Chromatin association and regulation of rDNA transcription by the Ras-family protein RasL11a

Abstract

RasL11a and RasL11b are Ras super-family proteins of unknown function. Here, we show that RasL11a is a chromatin-associated modulator of pre-ribosomal RNA (pre-rRNA) synthesis. RasL11a was found in the nucleolus of interphase mouse fibroblasts, where it co-localized with the RNA polymerase I-specific transcription factor UBF. Similar to UBF, RasL11a also marked the active subset of rDNA repeats (also called nucleolar organizers, or NORs) on mitotic chromosomes. In cells, RasL11a existed in stable complexes with UBF and, as shown by chromatin immunoprecipitation, distributed along the rDNA transcription unit. Upon treatment of cells with actinomycin D, RasL11a and UBF persisted on the transcription unit beyond the release of RNA polymerase I, and remained co-localized in peri-nucleolar cap structures. Ectopic expression of RasL11a enhanced pre-rRNA levels in cells, whereas RasL11a knockdown had the opposite effect. In transient transfection experiments, RasL11a enhanced the transcriptional activity of an RNA polymerase I-specific reporter controlled by the rDNA enhancer/promoter region. We speculate that RasL11a acts in concert with UBF to facilitate initiation and/or elongation by RNA polymerase I in response to specific upstream stimuli.

Figure 1

Subcellular localization of RasL11a. (A–C) Asynchronously proliferating NIH-3T3 cells were analysed by indirect immunofluorescence and examined under the confocal microscope. Nuclear DNA was counter-stained with DAPI. Representative confocal sections of a single nucleus are shown for interphase cells co-stained for (A) RasL11a (Ab49) and NPM, (B) RasL11a and nucleolin, (C) RasL11a and fibrillarin and (D) RasL11a and UBF.

Figure 2

Similar to UBF, RasL11a stains active NORs and is associated with rDNA. (A) Cells at the indicated stages of mitosis are shown from the same RasL11a/UBF co-localization experiment as in Figure 1D. (B) Immuno-FISH analysis was used to co-stain RasL11a (Ab49) and rDNA. The rDNA probe for FISH was located in the IGS (accession number AF441733). The successive panels (i–viii) show serial confocal sections of a representative mitotic cell. The six pairs of dots showing merged signals (yellow) are numbered 1–6. Other chromosomal dots are red, indicating the presence of rDNA without RasL11a, whereas the opposite (green chromosomal dots) is not observed. Note that the green cytosolic staining is due to the over-exposure required for FISH.

Figure 3

RasL11a is associated with rDNA in vivo. (A) Schematic representation of the mouse rDNA repeat. The sequence coordinates and annotation were taken from Genbank BK000964 (note that the promoter/enhancer region was moved from the 3′ to the 5′ end, for display). The position and numbering of all PCR amplicons used for qChIP analysis is indicated (amplicon #), with the corresponding PCR primers listed in Supplementary Table S1. All amplicons are 100–150 bp in length. qChIP analysis was performed with (B) the RPA antibodies H300 and TM, (C) anti-UBF or (D) the RasL11a antibodies Ab48 and Ab49. Data represent the average and standard deviation from two (B) or four (C, D) independent qChIP experiments with each antibody. All were quantified as percent of input DNA (Frank et al, 2001) and normalized to the maximum peak value in the rDNA profile. The maximum peak value for RPA194 was 1.27% of input with antibody H300 (on amplicon #18) and 3.24% with TM (on amplicon #19). For UBF, it was 1% of input (amplicon #1). For RasL11a it was 0,96% of input with Ab48 and 0.95% with Ab49 (both on amplicon #16).

Figure 4

RasL11a and UBF persist on the rDNA transcription unit after removal of RPI. Proliferating NIH-3T3 cells were treated for 1 h with the indicated concentrations of actinomycin D. qChIP analysis was performed with (A) the RPA194 antibodies H300 and TM, (B) anti-UBF or (C) the RasL11a antibody Ab48. nt, not treated.

Figure 5

RasL11a co-segregates with UBF after actinomycin D treatment. Proliferating NIH-3T3 cells were treated with actinomycin D as indicated to the left, stained by indirect immunofluorescence as indicated on top and examined under a confocal microscope.

Figure 6

RasL11a and UBF interact in cells. (A, B) NIH-3T3 nuclear lysates were immunoprecipitated (IP) with the antibodies indicated on top, and the precipitates analysed on immunoblots with the antibodies indicated to the left. BP6: anti-RasL11a monoclonal (see Materials and methods). Control mAb: non-specific monoclonal antibody of the same isotype. BP6 alone: mock IP with BP6 and no lysate. Supernatant BP6 IP: supernatant from the RasL11a immunoprecipitation.

Figure 7

RasL11a positively regulates RPI transcription. (A) RasL11a transactivates an RPI reporter. NIH-3T3 cell were co-transfected with the RPI reporter plasmid pMR5783-BH (Budde and Grummt, 1999) and increasing amounts of pCMV5-hRasL11a. The RNA produced from this reporter was quantified by RT–PCR, and normalized to the RNA from a co-transfected RPII-specific Renilla luciferase reporter, which was not influenced by RasL11a. All values are expressed relative to the control sample without RasL11a (black bar). The panels above the graph show RasL11a and vinculin levels from the same transfected cells, as analysed by immunoblotting. (B) NIH-3T3 cells were infected with retroviruses expressing human RasL11a, with or without a Flag epitope tag, as indicated. Top panels: immunoblot analysis of RasL11a and vinculin in the infected cells. Bar graph: pre-RNA levels in infected cells, measured by quantitative RT–PCR. The data are the average of four independent experiments. RasL11a mRNA levels were enhanced ca. six-fold in the pBabe-hRasL11a-infected cells, as assayed with primers complementary to both the mouse and human cDNAs (data not shown).

Figure 8

RasL11a is limiting for pre-rRNA synthesis in NIH-3T3 cells. (A) Cells were infected with a retrovirus expressing an RasL11a-specific shRNA hairpin (shL11a) or with a control vector, as indicated. RasL11a mRNA and pre-rRNA levels were measured by RT–PCR amplification (for the pre-rRNA 5′ ETS, amplicon #6′, Figure 3A) with total RNA as template, normalized to the TBP mRNA and expressed relative to the control (Vector). Note that knockdown of the RasL11a protein by shL11a is shown in Supplementary Figure S1B and D. (B) NIH-3T3 cells or (C) primary MEFs were transfected with two different siRNA oligonucleotides against the RasL11a mRNA (A13, A14), or with a control oligonucleotide (all from Dharmacon), as indicated. RasL11a mRNA and pre-rRNA levels were quantified as in (A). (D, E) NIH-3T3 cells were transfected with a control siRNA, the A13 siRNA against RasL11a, or a siRNA pool targeting the UBF mRNA. Pre-rRNA levels were analysed by RT–PCR with amplicons #6′ or #7 (see map, Figure 3A), with either total RNA (D), or the nascent (chromatin-associated) RNA fraction (E) as template.