© 2011 Nature America, Inc. All rights reserved. NATURE STRUCTURAL & MOLECULAR BIOLOGY ADVANCE ONLINE PUBLICATION 1 ARTICLES The initial primary transcripts synthesized by RNA polymerase II (RNAPII) in eukaryotic cells undergo a series of modifications before functional messenger RNAs (mRNAs) exit the nucleus to be translated in the cytoplasm. Many of the modifications to pre- cursor messenger RNAs (pre-mRNAs) occur co-transcriptionally, including 5′ end capping, intron removal by splicing, and 3′ end formation. Since the first report that alterations in the C-terminal domain (CTD) of RNAPII’s largest subunit inhibited splicing and polyadenylation 1 , multiple physical connections between the tran- scription and RNA processing machineries have been established 2 . As RNAPII progresses from initiation through elongation to termi- nation 3 , the CTD binds and recruits distinct sets of factors, thereby coordinating each stage of transcription with specific RNA process- ing events 4–6 . It has long been realized that introns stimulate mRNA produc- tion 7,8 , but precisely how splicing may influence transcription remains unclear. Early studies proposed that components of the splicing appa- ratus stimulate transcription elongation through direct interaction with elongation factors 9–13 . A more recent study suggests that splicing factors may facilitate transcriptional elongation even before partici- pating in spliceosome assembly 14 . Here, we set out to determine how inhibition of splicing affects transcription dynamics using a combination of live-cell imaging, chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qrtPCR) approaches. Our data reveal that correct spliceosome assembly is linked to RNAPII pausing for transcriptional termination and that uncoupling these processes results in leakage of unspliced transcripts to the nucleoplasm. RESULTS An assay to image b-globin transcription in living cells We have used the well characterized human β-globin (HBB) gene as a model system to study the dynamics of transcription by RNAPII in real time. We engineered human osteosarcoma–derived cells (U2OS Tet-On) to stably express tetracycline-inducible β-globin transgenes integrated in tandem in the genome (Fig. 1a). In the presence of doxycycline, the tetracycline-controlled transactivator (rtTA) binds to the tetracycline response element (TRE) and activates transcrip- tion from the minimal CMV promoter. Six MS2 coat protein binding sequences containing a single base change in the RNA stem-loop that enhances MS2 protein binding 15 were inserted in frame into exon 3 of the HBB gene (Fig. 1a). The resulting expression plasmid pTRE- βWT-MS2exon was cotransfected with a puromycin resistance plas- mid into U2OS Tet-On cells. We isolated and screened drug-resistant colonies by transient transfection with a plasmid expressing a fusion between MS2 coat protein, the red fluorescent protein mCherry and a nuclear localization signal (NLS) to target the chimeric protein to the nucleus 16 . We selected one clone (U2OS-βWT#9) for further analysis, because most cells contained a single, easily detectable nuclear focus of mCherry-MS2 upon induction with doxycycline. In the absence of doxycycline, mCherry-MS2 was diffusely distributed within the nucleus, whereas following transcriptional activation, the fluorescent protein accumulated at a single focal site in the nucleus (Fig. 1b). To further characterize the subcellular distribution of β-globin tran- scripts, we carried out RNA fluorescence in situ hybridization (FISH) using a probe that hybridizes to the β-globin pre-mRNA (Fig. 1c–e). In nontransfected cells (Fig. 1c), only background levels of hybridization 1 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. 2 Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Saitama, Japan. 3 These authors contributed equally to this work. Correspondence should be addressed to M.C.-F. (carmo.fonseca@fm.ul.pt). Received 24 January; accepted 19 July; published online 4 September 2011; doi:10.1038/nsmb.2124 Spliceosome assembly is coupled to RNA polymerase II dynamics at the 3′ end of human genes Sandra Bento Martins 1,3 , José Rino 1,3 , Teresa Carvalho 1 , Célia Carvalho 1 , Minoru Yoshida 2 , Jasmim Mona Klose 1 , Sérgio Fernandes de Almeida 1 & Maria Carmo-Fonseca 1 In the nucleus of higher eukaryotes, maturation of mRNA precursors involves an orderly sequence of transcription-coupled interdependent steps. Transcription is well known to influence splicing, but how splicing may affect transcription remains unclear. Here we show that a splicing mutation that prevents recruitment of spliceosomal snRNPs to nascent transcripts causes co-transcriptional retention of unprocessed RNAs that remain associated with polymerases stalled predominantly at the 3′ end of the gene. In contrast, treatment with spliceostatin A, which allows early spliceosome formation but destabilizes subsequent assembly of the catalytic complex, abolishes 3′ end pausing of polymerases and induces leakage of unspliced transcripts to the nucleoplasm. Taken together, the data suggest that recruitment of splicing factors and correct assembly of the spliceosome are coupled to transcription termination, and this might ensure a proofreading mechanism that slows down release of unprocessed transcripts from the transcription site.