TFIIH is an elongation factor of RNA polymerase I

Abstract

TFIIH is a multisubunit factor essential for transcription initiation and promoter escape of RNA polymerase II and for the opening of damaged DNA double strands in nucleotide excision repair (NER). In this study, we have analyzed at which step of the transcription cycle TFIIH is essential for transcription by RNA polymerase I. We demonstrate that TFIIH associates with the rDNA promoter and gene-internal sequences and leaves the rDNA promoter in a complex with RNA polymerase I after start of transcription. Moreover, mutations in the TFIIH subunits XPB and XPD found in Cockayne syndrome impair the interaction of TFIIH with the rDNA, but do not influence initiation complex formation or promoter escape of RNA polymerase I, but preclude the productivity of the enzyme by reducing transcription elongation in vivo and in vitro. Our results implicate that reduced RNA polymerase I transcription elongation and ribosomal stress could be one factor contributing to the Cockayne syndrome phenotype.

Figure 1.

TFIIH binds to the rDNA in vitro and in vivo. (A) One microgram of biotin labeled rDNA was bound to 20 µl of streptavidin magnetic beads and incubated with HeLa nuclear extract. After washing with buffer AM containing 100 mM KCl, bound proteins were eluted with 4 M urea and analyzed on western blots with the indicated antibodies. (Pol II-RNA polymerase II, Pol I-RNA polymerase I, TAF_I110/TAF_I68-subunits of selectivity factor 1, UBF-upstream binding factor, TIF-IA-transcription initiation factor IA, XPB/XPD/cdk7/p44-subunits of transcription factor of RNA polymerase II H). To control the specificity of binding, immobilized complexes formed on specific (pHrP₂) and unspecific (pUC) DNA were washed with buffer AM100 containing 0.125% sarcosyl. Binding of RNA polymerase I and TFIIH was stronger to the human rDNA promoter than to unspecific DNA. (B) ChIPanalysis with chromatin from HeLa cells expressing flag-tagged XPB (20). The antibodies are indicated above (α-TFIIH = α-flag). For PCR analysis, primers amplifying the IGS or promoter and gene-internal regions (H1, H4, H8) were used as depicted. Primer position is described in detail in O'Sullivan et al. (23). Promoter region and IGS from three independent experiments was analyzed by qPCR and normalized to the respective negative controls (***P < 0.001; *P < 0.05). Pictures are representatives from at least three independent experiments.

Figure 2.

TFIIH leaves the rDNA promoter with RNA polymerase I. (A) In vitro transcription experiment with HeLa nuclear extract titrating the sarcosyl concentration for single-round transcription. (B) Bead-bound immobilized complexes (IC HeLa) were challenged by the addition of nucleotides (NTPs). Transcription was restricted to a single round by sarcosyl and the remaining proteins analyzed after urea elution by western blots. Sarcosyl/NTPs denominates the single-round transcription experiment and sarcosyl the control reaction without nucleotides. RNA polymerase I and the XPB subunit of TFIIH were also monitored in the supernatants. (C) Supernatants of single-round transcriptions with immobilized-complex isolated transcription factors were subject to immunoprecipitation with control antibodies (IgG) or antibodies directed against RNA polymerase I (Pol I) and visualized by western blots. Pictures are representatives from at least three independent experiments.

Figure 3.

TFIIH associates with the rDNA throughout the transcription process. (A) Kinetics of single-round transcription with initiated complexes. Transcription was started after pre-incubation with the first 2 nt and restricted to a single round by addition of sarcosyl. Bars represent the mean value of three independent experiments. A typical autoradiograph of a single-round transcription from initiated complexes formed at the pHrP₂ template is shown below. (B) TIP experiments showing the time-course of the association of RNA polymerase I, TFIIH and TIF-IA with the rDNA in single-round transcriptions. Nuclear extracts were pre-incubated with rDNA template in the presence of the first 2 nt and reaction was started by addition of the lacking NTPs. After the indicated time-points formaldehyde was added to stop the reaction and to crosslink proteins to the DNA. These complexes were subsequent used for immunoprecipitation with the indicated antibodies and template association was detected in PCR reactions with primers directed against the human rDNA promoter. Pictures are representatives from at least three independent experiments.

Figure 4.

RNA polymerase I transcription is reduced in TFIIH/CS cells and can be rescued by wild-type TFIIH subunits in vivo and purified TFIIH in vitro. (A) Real-time PCR and northern blot analysis of 45S pre-rRNA expression of XPA cells as a wild-type control or CS cells harvested at equal density. Autoradiograph of the hybridized 45S pre-rRNA and ethidiumbromide stained 28S/18S rRNA as a loading control are shown below. (B) Real-time PCR analysis of pre-rRNA transcription and western blots of reconstituted lymphoblastoid cells with vector plasmid as a control, or wild-type XPB respective XPD. The XPB antibody recognizes only wild-type XPB, whereas the XPD antibody also detects mXPD. (C) Promoter-dependent RNA polymerase I in vitro transcription with the indicated nuclear extracts and purified TFIIH. Antibodies against the TFIIH subunit p44 inhibit the reconstitution (anti-TFIIH) and are inactivated by heat treatment (anti-TFIIH*). (D) Non specific RNA polymerase I transcription is stimulated by TFIIH. Sonified calf-thymus DNA was used as a template in the presence of α-amanitin with the indicated nuclear extracts and purified TFIIH. (E) The autoradiography shows end-to-end transcription of RNA polymerase I performed with a pUC template without rDNA promoter in the presence of α-amanitin. Pictures are representatives from at least three independent experiments.

Figure 5.

Reduced binding of mutant TFIIH to the rDNA in vitro and in vivo. (A) Quantitative PCR analysis of ChIP of the indicated cell lines with antibodies against RNA polymerase I and TFIIH (anti-cdk7) as indicated above. Primer were directed against the IGS, promoter or gene-internal regions (H4, H8). (B) Immobilized complexes were formed with the indicated nuclear extracts, washed with buffer AM50 and analyzed after urea elution on immunoblots. (C) Immobilized complexes from nuclear extracts as indicated above were challenged by addition of nucleotides and radioactive UTP. Sarcosyl was added to limit transcription to a single round. Synthesized RNA was isolated after 60 min and subject to eletrophoresis and autoradiography. Pictures are representatives from at least three independent experiments.

Figure 6.

Lack of TFIIH does not reduce residence time of the polymerase at the rDNA. TIP was performed by pre-incubation for 20 min of the indicated nuclear extracts with template and the first nucleotides to form initiated complexes. These complexes were challenged by the missing nucleotides and the reaction stopped after the indicated time points by addition of the crosslinking agent formaldehyde to 1%. Crosslinked proteins were immunoprecipitated with the indicated antibodies and associated template was amplified in semi-quantitative PCR. Pictures are representatives from at least three independent experiments.