RNA Polymerase I-Specific Subunit CAST/hPAF49 Has aRole in the Activation of Transcription by UpstreamBinding Factor

Abstract

Eukaryotic RNA polymerases are large complexes, 12 subunits of which are structurally or functionally homologous across the three polymerase classes. Each class has a set of specific subunits, likely targets of their cognate transcription factors. We have identified and characterized a human RNA polymerase I (Pol I)-specific subunit, previously identified as ASE-1 (antisense of ERCC1) and as CD3-associated signal transducer (CAST), and here termed CAST or human Pol I-associated factor of 49 kDa (hPAF49), after mouse orthologue PAF49. We provide evidence for growth-regulated Tyr phosphorylation of CAST/hPAF49, specifically in initiation-competent Pol I␤ complexes in HeLa cells, at a conserved residue also known to be important for signaling during T-cell activation. CAST/hPAF49 can interact with activator upstream binding factor (UBF) and, weakly, with selectivity factor 1 (SL1) at the rDNA (ribosomal DNA repeat sequence encoding the 18S, 5.8S, and 28S rRNA genes) promoter. CAST/hPAF49-specific antibodies and excess CAST/hPAF49 protein, which have no effect on basal Pol I transcription, inhibit UBF-activated transcription following functional SL1-Pol I-rDNA complex assembly and disrupt the interaction of UBF with CAST/hPAF49, suggesting that interaction of this Pol I-specific subunit with UBF is crucial for activation. Drawing on parallels between mammalian and Saccharomyces cerevisiae Pol I transcription machineries, we advance one model for CAST/ hPAF49 function in which the network of interactions of Pol I-specific subunits with UBF facilitates conformational changes of the polymerase, leading to stabilization of the Pol I-template complex and, thereby, activation of transcription.In eukaryotes, the three nuclear DNA-dependent RNA polymerases share a similar structural layout and catalytic activity but fulfill different specialist functions in cells: RNA polymerase I (Pol I) synthesizes rRNA precursors, Pol II synthesizes the pre-messenger RNAs, and Pol III synthesizes tRNAs and 5S rRNA, among other small RNAs. The ␣ 2 ␤␤Ј subunit composition of a prokaryotic core RNA polymerase is conserved for all three nuclear RNA polymerases from yeast to humans (reviewed in reference 8). The largest and second largest subunits of eukaryotic RNA polymerases share substantial homology with prokaryotic ␤Ј and ␤ subunits, respectively, and possess most of the enzymatic functions. Heterodimers AC40-AC19 of Pol I and Pol III and RPB3-RPB11 of Pol II are functional homologues of the prokaryotic ␣ 2 dimer, and the RPB6 (ABC23) subunit, shared between all three polymerases, is a structural and functional homologue of the bacterial subunit (Table 1).Eukaryotic RNA polymerases are more complex than their bacterial counterpart and require an additional four subunits to synthesize RNA from a nonspecific DNA template, which are shared between all three polymerases (RPB5, RPB8, RPB10, and RPB12). The unique subunits A12.2, RPB9, and C11 for yeast Pols I, II, and III, respectively, complete the 10

Two RNA Polymerase I Subunits Control the Binding and Release of Rrn3 during Transcription

“…N-terminal and C-terminal deletion forms of Rpa49 were cloned as Gal4 BD fusions into the pVV213 vector (54) and tested against Gal4 BD -Rpa34 in a two-hybrid assay. Proteins distantly related to Rpa34 have also been identified in fission yeast, mice, and humans (2,47). However, their sequence homology is restricted to two short conserved domains and to C-terminal KKE/D repeats often found in nucleolar proteins (21), and the mammalian subunit (PAF49) is much larger than its fungal counterpart (see Fig.…”

“…Seven others are related or even identical to subunits of the Pol II and Pol III core structures (1,16,29) and are homologous to archaeal RNA polymerase subunits (35,55). Rpa49 and Rpa34, on the other hand, are Pol I-specific subunits with unknown functions (20,36) that are also homologous to PAF53 and PAF49 in the mammalian Pol I system (47,56). We show here that these subunits bind each other in the Pol I structure and play an important role in transcription by improving the recruitment of the Rrn3-Pol I complex to the rDNA and by triggering the release of Rrn3 from the elongating Pol I.…”

Old Drug, New Target

“…Anti-FLAG M2 magnetic beads (Sigma) were used for immunoprecipitation (2 h at 4°C) from nuclear extracts, prepared as described (24) from U2OS or HeLa cells transfected (FuGENE HD reagent, Roche Applied Science) with one of the following expression vectors: FLAG-CAST expression vector pcFCAST (25), FLAG-RRN3 expression vector pcDNA3-FLAGhRRN3 (21), FLAG-TAF I 110 expression vector pcfTAF110 and FLAG-UBF expression vector pcfUBF. The beads were washed 3 times in TM10 buffer (50 mM Tris-HCl, pH 7.9, 12.5 mM MgCl 2 , 1 mM EDTA, 10% glycerol, 1 mM sodium metabisulfite, and 1 mM dithiothreitol), 0.15 M KCl.…”

Pharmacogenetics of Cancer and DNA Repair Enzymes

“…86 Later, this gene was identified as a subunit of RNA Polymerase I. 60 While the exact function of this Pol I-specific subunit is unknown, in mammalian cells the interaction with the activator of Pol I transcription, UBF, suggests a role for this subunit in facilitating the transition from transcription initiation to elongation at promoter escape. 59 In another model it has been proposed that conformational changes in the Pol I enzyme complex through CAST convert the Pol I into a processive enzyme complex.…”

“…59 In another model it has been proposed that conformational changes in the Pol I enzyme complex through CAST convert the Pol I into a processive enzyme complex. 60 Since the 3' NTR of ERCC1 overlaps with CAST open reading frame, rs3212986 causes an amino acid change in CAST in a putative nuclear localization signal. It is likely that this SNP affects CAST function instead of ERCC1 expression.…”