Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, requires transcription factor IIB (TFIIB). We have earlier demonstrated association of TFIIB with the distal ends of a gene in an activator-dependent manner (El Kaderi, B., Medler, S., Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015-25025). The presence of TFIIB at the 3' end of a gene required its interaction with cleavage factor 1 (CF1) 3' end processing complex subunit Rna15. Here, employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the entire CF1 complex in yeast cells. The factors required for general transcription such as TATA-binding protein, RNA polymerase II, and TFIIH are not a component of the TFIIB complex. This holo-TFIIB complex was resistant to MNase digestion. The complex was observed only in the looping-competent strains, but not in the looping-defective sua7-1 strain. The requirement of Rna15 in gene looping has been demonstrated earlier. Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes. Accordingly, cross-linking of TFIIB to the 3' end of genes was abolished in the mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1 exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and Pap1 is crucial for gene looping and transcriptional regulation.
"In yeast, gene loop formation requires an interaction between general transcription factors and 3 0 RNA processing factors (Ansari and Hampsey, 2005; Medler et al, 2011; Tan- Wong et al, 2012). However, mutations in Arabidopsis polyadenylation factors FCA and FPA did not disrupt the FI–FV interaction (Figure 2A) despite increasing FLC transcript levels 20-fold (Figure 2B). "
[Show abstract][Hide abstract] ABSTRACT: Gene activation in eukaryotes frequently involves interactions between chromosomal regions. We have investigated whether higher-order chromatin structures are involved in the regulation of the Arabidopsis floral repressor gene FLC, a target of several chromatin regulatory pathways. Here, we identify a gene loop involving the physical interaction of the 5' and 3' flanking regions of the FLC locus using chromosome conformation capture. The FLC loop is unaffected by mutations disrupting conserved chromatin regulatory pathways leading to very different expression states. However, the loop is disrupted during vernalization, the cold-induced, Polycomb-dependent epigenetic silencing of FLC. Loop disruption parallels timing of the cold-induced FLC transcriptional shut-down and upregulation of FLC antisense transcripts, but does not need a cold-induced PHD protein required for the epigenetic silencing. We suggest that gene loop disruption is an early step in the switch from an expressed to a Polycomb-silenced state.
The EMBO Journal 12/2012; 32(1). DOI:10.1038/emboj.2012.324 · 10.43 Impact Factor
"These include in mammals CPSF, CstF, and CFI (Glover-Cutter et al., 2008; Rozenblatt-Rosen et al., 2009; Venkataraman et al., 2005). PAP has long been known to associate only loosely with the other core polyadenylation factors (Takagaki et al., 1988), but recently it was reported to crosslink to both 5 0 and 3 0 end of genes in yeast, where it is necessary for gene looping (Medler et al., 2011). These data are consistent with our results showing that PAP can be recruited to the 3 0 ends of transcribed genes in human cells, implying that, even if polyadenylation occurs after release of the mRNA from RNAP II, PAP joins the 3 0 processing complex cotranscriptionally. "
[Show abstract][Hide abstract] ABSTRACT: The 3' ends of most eukaryotic mRNAs are produced by an endonucleolytic cleavage followed by synthesis of a poly(A) tail. Poly(A) polymerase (PAP), the enzyme that catalyzes the formation of the tail, is subject to tight regulation involving several posttranslational modifications. Here we show that the enzyme poly(ADP-ribose) polymerase 1 (PARP1) modifies PAP and regulates its activity both in vitro and in vivo. PARP1 binds to and modifies PAP by poly(ADP-ribosyl)ation (PARylation) in vitro, which inhibits PAP activity. In vivo we show that PAP is PARylated during heat shock, leading to inhibition of polyadenylation in a PARP1-dependent manner. The observed inhibition reflects reduced RNA binding affinity of PARylated PAP in vitro and decreased PAP association with non-heat shock protein-encoding genes in vivo. Our results provide direct evidence that PARylation can control processing of mRNA precursors, and also identify PARP1 as a regulator of polyadenylation during thermal stress.
"These proteins form several subcomplexes , such as cleavage factor IA (CF IA), cleavage factor II (CF II), and polyadenylation factor I (PF I). CF IA has four subunits, Rna14, Rna15, Clp1, and Pcf11, and this factor is also required for gene looping (Medler et al. 2011). Rna15 contains a RNA recognition module (RRM) at the N terminus (Pancevac et al. 2010), followed by a hinge region and a C-terminal domain (CTD) (Fig. 1A). "
[Show abstract][Hide abstract] ABSTRACT: A large protein machinery is required for 3'-end processing of mRNA precursors in eukaryotes. Cleavage factor IA (CF IA), a complex in the 3'-end processing machinery in yeast, contains four subunits, Rna14, Rna15, Clp1, and Pcf11. Rna14 has a HAT (half a TPR) domain at the N terminus and a region at the C terminus that mediates interactions with Rna15. Rna15 contains a RNA recognition module (RRM) at the N terminus, followed by a hinge region. These two proteins are homologs of CstF-77 and CstF-64 in the cleavage stimulation factor (CstF) of the mammalian 3'-end processing machinery. We report the first crystal structure of Rna14 in complex with the hinge region of Rna15, and the structures of the HAT domain of Rna14 alone in two different crystal forms. The complex of the C-terminal region of Rna14 with the hinge region of Rna15 does not have strong interactions with the HAT domain of Rna14, and this complex is likely to function independently of the HAT domain. Like CstF-77, the HAT domain of Rna14 is also a tightly associated dimer with a highly elongated shape. However, there are large variations in the organization of this dimer among the Rna14 structures, and there are also significant structural differences to CstF-77. These observations suggest that the HAT domain and especially its dimer may have some inherent conformational variability.
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