Rates of in situ transcription and splicing in large human genes

Abstract

Rates of in vivo transcription have proven hard to pin down, especially across long mammalian genes that can contain lengthy introns. Using DRB inhibition followed by release, the rates of transcription of multiple human genes are now measured and splicing rates of both U2- and U12-dependent introns are assessed. 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.