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Rates of in situ transcription and splicing in large human genes

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.

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Figure 1: DRB reversibly inhibits new transcription by RNAPII.
Figure 2: Kinetics of RNAPII-dependent transcription elongation.
Figure 3: Kinetics of splicing of U2-dependent introns.
Figure 4: Kinetics of splicing of U12-dependent introns.

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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.

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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.

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Correspondence to Richard A Padgett.

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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

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