NATURE | VOL 414 | 20/27 DECEMBER 2001 | www.nature.com 929 letters to nature ................................................................. Stimulatory effect of splicing factors on transcriptional elongation Yick W. Fong & Qiang Zhou Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720-3206, USA .............................................................................................................................................. Transcription and pre-mRNA splicing are tightly coupled gene expression events in eukaryotic cells 1,2 . An interaction between the carboxy-terminal domain of the largest subunit of RNA polymer- ase (Pol) II and components of the splicing machinery is postu- lated to mediate this coupling 3±5 . Here, we show that splicing factors function directly to promote transcriptional elongation, demonstrating that transcription is more intimately coupled to splicing than previously thought. The spliceosomal U small nuclear ribonucleoproteins (snRNPs) interact with human tran- scription elongation factor TAT-SF1 (refs 6±9) and strongly stimulate polymerase elongation when directed to an intron- free human immunode®ciency virus-1 (HIV-1) template. This effect is likely to be mediated through the binding of TAT-SF1 to elongation factor P-TEFb 10 , a proposed component of the tran- scription elongation complex 11,12 . Inclusion of splicing signals in the nascent transcript further stimulates transcription, support- ing the notion that the recruitment of U snRNPs near the elongating polymerase is important for transcription. Because the TAT-SF1±U snRNP complex also stimulates splicing in vitro, it may serve as a dual-function factor to couple transcription and splicing and to facilitate their reciprocal activation. The Pol II C-terminal domain (CTD) is hyperphosphorylated during the transcription cycle at a time coincident with processive polymerase elongation 13 . Phosphorylation of the CTD during elongation is carried out primarily by P-TEFb, a heterodimer of CDK9 and cyclin T1 (CYCT1) 10 . P-TEFb is also a cellular cofactor for the HIV-1 TAT protein, and the TAT±P-TEFb complex stimu- lates HIV-1 transcriptional elongation by interacting with the TAR RNA structure located at the 59 end of the nascent viral tran- script 14,15 . Although the cyclin box located in the amino-terminal half of CYCT1 is essential as it contacts CDK9, TATand TAR 16 , the CYCT1 C-terminal half has also been shown to contribute signi®- cantly to both basal and TAT-stimulated HIV-1 transcription 17,18 . The importance of the CYCT1 C-terminal domain prompted us to identify and analyse transcription factors that may associate with this domain. We therefore incubated nuclear extract of HeLa cells with immobilized GST or GST±CYCT1-C, which contained a CYCT1 C-terminal fragment (amino acids 402±701; ref. 18). Compared with the GST-depleted extract, extract depleted with GST±CYCT1-C showed a signi®cant decrease (about ninefold on average) in basal as well as TAT-dependent HIV-1 transcription from both templates: pHIV+TAR-G400, which contained the wild- type TAR element; and pHIVDTAR-G100, with a mutant TAR 19 (Fig. 1a). Because the level of TAT activation (about eightfold) was largely unaffected by the depletion, CYCT1-C probably removed general transcription activity. In nuclear extract of HeLa cells, CYCT1-C has been shown to interact with Pol II and TAT-SF1 18 , one or both of which may contribute to the activity depleted by CYCT1-C. TAT-SF1 has been identi®ed as a TAT cofactor as well as a general transcription elongation factor 6±9 . Because it is highly enriched in a partially puri®ed Q-Sepharose fraction of HeLa nuclear extract that contains a small portion of HeLa nuclear proteins (3±4%), including Pol II 8 , we examined whether this fraction can complement the CYCT1-C- depleted extract in transcription. HIV-1 transcription was restored by the addition of the Q fraction pre-depleted with GST, but not with GST±CYCT1-C (Fig. 1a), suggesting that the activity depleted from HeLa nuclear extract by CYCT1-C also exists in the Q fraction. Western analysis revealed a quantitative removalof TAT-SF1 from the CYCT1-C-depleted Q fraction (Fig. 1b). However, CYCT1-C only partially removed SPT5, an elongation factor reported to bind TAT-SF1 (ref. 6), and it removed very little Pol II or the TFIIF subunit RAP30. Thus, Pol II was probably not responsible for the activity depleted by CYCT1-C. Rather, the CYCT1-C-depleted activity seemed to associate with TAT-SF1, because the immunoprecipitation of TAT-SF1 and its associated factors from the Q fraction with anti-TAT-SF1, but not with preimmune antibodies, restored both basal and TAT-depen- dent HIV-1 transcription to a CYCT1-C-depleted nuclear extract (Fig. 1c). Because these reactions measured transcription from two G-less cassettes (G-400 and G-100) located about 1 kilobase (kb) downstream of the HIV-1 promoter 19 , the anti-TAT-SF1 immuno- precipitates were postulated to transactivate at the level of elongation. To con®rm this, we performed an assay involving discontinuous – – –– –+–+–+ –– ++ ++ – + –+ – + –+ c IP from Q fraction: IP from Q fraction: Pre- immune Anti- TAT-SF1 Anti-TAT-SF1 Preimmune NE depleted with CYCT1-C NE depleted with CYCT1-C TAT: 1–82 1,241–1,495 +TAR-G400 ∆TAR-G100 GST GST-CycT1-C GST GST– CYCT1-C Depleted NE: Depleted Q: TAT: a GST GST–CYCT1-C b TAR X +1 +955 +1,067 G-LESS pHIV∆TAR-G100 TATA Sp1 TATA TAR +1 +955 +1,340 pHIV+TAR-G400 G-LESS Transcription templates d ∆TAR-G100 +TAR-G400 Anti-Pol II Anti-SPT5 Anti-TAT-SF1 Anti-RAP30 Depleted Q: Figure 1 The CYCT1 C-terminal domain interacts with a TAT-SF1-associated transcription elongation activity. a, HeLa nuclear extract (NE) and the Q-Sepharose fraction (Q) were depleted with immobilized GST or GST±CYCT1-C and then incubated with templates pHIV+TAR-G400 and pHIVDTAR-G100 in reactions with or without Tat. The RNase T1-resistant G-less transcripts derived from the two templates are indicated. b, The levels of the indicated proteins in Q depleted with GST or GST±CYCT1-C were analysed by western blotting. c, The Q fraction was subjected to immunoprecipitation with preimmune or anti-TAT-SF1 antibody beads. The immunoprecipitates (IP) were analysed in reactions containing the CYCT1-C-depleted NE as in a. d, A discontinuous hybridization followed by RNase protection assay 20 was performed to analyse the level of transcription from template pHIV+TAR at two different distances from the initiation site. Reactions contained the CYCT1-C-depleted NE and the indicated immunoprecipitates. Numbers denote the 59 and 39 extent of the protected RNA fragments. © 2001 Macmillan Magazines Ltd