Abstract
Transcription and splicing must proceed over genomic distances of hundreds of kilobases in many human genes. However, the rates and mechanisms of these processes are poorly understood. We have used the compound 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB), which reversibly blocks gene transcription in vivo, combined with quantitative RT-PCR to analyze the transcription and RNA processing of several long human genes. We found that the rate of RNA polymerase II transcription over long genomic distances is about 3.8 kb min−1 and is similar whether transcribing long introns or exon-rich regions. We also determined that co-transcriptional pre-mRNA splicing of U2-dependent introns occurs within 5–10 min of synthesis, irrespective of intron length between 1 kb and 240 kb. Similarly, U12-dependent introns were co-transcriptionally spliced within 10 min of synthesis, confirming that these introns are spliced within the nuclear compartment. These results show that the expression of large genes is unexpectedly rapid and efficient.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
Purchase on Springer Link
Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Rasmussen, E.B. & Lis, J.T. In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes. Proc. Natl. Acad. Sci. USA 90, 7923–7927 (1993).
Proudfoot, N.J., Furger, A. & Dye, M.J. Integrating mRNA processing with transcription. Cell 108, 501–512 (2002).
Neugebauer, K.M. On the importance of being co-transcriptional. J. Cell Sci. 115, 3865–3871 (2002).
Bentley, D.L. Rules of engagement: co-transcriptional recruitment of pre-mRNA processing factors. Curr. Opin. Cell Biol. 17, 251–256 (2005).
Hirose, Y. & Manley, J.L. RNA polymerase II is an essential mRNA polyadenylation factor. Nature 395, 93–96 (1998).
Hirose, Y., Tacke, R. & Manley, J.L. Phosphorylated RNA polymerase II stimulates pre-mRNA splicing. Genes Dev. 13, 1234–1239 (1999).
de la Mata, M. et al. A slow RNA polymerase II affects alternative splicing in vivo. Mol. Cell 12, 525–532 (2003).
Cáceres, J.F. & Kornblihtt, A.R. Alternative splicing: multiple control mechanisms and involvement in human disease. Trends Genet. 18, 186–193 (2002).
Core, L.J. & Lis, J.T. Transcription regulation through promoter-proximal pausing of RNA polymerase II. Science 319, 1791–1792 (2008).
Batsché, E., Yaniv, M. & Muchardt, C. The human SWI/SNF subunit Brm is a regulator of alternative splicing. Nat. Struct. Mol. Biol. 13, 22–29 (2006).
Roberts, G.C., Gooding, C., Mak, H.Y., Proudfoot, N.J. & Smith, C.W. Co-transcriptional commitment to alternative splice site selection. Nucleic Acids Res. 26, 5568–5572 (1998).
Howe, K.J., Kane, C.M. & Ares, M. Jr. Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces cerevisiae. RNA 9, 993–1006 (2003).
Kornblihtt, A.R. Chromatin, transcript elongation and alternative splicing. Nat. Struct. Mol. Biol. 13, 5–7 (2006).
Tennyson, C.N., Klamut, H.J. & Worton, R.G. The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced. Nat. Genet. 9, 184–190 (1995).
O'Brien, T. & Lis, J.T. Rapid changes in Drosophila transcription after an instantaneous heat shock. Mol. Cell. Biol. 13, 3456–3463 (1993).
Femino, A.M., Fogarty, K., Lifshitz, L.M., Carrington, W. & Singer, R.H. Visualization of single molecules of mRNA in situ. Methods Enzymol. 361, 245–304 (2003).
Darzacq, X. et al. In vivo dynamics of RNA polymerase II transcription. Nat. Struct. Mol. Biol. 14, 796–806 (2007).
Boireau, S. et al. The transcriptional cycle of HIV-1 in real-time and live cells. J. Cell Biol. 179, 291–304 (2007).
Kessler, O., Jiang, Y. & Chasin, L.A. Order of intron removal during splicing of endogenous adenine phosphoribosyltransferase and dihydrofolate reductase pre-mRNA. Mol. Cell. Biol. 13, 6211–6222 (1993).
Audibert, A., Weil, D. & Dautry, F. In vivo kinetics of mRNA splicing and transport in mammalian cells. Mol. Cell. Biol. 22, 6706–6718 (2002).
Das, R. et al. Functional coupling of RNAP II transcription to spliceosome assembly. Genes Dev. 20, 1100–1109 (2006).
Listerman, I., Sapra, A.K. & Neugebauer, K.M. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells. Nat. Struct. Mol. Biol. 13, 815–822 (2006).
Kornblihtt, A.R., de la Mata, M., Fededa, J.P., Munoz, M.J. & Nogues, G. Multiple links between transcription and splicing. RNA 10, 1489–1498 (2004).
König, H., Matter, N., Bader, R., Thiele, W. & Muller, F. Splicing segregation: the minor spliceosome acts outside the nucleus and controls cell proliferation. Cell 131, 718–729 (2007).
Matera, A.G. & Ward, D.C. Nucleoplasmic organization of small nuclear ribonucleoproteins in cultured human cells. J. Cell Biol. 121, 715–727 (1993).
Pessa, H.K. et al. Minor spliceosome components are predominantly localized in the nucleus. Proc. Natl. Acad. Sci. USA 105, 8655–8660 (2008).
Friend, K., Kolev, N.G., Shu, M.D. & Steitz, J.A. Minor-class splicing occurs in the nucleus of the Xenopus oocyte. RNA 14, 1459–1462 (2008).
König, H. & Muller, F. Minor splicing: nuclear dogma still in question. Proc. Natl. Acad. Sci. USA 105, E37 (2008).
Steitz, J.A. et al. Where in the cell is the minor spliceosome? Proc. Natl. Acad. Sci. USA 105, 8485–8486 (2008).
Marshall, N.F. & Price, D.H. Control of formation of two distinct classes of RNA polymerase II elongation complexes. Mol. Cell. Biol. 12, 2078–2090 (1992).
Price, D.H. P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol. Cell. Biol. 20, 2629–2634 (2000).
Cheng, B. & Price, D.H. Properties of RNA polymerase II elongation complexes before and after the P-TEFb-mediated transition into productive elongation. J. Biol. Chem. 282, 21901–21912 (2007).
Chodosh, L.A., Fire, A., Samuels, M. & Sharp, P.A. 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro. J. Biol. Chem. 264, 2250–2257 (1989).
Gomes, N.P. et al. Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program. Genes Dev. 20, 601–612 (2006).
Capranico, G. et al. The effects of camptothecin on RNA polymerase II transcription: roles of DNA topoisomerase I. Biochimie 89, 482–489 (2007).
Mondal, N. & Parvin, J.D. DNA topoisomerase IIα is required for RNA polymerase II transcription on chromatin templates. Nature 413, 435–438 (2001).
Khobta, A. et al. Early effects of topoisomerase I inhibition on RNA polymerase II along transcribed genes in human cells. J. Mol. Biol. 357, 127–138 (2006).
Der, S.D., Zhou, A., Williams, B.R. & Silverman, R.H. Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95, 15623–15628 (1998).
Patel, A.A., McCarthy, M. & Steitz, J.A. The splicing of U12-type introns can be a rate-limiting step in gene expression. EMBO J. 21, 3804–3815 (2002).
Ucker, D.S. & Yamamoto, K.R. Early events in the stimulation of mammary tumor virus RNA synthesis by glucocorticoids. Novel assays of transcription rates. J. Biol. Chem. 259, 7416–7420 (1984).
Burnette, J.M., Miyamoto-Sato, E., Schaub, M.A., Conklin, J. & Lopez, A.J. Subdivision of large introns in Drosophila by recursive splicing at nonexonic elements. Genetics 170, 661–674 (2005).
Fong, N., Bird, G., Vigneron, M. & Bentley, D.L. A 10 residue motif at the C-terminus of the RNA Pol II CTD is required for transcription, splicing and 3′ end processing. EMBO J. 22, 4274–4282 (2003).
Zeng, C. & Berget, S.M. Participation of the C-terminal domain of RNA polymerase II in exon definition during pre-mRNA splicing. Mol. Cell. Biol. 20, 8290–8301 (2000).
Hirose, Y. & Ohkuma, Y. Phosphorylation of the C-terminal domain of RNA polymerase II plays central roles in the integrated events of eucaryotic gene expression. J. Biochem. 141, 601–608 (2007).
Dye, M.J., Gromak, N. & Proudfoot, N.J. Exon tethering in transcription by RNA polymerase II. Mol. Cell 21, 849–859 (2006).
Rosonina, E. & Blencowe, B.J. Analysis of the requirement for RNA polymerase II CTD heptapeptide repeats in pre-mRNA splicing and 3′-end cleavage. RNA 10, 581–589 (2004).
West, A.B. et al. N-myc regulates parkin expression. J. Biol. Chem. 279, 28896–28902 (2004).
Acknowledgements
We wish to thank R. Dietrich and S. Saikia for technical assistance, J. Shohet (Baylor College of Medicine) and M. Schwab (Deutsches Krebsforschungszentrum) for cell lines used in this study, D. Price for suggesting the use of DRB and D. Luse for careful reading of the manuscript. This work was supported by grants to R.A.P. from the US National Institutes of Health and the Ralph Wilson Medical Research Foundation.
Author information
Authors and Affiliations
Contributions
R.A.P. conceived and coordinated the project. J.S. performed all experimental work and compiled the data. Both authors analyzed the data and wrote the manuscript.
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 and Supplementary Table 1 (PDF 701 kb)
Rights and permissions
About this article
Cite this article
Singh, J., Padgett, R. Rates of in situ transcription and splicing in large human genes. Nat Struct Mol Biol 16, 1128–1133 (2009). https://doi.org/10.1038/nsmb.1666
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.1666
This article is cited by
-
The NELF pausing checkpoint mediates the functional divergence of Cdk9
Nature Communications (2023)
-
The long transcript of lncRNA TMPO-AS1 promotes bone metastases of prostate cancer by regulating the CSNK2A1/DDX3X complex in Wnt/β-catenin signaling
Cell Death Discovery (2023)
-
Temporal-iCLIP captures co-transcriptional RNA-protein interactions
Nature Communications (2023)
-
Ageing-associated changes in transcriptional elongation influence longevity
Nature (2023)
-
RNA export through the nuclear pore complex is directional
Nature Communications (2022)