RRM
2010 Rustbelt RNA Meeting
RRM
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Talk abstracts
Abstract:
We have measured the rates of transcription and splicing of genes in their natural in vivo chromatin environment. We used DRB to reversibly block new rounds of transcription in cultured cells. During DRB treatment, elongating RNA polymerases exited the genes and pre-mRNA was processed. Upon release from DRB, new rounds of transcription began within minutes and could be monitored using qRT-PCR of pre-mRNAs. Using this method, we found that transcription elongation proceeded at a rate of about 3.8 kb/min and that splicing occurred co-transcriptionally within 5-10 minutes of synthesis of the downstream exon. Remarkably, this splicing rate was independent of intron length between 1 kb and 250 kb. We have further shown that these results are essentially the same for short (30 min) and long (3 hr) DRB treatments as well as when flavopiridol is used in place of DRB. We also see similar results using interferon induction of genes instead of DRB treatment (1).
We have also used this method to follow the kinetics of transcription and splicing of long introns. In Drosophila, long introns are spliced in multiple steps by a recursive mechanism in a 5’ to 3’ order. It has been unclear whether long mammalian introns are spliced via a single event or multiple events. When we analyzed a 107 kb human intron, we found that transcription proceeded uniformly through the intron. Multiple sites within the intron were monitored for the kinetics of appearance and disappearance of intronic RNA. We found that all intronic RNA signals increased from the time of synthesis until 5 minutes after synthesis of the downstream exon and appearance of spliced RNA. The signal at the 5’ exon/intron junction was stable until splicing occurred at the normal 3’ splice site showing that recursive splicing did not occur. No evidence was seen for internal splicing events within the intron. This data strongly suggests that this intron is spliced out in a single event. Remarkably, the entire 107 kb intron appeared to be stable until shortly after splicing at which point all the intronic signals decreased in parallel consistent with rapid degradation.
References:
1. Singh, J. and Padgett, R.A. (2009) Rates of in situ transcription and splicing of large human genes. Nat. Struct. Molec. Biol. 16: 1128-1133.
Keywords: Splicing, Transcription, Chromatin
