Transcription Elongation Rate Research Articles

SWI/SNF associates with nascent pre-mRNPs and regulates alternative pre-mRNA processing.

The SWI/SNF chromatin remodeling complexes regulate the transcription of many genes by remodeling nucleosomes at promoter regions. In Drosophila, SWI/SNF plays an important role in ecdysone-dependent transcription regulation. Studies in human cells suggest that Brahma (Brm), the ATPase subunit of SWI/SNF, regulates alternative pre-mRNA splicing by modulating transcription elongation rates. We describe, here, experiments that study the association of Brm with transcribed genes in Chironomus tentans and Drosophila melanogaster, the purpose of which was to further elucidate the mechanisms by which Brm regulates pre-mRNA processing. We show that Brm becomes incorporated into nascent Balbiani ring pre-mRNPs co-transcriptionally and that the human Brm and Brg1 proteins are associated with RNPs. We have analyzed the expression profiles of D. melanogaster S2 cells in which the levels of individual SWI/SNF subunits have been reduced by RNA interference, and we show that depletion of SWI/SNF core subunits changes the relative abundance of alternative transcripts from a subset of genes. This observation, and the fact that a fraction of Brm is not associated with chromatin but with nascent pre-mRNPs, suggest that SWI/SNF affects pre-mRNA processing by acting at the RNA level. Ontology enrichment tests indicate that the genes that are regulated post-transcriptionally by SWI/SNF are mostly enzymes and transcription factors that regulate postembryonic developmental processes. In summary, the data suggest that SWI/SNF becomes incorporated into nascent pre-mRNPs and acts post-transcriptionally to regulate not only the amount of mRNA synthesized from a given promoter but also the type of alternative transcript produced.

Shedding UV Light on Alternative Splicing

After DNA damage, cells modulate pre-messenger RNA (pre-mRNA) splicing to induce an anti- or proapoptotic response. In this issue, Muñoz et al. (2009) uncover a cotranscriptional mechanism for activating alternative pre-mRNA splicing after ultraviolet irradiation that depends unexpectedly on hyperphosphorylation of the RNA polymerase II C-terminal domain and decreased rates of transcription elongation.

Mammalian Elongin A complex mediates DNA‐damage‐induced ubiquitylation and degradation of Rpb1

The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II (pol II) by suppressing transient pausing of the pol II at many sites along the DNA. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which can form an isolable Elongin BC subcomplex. Here, we have shown that both the ubiquitylation and proteasomal degradation of the largest subunit of pol II (Rpb1) following UV‐irradiation are significantly suppressed in Elongin A‐deficient cells; however, in both cases suppression is rescued by transfection of wild‐type Elongin A. Moreover, we have demonstrated that the Elongin A‐Elongin BC complex is capable of assembling with the Cul5/Rbx2 module, and that this hetero‐pentamer complex efficiently ubiquitylates Rpb1 in vitro. Mechanistic studies indicate that colocalization of Elongin A and Cul5 in cells and the interaction of Elongin A with the Ser5‐phosphorylated form of Rpb1 are strongly enhanced following UV‐irradiation. Taken together, our results suggest that mammalian Elongin A is directly involved in ubiquitylation and degradation of Rpb1 following DNA damage.

Spt6 enhances the elongation rate of RNA polymerase II in vivo

Several eukaryotic transcription factors have been shown to modulate the elongation rate of RNA polymerase II (Pol II) on naked or chromatin-reconstituted templates in vitro. However, none of the tested factors have been shown to directly affect the elongation rate of Pol II in vivo. We performed a directed RNAi knock-down (KD) screen targeting 141 candidate transcription factors and identified multiple factors, including Spt6, that alter the induced Hsp70 transcript levels in Drosophila S2 cells. Spt6 is known to interact with both nucleosome structure and Pol II, and it has properties consistent with having a role in elongation. Here, ChIP assays of the first wave of Pol II after heat shock in S2 cells show that KD of Spt6 reduces the rate of Pol II elongation. Also, fluorescence recovery after photobleaching assays of GFP-Pol II in salivary gland cells show that this Spt6-dependent effect on elongation rate persists during steady-state-induced transcription, reducing the elongation rate from approximately 1100 to 500 bp/min. Furthermore, RNAi depletion of Spt6 reveals its broad requirement during different stages of development.

Mammalian Elongin A complex mediates DNA-damage-induced ubiquitylation and degradation of Rpb1

The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II (pol II) by suppressing transient pausing of the pol II at many sites along the DNA. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which can form an isolable Elongin BC subcomplex. Here, we have shown that both the ubiquitylation and proteasomal degradation of the largest subunit of pol II (Rpb1) following UV-irradiation are significantly suppressed in Elongin A-deficient cells; however, in both cases suppression is rescued by transfection of wild-type Elongin A. Moreover, we have demonstrated that the Elongin A-Elongin BC complex is capable of assembling with the Cul5/Rbx2 module, and that this hetero-pentamer complex efficiently ubiquitylates Rpb1 in vitro. Mechanistic studies indicate that colocalization of Elongin A and Cul5 in cells and the interaction of Elongin A with the Ser5-phosphorylated form of Rpb1 are strongly enhanced following UV-irradiation. Taken together, our results suggest that mammalian Elongin A is directly involved in ubiquitylation and degradation of Rpb1 following DNA damage.

Kinetic Analysis of the Interactions Between the Different ELL and EAF Proteins Involved in Acute Myeloid Leukemia

The ELL gene was first cloned as a fusion partner of MLL in the (11;19)(q23;p13.1) translocation that occurs in acute myeloid leukemia. Subsequently, the ELL2 gene was cloned on the basis of its sequence homology to ELL. Both proteins stimulate the rate of transcript elongation by RNA polymerase II. Previously, we isolated two closely related proteins, EAF1 and EAF2, which interact with ELL and ELL2. Deletion mapping studies carried out to delineate the domain(s) of ELL involved in its interaction with either EAF1 or EAF2 showed that the N-terminus (amino acids 1–207) of ELL binds to both EAF1 and EAF2. In comparison, the middle region (207–411 amino acids) does not bind to either of the two EAF proteins and the C-terminus region (411–621 amino acids) binds only to the EAF1 protein. Biochemical studies have revealed that EAF1 and EAF2 enhance the rate of mRNA chain elongation by the ELL proteins in vitro. Although both ELL and ELL2 have similar roles in transcriptional elongation, ELL2 has not been shown to be involved in any hematological abnormality so far. In an attempt to gain a deeper understanding of the biology and functions of the interactions between these different proteins, we determined the kinetic properties of these interactions using the biophysical techniques of surface plasmon resonance (SPR) and isothermal calorimetry (ITC). SPR detects complex formation in real time and provides a better comprehension of the dynamics of association and dissociation of an interaction, and ITC is used to determine the thermodynamics of the interaction. Our SPR analysis has provided novel insights into the nature of the binding of the ELL proteins to the EAF proteins. We observed that both ELL and ELL2 bind to EAF1 and EAF2 with a high affinity, but the binding affinity of ELL2 for both EAF1 and EAF2 is almost twelve-fold greater than the affinity of ELL for both the EAF proteins. The higher affinity of ELL2 is due to much slower uptake and release kinetics reflected by the low association and dissociation rate constants of ELL2 compared to ELL. The stoichiometry of ELL, ELL2, EAF1 and EAF2 in the ELL-EAF1, ELL-EAF2, ELL2-EAF1 and ELL2-EAF2 complexes was estimated to be 1:1 after fitting the respective sensorgrams obtained by SPR analysis to the Langmuir's bimolecular model. Interestingly, we did not observe any difference in the affinity of either ELL or ELL2 for binding to EAF1 or EAF2. We used SPR-based competition experiments to show that ELL and ELL2 bind to the same sites on the EAF proteins. We have also investigated the characteristics of binding of the various ELL domains to the EAF1/2 proteins. In the (11;19)(q23;p13.1) translocation, the C-terminus of ELL fuses to the N-terminus of MLL to generate a chimeric protein that interacts with EAF1 and this interaction is critical for the role of ELL in cell immortalization in vitro and leukemogenesis in vivo. In agreement with this observation, we found that the C-terminus of ELL binds EAF1 with a higher affinity than EAF2, while the N-terminus of ELL binds with similar affinities and displays similar kinetics of binding to both EAF1 and EAF2. We also found that the individual binding sites on the ELL N-terminus and the C-terminus exhibited a lower affinity for the EAF proteins, but the affinity increases when the two sites function together in the context of the full-length protein, suggesting that the two sites co-operate with each other to increase the affinity for the full-length ELL protein. Taken together, these observations suggest that although ELL and ELL2 share many similarities in terms of their sequence and function in transcription elongation, they bind to the EAF proteins with different affinities and kinetics. Alternative interaction dynamics and the interplay between the different ELL and EAF proteins permit distinct functional regulation of transcriptional elongation in normal and leukemic cells.

Histone N-terminal Tails Interfere with Nucleosome Traversal by RNA Polymerase II

We determined the effect of the N-terminal histone tails on nucleosome traversal by yeast and human RNA polymerase II (pol II). Removal of H2A/H2B tails, H3/H4 tails, or all tails increased complete traversal of the nucleosome by human pol II, although the increase varied considerably depending on the template and on which tails were removed. Human pol II achieved >80% traversal of one nucleosomal template lacking the H2A/H2B tails, but even in those reactions, the transcript elongation rate was lower than the rate on pure DNA templates. For yeast pol II, transcription proceeded much farther into the nucleosome in the absence of tails, but complete read-through was not substantially increased by tail removal. Transcription factor IIS provided roughly the same level of read-through stimulation for transcript elongation in the presence or absence of tails. FACT also stimulated elongation on nucleosomal templates, and this effect was similar regardless of the presence of tails. For both polymerases, removal of the H2A/H2B tails reduced pausing throughout the nucleosome, suggesting that histone tails affect a common step at most points during nucleosome traversal. We conclude that histone tails provide a significant part of the nucleosomal barrier to pol II transcript elongation.

Determinants of vaccinia virus early gene transcription termination

Vaccinia virus early gene transcription requires the vaccinia termination factor, VTF, nucleoside triphosphate phosphohydrolase I, NPH I, ATP, the virion RNA polymerase, and the motif, UUUUUNU, in the nascent RNA, found within 30 to 50 bases from the poly A addition site, in vivo. In this study, the relationships among the vaccinia early gene transcription termination efficiency, termination motif specificity, and the elongation rate were investigated. A low transcription elongation rate maximizes termination efficiency and minimizes specificity for the UUUUUNU motif. Positioning the termination motif over a 63 base area upstream from the RNA polymerase allowed efficient transcript release, demonstrating a remarkable plasticity in the transcription termination complex. Efficient transcript release was observed during ongoing transcription, independent of VTF or UUUUUNU, but requiring both NPH I and either ATP or dATP. This argues for a two step model: the specifying step, requiring both VTF and UUUUUNU, and the energy-dependent step employing NPH I and ATP. Evaluation of NPH I mutants for the ability to stimulate transcription elongation demonstrated that ATPase activity and a stable interaction between NPH I and the Rap94 subunit of the viral RNA polymerase are required. These observations demonstrate that NPH I is a component of the elongating RNA polymerase, which is catalytically active during transcription elongation.

A Sequence Motif in the Simian Virus 40 (SV40) Early Core Promoter Affects Alternative Splicing of Transcribed mRNA

To identify new sequence elements in the promoter that affect splicing patterns of pre-mRNAs, we analyzed effects of different promoters on alternative splicing of model reporter genes. We compared the E1a alternative splicing pattern in transcripts expressed from the full-length cytomegalovirus, SV40 early, or a hybrid cytomegalovirus/SV40 early promoter and found that the hybrid promoter improved selection of the suboptimal E1a 5'SS-1. Expressing RNA from the hybrid promoter also enhanced selection of suboptimal splice sites in other alternatively spliced reporter genes, demonstrating the generality of this effect. Unlike previously defined promoter elements shown to affect alternative splicing, which were located in the enhancer/upstream activating sequences, the motif identified in this work is positioned within the core promoter; it is comprised of eight T-residues directly upstream of the SV40 early TATA box. This motif was previously implicated in DNA bending and negative regulation of transcription. Together, these results suggest that the identity of transcription complex assembled in the core promoter-dependent fashion can affect splice site selection during pre-mRNA splicing, perhaps by influencing the processivity of transcription elongation.

Identification and Characterization of a Schizosaccharomyces pombe RNA Polymerase II Elongation Factor with Similarity to the Metazoan Transcription Factor ELL

ELL family transcription factors activate the rate of transcript elongation by suppressing transient pausing by RNA polymerase II at many sites along the DNA. ELL-associated factors 1 and 2 (EAF1 and EAF2) bind stably to ELL family members and act as strong positive regulators of their transcription activities. Orthologs of ELL and EAF have been identified in metazoa, but it has been unclear whether such RNA polymerase II elongation factors are utilized in lower eukaryotes. Using bioinformatic and biochemical approaches, we have identified a new Schizosaccharomyces pombe RNA polymerase II elongation factor that is composed of two subunits designated SpELL and SpEAF, which share weak sequence similarity with members of the metazoan ELL and EAF families. Like mammalian ELL-EAF, SpELL-SpEAF stimulates RNA polymerase II transcription elongation and pyrophosphorolysis. In addition, like many yeast RNA polymerase II elongation factors, deletion of the SpELL gene renders S. pombe sensitive to the drug 6-azauracil. Finally, phylogenetic analyses suggest that the SpELL and SpEAF proteins are evolutionarily conserved in many fungi but not in Saccharomyces cerevisiae.

The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export.

Spt6 promotes transcription elongation at many genes and functions as a histone H3 chaperone to alter chromatin structure during transcription. We show here that mammalian Spt6 binds Ser2-phosphorylated (Ser2P) RNA polymerase II (RNAPII) through a primitive SH2 domain, which recognizes phosphoserine rather than phosphotyrosine residues. Surprisingly, a point mutation in the Spt6 SH2 domain (R1358K) blocked binding to RNAPIIo without affecting transcription elongation rates in vitro. However, HIV-1 and c-myc RNAs formed in cells expressing the mutant Spt6 protein were longer than normal and contained splicing defects. Ectopic expression of the wild-type, but not mutant, Spt6 SH2 domain, caused bulk poly(A)+ RNAs to be retained in the nucleus, further suggesting a widespread role for Spt6 in mRNA processing or assembly of export-competent mRNP particles. We cloned the human Spt6-interacting protein, hIws1 (interacts with Spt6), and found that it associates with the nuclear RNA export factor, REF1/Aly. Depletion of endogenous hIws1 resulted in mRNA processing defects, lower levels of REF1/Aly at the c-myc gene, and nuclear retention of bulk HeLa poly(A)+ RNAs in vivo. Thus binding of Spt6 to Ser2-P RNAPII provides a cotranscriptional mechanism to recruit Iws1, REF1/Aly, and associated mRNA processing, surveillance, and export factors to responsive genes.

Mutations in the SF1-U2AF59-U2AF23 Complex Cause Exon Skipping in Schizosaccharomyces pombe

To identify genes involved in the mechanism to ensure ordered 5' to 3' exon joining in constitutively spliced pre-mRNAs, we screened for mutants that cause exon skipping in the fission yeast Schizosaccharomyces pombe using a reporter plasmid, which contains the ura4+ gene with the nda3 intron 1-exon 2-intron 2 sequence. The reporter plasmid was designed to produce the functional ura4+ mRNA, when the central nda3 exon is skipped during the splicing reaction. We mutagenized cells harboring the plasmid by UV irradiation and isolated 34 ura+ mutants that grew on minimal medium. Of those, eight mutants were found to be temperature sensitive (ts) for growth. Complementation analyses revealed that the ts mutants belong to three distinct complementation groups named ods (ordered splicing) 1, 2, and 3. RT-PCR analyses showed that products of exon skipping were actually generated in the ods mutants. We cloned the genes responsible for the ods mutations, and found that ods1+, ods2+, and ods3+ encode splicing factors Prp2p/U2AF59, U2AF23, and SF1, respectively, which form a SF1-U2AF59-U2AF23 complex involved in recognition of the branch-point and 3' splice site sequences in a pre-mRNA. We also showed that mutations in the SF1-U2AF59-U2AF23 binding sequences in the reporter plasmid result in exon skipping in wild-type S. pombe cells. In addition, drugs that decrease the rate of transcription elongation were found to suppress the exon skipping in the ods mutants. These results suggest that co-transcriptional recognition of a nascent pre-mRNA by the SF1-U2AF59-U2AF23 complex is essential for ordered exon joining in constitutive splicing in S. pombe.

Pulling on the Nascent RNA during Transcription Does Not Alter Kinetics of Elongation or Ubiquitous Pausing

Transcriptional elongation and termination by RNA polymerase (RNAP) are controlled by interactions among the nascent RNA, DNA, and RNAP that comprise the ternary transcription elongation complex (TEC). To probe the effects of cotranscriptionally folded RNA hairpins on elongation as well as the stability of the TEC, we developed a single-molecule assay to monitor RNA elongation by Escherichia coli RNAP molecules while applying controlled loads to the nascent RNA that favor forward translocation. Remarkably, forces up to 30 pN, twice those required to disrupt RNA secondary structure, did not significantly affect enzyme processivity, transcription elongation rates, pause frequencies, or pause lifetimes. These results indicate that ubiquitous transcriptional pausing is not a consequence of the formation of hairpins in the nascent RNA. The ability of the TEC to sustain large loads on the transcript reflects a tight binding of RNA within the TEC and has important implications for models of transcriptional termination.

Growth Factor Signaling Regulates Elongation of RNA Polymerase I Transcription in Mammals via UBF Phosphorylation and r-Chromatin Remodeling

Synthesis of the 45S rRNA by RNA polymerase I limits cell growth. Knowledge of the mechanism of its regulation is therefore key to understanding growth control. rRNA transcription is believed to be regulated solely at initiation/promoter release. However, we found that stimulation of endogenous 45S rRNA synthesis by epidermal growth factor (EGF) and serum failed to induce an increase in RNA polymerase I engagement on the rRNA genes, despite robust enhancement of 45S rRNA synthesis. Further, endogenous transcription elongation rates were measured and found to be directly proportional to 45S rRNA synthesis. Thus, elongation is a rate-limiting step for rRNA synthesis in vivo. ERK phosphorylation of the HMG boxes of UBF, an RNA polymerase I factor essential for transcription enhancement, was shown to directly regulate elongation by inducing the remodeling of ribosomal gene chromatin. The data suggest a mechanism for coordinating the cotranscriptional assembly of preribosomal particles.

A Polar Mechanism Coordinates Different Regions of Alternative Splicing within a Single Gene

Alternative splicing plays a key role in generating protein diversity. Transfections with minigenes revealed coordination between two distant, alternatively spliced exons in the same gene. Mutations that either inhibit or stimulate inclusion of the upstream alternative exon deeply affect inclusion of the downstream one. However, similar mutations at the downstream alternative exon have little effect on the upstream one. This polar effect is promoter specific and is enhanced by inhibition of transcriptional elongation. Consistently, cells from mutant mice with either constitutive or null inclusion of a fibronectin alternative exon revealed coordination with a second alternative splicing region, located far downstream. Using allele-specific RT-PCR, we demonstrate that this coordination occurs in cis and is also affected by transcriptional elongation rates. Bioinformatics supports the generality of these findings, indicating that 25% of human genes contain multiple alternative splicing regions and identifying several genes with nonrandom distribution of mRNA isoforms at two alternative regions.

Identification of a structural element that is essential for two functions of transcription factor NusG

The transcription factor NusG from Escherichia coli modulates the rate of transcript elongation by RNA polymerase and the efficiency of Rho-dependent transcript termination. It consists of two globular domains with an extra loop extending out of the amino-terminal domain in the position that is occupied by a third globular domain in some NusG homologues. We have tested the role of this appended mini-domain by assaying the elongation and termination enhancement activities of variants. The results show that variants with changes in their sequence do not cause a loss of functions, whereas variants with the deletions of the residues in that domain are much less active for both functions. This finding suggests that the mini-domain serves as a structural element for an interaction rather than as a site for residue-specific contacts.

In vitro regulation of reporter gene transcription by the androgen receptor AF1 domain

The androgen receptor (AR) is a ligand-activated transcription factor that regulates gene expression in response to the steroids testosterone and dihydrotestosterone. AR-dependent gene expression is likely to play an important role in a number of receptor-associated disorders, such as prostate cancer, spinal bulbar muscular atrophy, male type baldness and hirsutism. The AR contains two transactivation domains, termed AF1 (activation function 1) located in the N-terminus and AF2 (activation function 2) in the C-terminal ligand-binding domain. AF2 exhibits weak transcriptional activity, whereas AF1 is a strong regulator of transcription. Transcriptional regulation by AF1 is thought to be modulated by a number of proteins that interact with this region, and by post-translational modifications. Our focus is on the N-terminal-interacting proteins and their regulation of transcription via interaction with the receptor. To better understand the mechanism of AR-AF1 action, we have reconstituted AR activity in HeLa nuclear extracts using a unique dual reporter gene assay. Multiple LexA-binding sites in the promoter allow transcription to be driven by a recombinant AR-AF1-Lex fusion protein. The findings from initial experiments suggest an increase in transcription initiation and elongation rates by AR-AF1-Lex. The role of protein-protein interactions involving co-activators and basal transcription factors and AR-AF1 activity are discussed.

Codon optimization of the HIV-1 vpu and vif genes stabilizes their mRNA and allows for highly efficient Rev-independent expression

Two HIV-1 accessory proteins, Vpu and Vif, are notoriously difficult to express autonomously in the absence of the viral Tat and Rev proteins. We examined whether the codon bias observed in the vpu and vif genes relative to highly expressed human genes contributes to the Rev dependence and low expression level outside the context of the viral genome. The entire vpu gene as well as the 5′ half of the vif gene were codon optimized and the resulting open reading frames (ORFs) ( vphu and hvif, respectively) were cloned in autonomous expression vectors under the transcriptional control of the CMV promoter. Codon optimization efficiently removed the expression block observed in the native genes and allowed high levels of Rev- and Tat-independent expression of Vpu and Vif. Most of the higher protein levels detected are accounted for by enhanced steady-state levels of the mRNA encoding the optimized species. Nuclear run-on experiments show for the first time that codon optimization has no effect on the rate of transcriptional initiation or elongation of the vphu mRNA. Instead, optimization of the vpu gene was found to stabilize the vphu mRNA in the nucleus and enhance its export to the cytoplasm. This was achieved by allowing the optimized mRNA to use a new CRM1-independent nuclear export pathway. This work provides a better understanding of the molecular mechanisms underlying the process of codon optimization and introduces novel tools to study the biological functions of the Vpu and Vif proteins independently of other viral proteins.

Identification of EloA-BP1, a novel Elongin A binding protein with an exonuclease homology domain

The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites along the DNA template. Elongin is composed of a transcriptionally active A subunit, and two positive regulatory B and C subunits. Although the NH 2-terminal ∼120 amino acid region of Elongin A is dispensable for its transcriptional activity in vitro, it shares significant sequence similarity with the NH 2-terminus of other class of transcription factors SII and CRSP70, suggesting that the NH 2-terminus mediates interactions important for the regulation of transcription in vivo. To identify proteins that can bind to these conserved sequences, a human B cell cDNA library was screened using the NH 2-terminus of Elongin A as bait in a yeast two-hybrid system. Here, we report on the cloning and characterization of a novel human exonuclease domain-containing protein, Elongin A-binding protein 1 (EloA-BP1). EloA-BP1 is composed of 1221 amino acids and its mRNA is ubiquitously expressed. Double immunofluorescence labeling in COS7 cells suggested that EloA-BP1 and Elongin A are colocalized to the cell nucleus. By using an in vitro binding assay, we show that EloA-BP1 is capable of binding not only the NH 2-terminal ∼120 amino acid region of Elongin A, but also that of SII. Although the purified EloA-BP1 had no detectable effect on the rate of transcription elongation in vitro, it may play some role in the regulation of elongation in vivo.

Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces cerevisiae.

Unknown mechanisms exist to ensure that exons are not skipped during biogenesis of mRNA. Studies have connected transcription elongation with regulated alternative exon inclusion. To determine whether the relative rates of transcription elongation and spliceosome assembly might play a general role in enforcing constitutive exon inclusion, we measured exon skipping for a natural two-intron gene in which the internal exon is constitutively included in the mRNA. Mutations in this gene that subtly reduce recognition of the intron 1 branchpoint cause exon skipping, indicating that rapid recognition of the first intron is important for enforcing exon inclusion. To test the role of transcription elongation, we treated cells to increase or decrease the rate of transcription elongation. Consistent with the "first come, first served" model, we found that exon skipping in vivo is inhibited when transcription is slowed by RNAP II mutants or when cells are treated with inhibitors of elongation. Expression of the elongation factor TFIIS stimulates exon skipping, and this effect is eliminated when lac repressor is targeted to DNA encoding the second intron. A mutation in U2 snRNA promotes exon skipping, presumably because a delay in recognition of the first intron allows elongating RNA polymerase to transcribe the downstream intron. This indicates that the relative rates of elongation and splicing are tuned so that the fidelity of exon inclusion is enhanced. These findings support a general role for kinetic coordination of transcription elongation and splicing during the transcription-dependent control of splicing.