Transitions in RNA polymerase II elongation complexes at the 3′ ends of genes

Abstract

To understand the factor interactions of transcribing RNA polymerase II (RNApII) in vivo, chromatin immunoprecipitations were used to map the crosslinking patterns of multiple elongation and polyadenylation factors across transcribed genes. Transcription through the polyadenylation site leads to a reduction in the levels of the Ctk1 kinase and its associated phosphorylation of the RNApII C-terminal domain. One group of elongation factors (Spt4/5, Spt6/Iws1, and Spt16/Pob3), thought to mediate transcription through chromatin, shows patterns matching that of RNApII. In contrast, the Paf and TREX/THO complexes partially overlap RNApII, but do not crosslink to transcribed regions downstream of polyadenylation sites. In a complementary pattern, polyadenylation factors crosslink strongly at the 3 0 ends of genes. Mutation of the 3 0 polyadenylation sequences or the Rna14 protein causes loss of polyadenylation factor crosslinking and readthrough of termination sequences. Therefore, transcription termination and polyadenylation involve transitions at the 3 0 end of genes that may include an exchange of elongation and polyadenylation/termination factors.

Localization of RNAPII and 3′ end formation factor CstF subunits onC. elegansgenes and operons

“…1A), as expected based on its presumed function. 28,53,55 Metagene analysis on isolated/ expressed genes shows that both CstF subunits are low at the 5 0 end and throughout the body of genes (Fig. 1C).…”

Transcription termination by nuclear RNA polymerases

“…In yeast, mutations and conditional depletion of factors involved in pre-mRNA cleavage and polyadenylation, including homologs of CstF-64 (Rna15), CstF-77 (Rna14), Pcf11, CPSF160 (Yhh1), CPSF-73 (Ysh1), and Ssu72, result in read-through at the 39-end of protein-coding genes (Birse et al 1998;Dichtl et al 2002;Steinmetz and Brow 2003;Garas et al 2008). Accordingly, ChIP assays suggest that 39-end processing factors, including Pcf11, Rna14, and Rna15, become associated with the RNAPII EC at the poly(A) site, in a Ser2 phosphorylated CTD-dependant manner (Ahn et al 2004;Kim et al 2004a;Luo et al 2006). Other components of the yeast polyadenylation machinery, including homologs of CPSF-100, CPSF-30, and symplekin, are also associated with the 39-ends of protein-coding genes (Kim et al 2004a).…”

“…Accordingly, ChIP assays suggest that 39-end processing factors, including Pcf11, Rna14, and Rna15, become associated with the RNAPII EC at the poly(A) site, in a Ser2 phosphorylated CTD-dependant manner (Ahn et al 2004;Kim et al 2004a;Luo et al 2006). Other components of the yeast polyadenylation machinery, including homologs of CPSF-100, CPSF-30, and symplekin, are also associated with the 39-ends of protein-coding genes (Kim et al 2004a). ChIP analysis in human cells also revealed the association of components of the 39-end processing complex, including CPSF-73 and CstF-64 at the 39-end of genes (Swinburne et al 2006;Glover-Cutter et al 2008).…”

“…In ChIP experiments, factors such as the Paf1C and the TREX/THO complex, which functions in mRNP biogenesis and maintenance of genome integrity (Garcia-Rubio et al 2008), cross-link throughout genes, but their levels are reduced on passage through the poly(A) site, suggesting they are released from the EC (Ahn et al 2004;Kim et al 2004a). Dissociation of a CstF64-interacting protein, the transcriptional coactivator PC4 (human homolog of Sub1), at the poly(A) site renders RNAPII competent for termination, and genetic evidence in yeast suggests that Sub1/PC4 has an evolutionarily conserved anti-terminator activity (Calvo and Manley 2001).…”

Synchronicity: policing multiple aspects of gene expression by Ctk1: Figure 1.

“…Pol II is recruited to the promoter in an unphosphorylated form (Pol IIA) that becomes extensively phosphorylated at Ser2 and Ser5 during different stages of the transcription cycle. Differential CTD phosphorylation promotes the exchange of initiation and elongation factors at promoter clearance (Pokholok et al 2002) and the exchange of elongation and 3Ј-end processing factors at termination (Kim et al 2004).…”