A genomic analysis of RNA polymerase II modification and chromatin architecture related to 3′ end RNA polyadenylation

Abstract

Genomic analyses have been applied extensively to analyze the process of transcription initiation in mammalian cells, but less to transcript 3Ј end formation and transcription termination. We used a novel approach to prepare 3Ј end fragments from polyadenylated RNA, and mapped the position of the poly(A) addition site using oligonucleotide arrays tiling 1% of the human genome. This approach revealed more 3Ј ends than had been annotated. The distribution of these ends relative to RNA polymerase II (PolII) and di-and trimethylated lysine 4 and lysine 36 of histone H3 was compared. A substantial fraction of unannotated 3Ј ends of RNA are intronic and antisense to the embedding gene. Poly(A) ends of annotated messages lie on average 2 kb upstream of the end of PolII binding (termination). Near the termination sites, and in some internal sites, unphosphorylated and C-terminal domain (CTD) serine 2 phosphorylated PolII (POLR2A) accumulate, suggesting pausing of the polymerase and perhaps dephosphorylation prior to release. Lysine 36 trimethylation occurs across transcribed genes, sometimes alternating with stretches of DNA in which lysine 36 dimethylation is more prominent. Lysine 36 methylation decreases at or near the site of polyadenylation, sometimes disappearing before disappearance of phosphorylated RNA PolII or release of PolII from DNA. Our results suggest that transcription termination loss of histone 3 lysine 36 methylation and later release of RNA polymerase. The latter is often associated with polymerase pausing. Overall, our study reveals extensive sites of poly(A) addition and provides insights into the events that occur during 3Ј end formation.

Transcription termination by nuclear RNA polymerases

“…The H3-K36 methyltransferase Set2 binds elongating RNAPII, recognizing the doubly phosphorylated CTD (Ser5/Ser2) (Hampsey and Reinberg 2003;Kizer et al 2005). Interestingly, a genomic analysis employing tilling arrays in mammalian cells showed that H3-K36 methylation often decreases near the poly(A) site during or prior to RNAPII release, leading to the intriguing possibility that H3-K36 methylation plays a role in transcription termination (Lian et al 2008).…”

“…Dephosphorylation of Ser2 is also critical for RNAPII recycling (Cho et al 1999). Both unphosphorylated and Ser2 phosphorylated CTD accumulate near transcription termination sites, suggesting that CTD dephosphorylation occurs during or prior to RNAPII release (Lian et al 2008). Chromatin immunoprecipitation (ChIP) experiments localized Ssu72 predominantly to the 39-ends of genes (Nedea et al 2003), but it was also detected at promoter regions (Ansari and Hampsey 2005).…”

Poly(A) signal-dependent degradation of unprocessed nascent transcripts accompanies poly(A) signal-dependent transcriptional pausing in vitro

“…This poly(A) signaldependent pausing can be seen in the 39 flanks of genes across the genome (Gromak et al 2006;Boireau et al 2007;Glover-Cutter et al 2008;Lian et al 2008) and has been proposed to coincide with checkpoint activity that leads either to cleavage at the poly(A) site or, alternatively, to continued transcription, or to degradation of the transcript (Orozco et al 2002;Rigo et al 2005;Nag et al 2007). In yeast, surveillance during the polyadenylation phase of 39-end processing is well documented for situations in which the polyadenylation has been compromised by defects in mRNP maturation factors or by the action of special regulatory elements Roth et al 2009).…”

An alternative polyadenylation mechanism coopted to the Arabidopsis RPP7 gene through intronic retrotransposon domestication

“…Intriguingly, polyadenylation sites were shown to be depleted of nucleosomes in Saccharomyces cerevisiae (20). In addition, differential distribution of PHMs along genes possibly marking distinct architectural features of their sequence has been reported in humans (21).…”