Nucleolin is required for RNA polymerase I transcription in vivo

Abstract

Eukaryotic genomes are packaged with histones and accessory proteins in the form of chromatin. RNA polymerases and their accessory proteins are sufficient for transcription of naked DNA, but not of chromatin, templates in vitro. In this study, we purified and identified nucleolin as a protein that allows RNA polymerase II to transcribe nucleosomal templates in vitro. As immunofluorescence confirmed that nucleolin localizes primarily to nucleoli with RNA polymerase I, we demonstrated that nucleolin allows RNA polymerase I transcription of chromatin templates in vitro. The results of chromatin immunoprecipitation experiments established that nucleolin is associated with chromatin containing rRNA genes transcribed by RNA polymerase I but not with genes transcribed by RNA polymerase II or III. Knockdown of nucleolin by RNA interference resulted in specific inhibition of RNA polymerase I transcription. We therefore propose that an important function of nucleolin is to permit RNA polymerase I to transcribe nucleolar chromatin.

FIG. 1.

FACT-dependent RNA polymerase II transcription of a chromatin template. (A) In vitro chromatin assembly. The proteins used for chromatin assembly are shown after electrophoresis in polyacrylamide gels containing SDS and Coomassie blue staining. (B) Chromatin assembled onto the pG5MLP plasmid was digested with two concentrations of micrococcal nuclease (MNase), resolved by electrophoresis in a 1.3% agarose gel, and visualized by ethidium bromide staining. The arrow indicates an increasing concentration of micrococcal nuclease. (C) Transcription reaction mixtures contained naked (left side) or chromatin-assembled (middle) pG5MLP and the transcriptional components and concentrations of FACT indicated. RNA labeled in vitro was purified and visualized as described in Materials and Methods. The right side shows purified FACT analyzed by SDS-PAGE and Coomassie blue staining. Ad MLP, adenovirus major late promoter.

FIG. 2.

Purification of proteins that stimulate transcription of chromatin templates by RNA polymerase II. (A) Transcription reaction mixtures contained the chromatin template described in the legend to Fig. 1B, the quantities of FACT indicated at the top, and equal volumes of the P11 fractions indicated. The bottom part shows the immunoblotting of these fractions for the hSpt16 subunit of FACT. (B) Summary of purification of stimulatory activities from the 0.1 and 0.35 M P11 fractions. NE, nuclear extract; ppt., precipitation. Panels C and D show the results of the final steps in purification of the activities detected in the 0.1 and 0.35 M P11 fractions. In each case, the top and bottom parts show the results of assaying the fractions indicated under the conditions described for panel A and of analysis of the proteins present in the fractions by SDS-PAGE and Coomassie blue staining, respectively. The proteins marked by the arrows were identified as nucleolin by mass spectroscopy. nt, nucleotides; M, markers.

FIG. 3.

Nucleolin is the protein responsible for chromatin transcription activity. (A) Chromatin templates were transcribed in the presence of a limiting concentration (0.5×) of FACT (all lanes) and purified nucleolin (lane 2) or purified nucleolin that had been mock depleted with beads alone (M) (lane 3) or incubated with control antibody bound to beads (C) (lane 4) or with antinucleolin antibody bound to beads (Nuc) (lane 5). nt, nucleotides. (B) Immunoblotting of these same nucleolin preparations with an antinucleolin antibody.

FIG. 4.

Nucleolin specifically stimulates transcription of a chromatin template. (A) Coomassie blue staining of SDS-PAGE of purified FLAG-tagged nucleolin. (B) FLAG-nucleolin was assayed in chromatin transcription assays. (C) The naked DNA template was transcribed in the presence of FLAG-nucleolin as indicated. nt, nucleotides; M, markers.

FIG. 5.

Nucleolin and RNA polymerase I localize to nucleoli. HeLa cells were fixed and stained (red) with antibodies against nucleolin (A), RNA polymerase I (B), TFIIF (C), or RNA polymerase II (D). In all cases, DNA was stained with DAPI (blue).

FIG. 6.

Nucleolin facilitates RNA polymerase (pol) I transcription of chromatin templates. (A) Coomassie blue staining of SDS-polyacrylamide gels containing the purified proteins used to reconstitute RNA polymerase I transcription. (B) RNA polymerase I, SL-1, and UBF-1 were added to transcription reaction mixtures containing an rRNA gene template as indicated at the top of the panel. The transcripts were amplified by one-step RT-PCR as described in Materials and Methods. No products were observed in reaction mixtures that lacked SL1 (lane 2) or contained only nucleolin (data not shown), establishing the specificity for rRNA transcripts and the absence of rRNA bound to nucleolin, respectively. (C) Nucleolin does not stimulate RNA polymerase I transcription from a naked template. Proteins were added as indicated at the top of the panel. All reaction mixtures contained RNA polymerase I, SL-1, and UBF-1. (D) Transcription reaction mixtures contained the components shown in panel A, the rRNA gene template assembled into chromatin and FLAG-nucleolin, or FACT, as indicated at the top.

FIG. 7.

Nucleolin binds to chromatin containing rRNA. (A) The association of FLAG-nucleolin with chromosomal histones was examined by immunoprecipitation. The protein present in input chromatin (I) and the flowthrough (FT) and peptide-eluted (E) fractions was examined by SDS-PAGE and Coomassie blue staining. Nucleophosphin (fraction E) was identified by mass spectrometry. M, markers (the values on the left are sizes in kilodaltons). (B) Chromatin immunoprecipitations of cross-linked HeLa cell chromatin were performed with control beads (lanes 1) or with 2 μg of nucleolin antibody conjugated to beads (lanes 2). DNA isolated from the immunoprecipitates was analyzed by PCR for the genes indicated. Signals were quantified with Image J software and used to calculate the DNA concentration in the nucleolin immunoprecipitate relative to that of the control. The values shown are the means of four independent experiments. mAb, monoclonal antibody.

FIG. 8.

Nucleolin is required for RNA polymerase I transcription. (A) Immunoblotting of proteins isolated from cells treated with siRNA for nucleolin, as indicated. (B) RNA isolated from cells treated with siRNA for nucleolin, as indicated, was amplified by one-step RT-PCR as described in Materials and Methods.