Talk abstracts

Talk on Friday 05:00-05:15pm submitted by Jarnail Singh

Rates of in situ transcription and splicing in large human genes

Jarnail Singh (Dept. of Molecular Genetics, Cleveland Clinic), Richard A. Padgett (Dept. of Molecular Genetics, Cleveland Clinic)

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-b-D-ribofuranoside (DRB) that 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 per minute and is nearly the same 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 minutes of synthesis irrespective of intron length between 1 kb and 240 kb. Similarly, U12-dependent introns were co-transcriptionally spliced within 10 minutes of synthesis confirming that these introns are spliced within the nuclear compartment. These results show that the expression of large genes is surprisingly rapid and efficient. In summary, we have developed a useful technique to follow the transcription elongation by RNAPII over hundreds of kilobases of DNA in large genes in a highly physiological setting. This system is thus well suited for the investigation of the effects of perturbation of the various components on transcription elongation and RNA processing.

Keywords: RNA Polymerase II, Transcription rate, splicing