb and Tat:P-TEFb complexes in nuclear extracts and interacts with recombinant Tat:P-TEFb:TAR RNA complexes in vitro, indicating that it may act through nascent RNA to overcome pausing by RNAPII. SKIP also associates with U5snRNP proteins and tri-snRNP110K in nuclear extracts, and facilitates recognition of an alternative Tat-specific splice site in vivo. The effects of SKIP on transcription elongation, binding to P-TEFb, and splicing are mediated through the SNW domain. HIV-1 Tat transactivation is accompanied by the recruitment of P-TEFb, SKIP, and tri-snRNP110K to the integrated HIV-1 promoter in vivo, whereas the U5snRNPs associate only with the transcribed coding region. These findings suggest that SKIP plays independent roles in transcription elongation and pre-mRNA splicing.

[1]  W. Bickmore,et al.  Mammalian PRP4 Kinase Copurifies and Interacts with Components of Both the U5 snRNP and the N-CoR Deacetylase Complexes , 2002, Molecular and Cellular Biology.

[2]  F. Kashanchi,et al.  The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[3]  A. F. Neuwald,et al.  Purification and biochemical characterization of interchromatin granule clusters , 1999, The EMBO journal.

[4]  X. Y. Li,et al.  The HIV-1 Tat cellular coactivator Tat-SF1 is a general transcription elongation factor. , 1998, Genes & development.

[5]  D. Bentley,et al.  RNA Polymerase II Carboxy-Terminal Domain Phosphorylation Is Required for Cotranscriptional Pre-mRNA Splicing and 3′-End Formation , 2004, Molecular and Cellular Biology.

[6]  W. Craigen,et al.  Activation of cardiac Cdk9 represses PGC‐1 and confers a predisposition to heart failure , 2004, The EMBO journal.

[7]  F. Kashanchi,et al.  Coordination of Transcription Factor Phosphorylation and Histone Methylation by the P-TEFb Kinase during Human Immunodeficiency Virus Type 1 Transcription , 2004, Journal of Virology.

[8]  M. Garber,et al.  CDK9 Autophosphorylation Regulates High-Affinity Binding of the Human Immunodeficiency Virus Type 1 Tat–P-TEFb Complex to TAR RNA , 2000, Molecular and Cellular Biology.

[9]  A. Shilatifard,et al.  The RNA polymerase II elongation complex. , 2003, Annual review of biochemistry.

[10]  J. Lis,et al.  Coordination of transcription, RNA processing, and surveillance by P-TEFb kinase on heat shock genes. , 2004, Molecular cell.

[11]  M. Hayman,et al.  Differential effects of the Ski-interacting protein (SKIP) on differentiation induced by transforming growth factor-beta1 and bone morphogenetic protein-2 in C2C12 cells. , 2004, Experimental cell research.

[12]  P. Folk,et al.  Transcriptional coregulator SNW/SKIP: the concealed tie of dissimilar pathways , 2004, Cellular and Molecular Life Sciences CMLS.

[13]  Juri Rappsilber,et al.  Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex , 1998, Nature Genetics.

[14]  T. Rana,et al.  Tat stimulates cotranscriptional capping of HIV mRNA. , 2002, Molecular cell.

[15]  B. O’Malley,et al.  Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Mark Gerber,et al.  Transcriptional Elongation by RNA Polymerase II and Histone Methylation* , 2003, Journal of Biological Chemistry.

[17]  S. Dowdy,et al.  TAT-mediated protein transduction into mammalian cells. , 2001, Methods.

[18]  P. Puigserver,et al.  Direct coupling of transcription and mRNA processing through the thermogenic coactivator PGC-1. , 2000, Molecular cell.

[19]  Takashi Ito,et al.  SKIP modifies gene expression by affecting both transcription and splicing. , 2004, Biochemical and biophysical research communications.

[20]  P. Charneau,et al.  Fusion from without directed by human immunodeficiency virus particles , 1994, Journal of virology.

[21]  Tamás Kiss,et al.  7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes , 2001, Nature.

[22]  J. Beggs,et al.  Identification and characterization of Prp45p and Prp46p, essential pre-mRNA splicing factors. , 2003, RNA.

[23]  Qiang Zhou,et al.  The 7SK small nuclear RNA inhibits the CDK9/cyclin T1 kinase to control transcription , 2001, Nature.

[24]  S. Buratowski,et al.  Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing. , 2004, Molecular cell.

[25]  Or Gozani,et al.  A Potential Role for U2AF-SAP 155 Interactions in Recruiting U2 snRNP to the Branch Site , 1998, Molecular and Cellular Biology.

[26]  M. Giacca,et al.  Regulation of HIV‐1 gene expression by histone acetylation and factor recruitment at the LTR promoter , 2003, The EMBO journal.

[27]  R. Young,et al.  CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in Tat-activated human immunodeficiency virus type 1 transcription , 1997, Molecular and cellular biology.

[28]  Henning Urlaub,et al.  Small Nuclear Ribonucleoprotein Remodeling During Catalytic Activation of the Spliceosome , 2002, Science.

[29]  M. Mancini,et al.  The Cdk9 and cyclin T subunits of TAK/P-TEFb localize to splicing factor-rich nuclear speckle regions. , 2001, Journal of cell science.

[30]  Ping Wei,et al.  A Novel CDK9-Associated C-Type Cyclin Interacts Directly with HIV-1 Tat and Mediates Its High-Affinity, Loop-Specific Binding to TAR RNA , 1998, Cell.

[31]  K. Jones,et al.  Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. , 2004, Molecular cell.

[32]  V. Saudek,et al.  SKIP is an indispensable factor for Caenorhabditis elegans development , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[33]  E. Makarov,et al.  The 65 and 110 kDa SR‐related proteins of the U4/U6·U5 tri‐snRNP are essential for the assembly of mature spliceosomes , 2001, The EMBO journal.

[34]  D. Chen,et al.  Tat Activates Human Immunodeficiency Virus Type 1 Transcriptional Elongation Independent of TFIIH Kinase , 1999, Molecular and Cellular Biology.

[35]  Qiang Zhou,et al.  Stimulatory effect of splicing factors on transcriptional elongation , 2001, Nature.

[36]  J. Eisman,et al.  Interactions of SKIP/NCoA-62, TFIIB, and Retinoid X Receptor with Vitamin D Receptor Helix H10 Residues* , 2003, The Journal of Biological Chemistry.

[37]  D. Bentley,et al.  The link between mRNA processing and transcription: communication works both ways. , 2004, Experimental cell research.

[38]  Y. Fong,et al.  Relief of Two Built-In Autoinhibitory Mechanisms in P-TEFb Is Required for Assembly of a Multicomponent Transcription Elongation Complex at the Human Immunodeficiency Virus Type 1 Promoter , 2000, Molecular and Cellular Biology.

[39]  D. Price,et al.  Flavopiridol Inactivates P-TEFb and Blocks Most RNA Polymerase II Transcription in Vivo * , 2001, The Journal of Biological Chemistry.

[40]  Gary S Stein,et al.  Nuclear Coactivator-62 kDa/Ski-interacting Protein Is a Nuclear Matrix-associated Coactivator That May Couple Vitamin D Receptor-mediated Transcription and RNA Splicing* , 2003, Journal of Biological Chemistry.

[41]  Danny Reinberg,et al.  Histone lysine methylation: a signature for chromatin function. , 2003, Trends in genetics : TIG.

[42]  D. Chen,et al.  Transcription elongation factor P‐TEFb mediates Tat activation of HIV‐1 transcription at multiple stages , 1998, The EMBO journal.

[43]  D. Lockshon,et al.  Cotranscriptional Recruitment of the U1 snRNP to Intron-Containing Genes in Yeast , 2003, Molecular and Cellular Biology.

[44]  Michael R Green,et al.  HIV-1 Tat Stimulates Transcription Complex Assembly through Recruitment of TBP in the Absence of TAFs , 2005, PLoS biology.

[45]  Junmin Peng,et al.  Tat Modifies the Activity of CDK9 To Phosphorylate Serine 5 of the RNA Polymerase II Carboxyl-Terminal Domain during Human Immunodeficiency Virus Type 1 Transcription , 2000, Molecular and Cellular Biology.

[46]  Danny Reinberg,et al.  Elongation by RNA polymerase II: the short and long of it. , 2004, Genes & development.

[47]  S. Kameoka,et al.  p54nrb associates with the 5′ splice site within large transcription/splicing complexes , 2004, The EMBO journal.

[48]  T. Wada,et al.  The regulation of elongation by eukaryotic RNA polymerase II: a recent view. , 2001, Molecules and cells.

[49]  D. Luse,et al.  Newly Initiated RNA Encounters a Factor Involved in Splicing Immediately upon Emerging from within RNA Polymerase II* , 2004, Journal of Biological Chemistry.

[50]  J. Eisman,et al.  Ski-interacting protein, a bifunctional nuclear receptor coregulator that interacts with N-CoR/SMRT and p300. , 2004, Biochemical and biophysical research communications.

[51]  S. Hayward,et al.  Viral interactions with the Notch pathway. , 2004, Seminars in cancer biology.

[52]  T. Komori,et al.  Mammalian polycomb-mediated repression of Hox genes requires the essential spliceosomal protein Sf3b1. , 2005, Genes & development.

[53]  Ed Hurt,et al.  Cotranscriptional recruitment of the serine-arginine-rich (SR)-like proteins Gbp2 and Hrb1 to nascent mRNA via the TREX complex , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[54]  D. Ropers,et al.  Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA* , 2004, Journal of Biological Chemistry.