1. CSB Is a Component of RNA Pol I Transcription
John Bradsher, Jerome Auriol, Luca Proietti de Santis, Sebastian Iben, Jean-Luc Vonesch, Ingrid Grummt, Jean-Marc Egly 
Molecular Cell 
Volume 10, Issue 4, Pages (October 2002)
DOI: /S (02)

2. Figure 1 CSB Is Found in the Nucleolus
Confocal images using CSB, CSA, BrUTP, and RNA pol II antibodies (as described in Experimental Procedures), of untreated (A–E), Dichlororibofuranosyl benzimidazole- (DRB) (F–J), or actinomycin D (Actino. D)-treated (K and L) C5Ro cells. CSB colocalizes with RNA pol I (M–P): nuclear matrix from C5Ro cells was described in Experimental Procedures, fixed and stained for CSB, then counterstained for RNA pol I. CSB colocalizes with TFIIH (Q–T): nuclear matrix from C5Ro cells was described above. CSB and CSA protein are distributed distinctly (U–W): C5Ro cells were stained for CSB, counterstained for CSA. H stained cells: Hoechst In (E), (J), (P), and (T), nucleolar images are presented at higher magnification (H.M.).
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2 CSB Stimulates Transcription from an rDNA Template
In vitro transcription was performed with recombinant UBF plus partially purified fractions of TIF-1A, TIF-1B, TIF-1C, and RNA pol I and increasing amounts of recombinant CSB (4, 16, and 40 fmoles) in which about 5 fm of TFIIH was present (lanes 1–4). The size of the run-off transcript is indicated at the right of the figure.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3 CSB Is Found in a Multifunctional Enzyme Complex
(A) Immunoprecipitates were prepared from the H0.6 fractions at physiological salt concentration using antibodies raised against CSB (CSB IP/150), eluted in the presence of peptide competitor, resolved on SDS-PAGE, and immuno-probed for the proteins as indicated at the right of each panel.
(B) CSB IP/150 was also tested for rRNA synthesis activity, in an assay that lacks either RNA pol I (left panel) or TIF-1B (right panel).
(C) CSB IP/150 was tested in a complete reconstituted RNA pol II transcription system (Gerard et al., 1991) containing (lane 1) or lacking either TFIIF, TFIIH, or RNA pol II, as indicated at the top of the panel. When indicated (lanes 5–8), CSB IP/150 was added in the incubation mixture.
(D) CSB IP/150 (lanes 8–13) was also tested in a reconstituted incision-excision assay (Araujo et al., 2000), in which XPA, RPA, XPC, ERCC1/XPF, TFIIH, or XPG repair was individually omitted as shown at the top of the panel.
(E and F) CSB IP/50 fraction was prepared as described above, except that the immunoprecipitate was washed at 50 mM KCl, resolved, and Western blotted for the proteins as indicated (E) or tested for the activity of RNA pol I and RNA pol II (F).
Abbreviations: L, load; FT, flowthrough; XPB and XPD are subunits of TFIIH; RPA116, the second largest subunit of RNA pol I; RPB1, the largest subunit of RNA pol II; TAF68 and TAF250 are subunits of TIF-1B and TFIID, respectively; CSA, Cockayne syndrome protein A. The size of the RNA pol I (300 nt) transcript, the RNA pol II (309 nt) transcript, and the excised DNA (26–32 nt) fragments are indicated at the right of each panel.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4 CSB Cofractionates with TFIIH, RNA Pol I, and XPG
H0.6 fraction derived from HeLa WCE was applied either to a Sephacryl S400 gel filtration or MonoQ anion exchange column as detailed in Experimental Procedures. Column calibration is indicated at the top of the panel: the void volume (Vo), the elution of the 70S bacterial ribosome (2.5 mD), the ferritin (0.44 mD). p89/XPB, p62, and p44 are subunits of TFIIH whereas RPA116 represents RNA pol I.
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5 rRNA Transcription is Deficient in CSB Patient-Derived Cells
(A) In CSB patient-derived cells, rRNA transcription is deficient: Unaffected patient-derived (C5Ro), CSB-deficient (CS1AN), and CSB-deficient cells transfected to express the wild-type CSB (CS1AN/CSB) were either untreated (a–c) or DRB treated (d–f). After BrUTP incorporation, cells were fixed and stained with anti-BrUTP antibody and examined by confocal microscopy. (g–i) H33258 counterstaining of the (d–f) images.
(B) rRNA incorporation: Cells were grown in 32P phosphoric acid, then lysed, and total RNA was recovered prior to formaldehyde agarose electrophoresis. The presence of similar levels of total 28S and 18S rRNA among these cells is documented in the lower EtBr-stained panel, whereas newly synthesized radiolabeled 45S rRNA is observed in the upper panel.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6 CSB Maintains RNA Pol I and TFIIH in a Protein Complex
Heparin fractions (500 μg) from CS1AN and CS1AN/CSB cells were applied and resolved on MonoQ as described (Experimental Procedures). In the lower panel, we have reconstituted the multifunctional protein complex by addition of recombinant highly purified CSB (60 ng) to the CS1AN extract. Fractions from each column were analyzed by immunoblotting for CSB, TFIIH (p44), and RNA pol I (RPA116).
Molecular Cell  , DOI: ( /S (02) )

8. Figure 7
The CSB-IP Complex Is Deficient in Cells of Patients with CS-Associated Mutations in CSB, XPB, and XPD
(A) MRC5, CS1AN, and CS1AN/CSB H0.6 fractions derived from the corresponding cell extracts (1 mg) were immunoprecipitated at 150 mM KCl using antibodies raised against the p44 subunit of TFIIH.
(B) Extracts (1 mg) from either the GM2246/XPC-deficient (as a control), GM2252/XPB-deficient (XPB/CS), or GM3249/XPD-deficient (XPD/CS) cells were precipitated with CSB antibodies. The load (L), the flowthrough (FT), as well as the immunoprecipitates (Bound) were resolved on SDS-PAGE followed by immunoblotting using antibodies directed toward TFIIH (p62 subunit), RNA pol I (RPA116), RNA pol II (RPB1), and CSB.
Molecular Cell  , DOI: ( /S (02) )