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

Abstract

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.[Keywords: HIV-1 Tat; CycT1:CDK9/P-TEFb; c-Ski-interacting protein; transcription elongation; alternative splicing; HIV-1 TAR RNA] Supplemental material is available at http://www.genesdev.org.

The Werner Syndrome Helicase Is a Cofactor for HIV-1 Long Terminal Repeat Transactivation and Retroviral Replication

“…The Tat protein is acetylated on Lys 28 , Lys 50 , and Lys 51 by the transcriptional coactivators/acetyltransferases, p300/CBP and PCAF/hGCN5 (20 -22, 24, 27-30, 38), which has been shown to modulate Tat interactions with P-TEFb and Brm, as well as the ability of Tat for binding TAR-RNA (20,30,35,36,39). The formation of Tat/TAR-RNA/P-TEFb/PCAF complexes on the HIV-1 LTR stimulates Ser 2 -Ser 5 -phosphorylation of the RNA pol II carboxyl-terminal domain associated with increased transcriptional elongation (30,37,40). Importantly, our results demonstrate that the WRN helicase interacts and cooperates with Tat to transactivate the HIV-1 LTR to promote retroviral replication through the stable recruitment of PCAF/P-TEFb to Tat/ TAR-RNA transcription complexes.…”

“…NF-B and SP1) that synergize with the transactivator protein, Tat, bound to TAR-RNA, to promote retroviral gene expression in HIV-1-infected tissues, macrophages/monocytes, and CD4 ϩ T-lymphocytes (6 -17). The mechanism by which Tat/TAR-RNA complexes regulate transcription from the HIV-1 LTR involves the concerted recruitment of a plethora of cellular factors, including p300/CREB-binding protein (p300/CBP) (18 -25), PCAF/hGCN5 (20 -22, 26 -30), P-TEFb (30 -33), SET7/SET9 methyltransferases (34), SIRT1 (35), the Brm component of the SWI/SNF chromatin-remodeling com-plex (36), and the splicing factor, SKIP (37). The Tat protein is acetylated on Lys 28 , Lys 50 , and Lys 51 by the transcriptional coactivators/acetyltransferases, p300/CBP and PCAF/hGCN5 (20 -22, 24, 27-30, 38), which has been shown to modulate Tat interactions with P-TEFb and Brm, as well as the ability of Tat for binding TAR-RNA (20,30,35,36,39).…”

Stress-induced expression of p53 target genes is insensitive to SNW1/SKIP downregulation

“…The protein was found as part of interchromatin granule clusters in interphase nuclei [30] and its nuclear distribution paralleled the speckled pattern of the SR protein SC-35 [31]. Since SNW1 was shown to be associated with both ongoing transcription, through its association with P-TEFb [21], and splicing, through its participation in spliceosomal complexes [23], we were interested in obtaining a more detailed view of its localization in relation to sites of active transcription. Using confocal microscopy, we analyzed HeLa cell nuclei double stained for SNW1 and Pol II (Fig.…”

“…SNW1 was classified as a transcriptional co-regulator in a number of instances including the pathways of nuclear hormone receptors, Notch/CBF1, TGFβ/Smad2/3, MyoD, or pRb [22]. At the same time, it was shown to be part of catalytically competent spliceosomes [23] and to affect the splicing of both HIV-1 and endogenous transcripts [21,24]. In this report, we have asked whether SNW1 is required for the transcription of stress-induced genes of the p53 transcriptional program.…”

“…It is intriguing to ask whether any of the P-TEFb partners can substitute its function in cases of stress-induced Cdk9/CycT-independent gene expression. One such factor is SNW1, which was found to bind P-TEFb in nuclear extracts and Tat:P-TEFb on the HIV-1 promoter [21]. SNW1 was classified as a transcriptional co-regulator in a number of instances including the pathways of nuclear hormone receptors, Notch/CBF1, TGFβ/Smad2/3, MyoD, or pRb [22].…”