Ribin, a protein encoded by a message complementary to rRNA, modulates ribosomal transcription and cell proliferation

Abstract

The control of rRNA transcription, tightly coupled to the cell cycle and growth state of the cell, is a key process for understanding the mechanisms that drive cell proliferation. Here we describe a novel protein, ribin, found in rodents, that binds to the rRNA promoter and stimulates its activity. The protein also interacts with the basal rRNA transcription factor UBF. The open reading frame encoding ribin is 96% complementary to a central region of the large rRNA. This demonstrates that ribosomal DNA-related sequences in higher eukaryotes can be expressed as protein-coding messages. Ribin contains two predicted nuclear localization sequence elements, and green fluorescent protein-ribin fusion proteins localize in the nucleus. Cell lines overexpressing ribin exhibit enhanced rRNA transcription and faster growth. Furthermore, these cells significantly overcome the suppression of rRNA synthesis caused by serum deprivation. On the other hand, the endogenous ribin level correlates positively with the amount of serum in the medium. The data show that ribin is a limiting stimulatory factor for rRNA synthesis in vivo and suggest its involvement in the pathway that adapts ribosomal transcription and cell proliferation to physiological changes.

FIG. 1

Amino acid sequence of ribin ORF, rDNA homology region, and PCR detection of the cloned gene. (A) Amino acid sequence of the 295-amino-acid ribin ORF. NLS-type sequences are underlined. (B) The ribin gene is homologous to an internal region of 28S rDNA; the ribin ORF is oriented opposite to rRNA polarity. About half of this ORF overlaps the D8 variable domain of the rRNA gene. (C) Rat, mouse, and human DNAs are PCR amplified with primers A plus B (lanes 1 and 3) or A plus C (lanes 2 and 4) (primer positions are shown in panel B). Primers A and C are derived from rDNA, where A is within the ribin coding region, while C is located beyond the 3′ end of ribin cDNA. Primer B ends in a ribin-specific BglII site at the 3′ terminus of the gene (shown by the asterisk in panel B). The AB and AC products should therefore represent ribin and the bulk rRNA genes, respectively. In lanes 3 and 4, the template DNA was digested with BglII prior to PCR. Amplified products (386 and 450 bp, shown by arrows) were resolved in a 2% ethidium bromide-stained agarose gel, along with a 100-bp DNA ladder (lanes M).

FIG. 2

Binding of ribin to rRNA core promoter, effect on Pol I transcription, and interaction of the protein with UBF. (A) SWA of extracts from rat N1S1 cells (lanes 1 and 2) or bacterial cells induced with IPTG to express the GST-ribin fusion (lanes 4 and 6) or uninduced (lanes 3 and 5). Duplicate samples of each extract were probed with the wild-type (WT; rCPr, lanes 1, 3, and 4) or point-mutated (M; rCPrM, lanes 2, 5, and 6) rat rRNA core promoter probe. The binding signals corresponding to ribin and the GST fusion are marked by black and white arrowheads, respectively, and the positions of protein standards are shown (in kilodaltons) on the right. (B) Ribin was translated (trl) in the presence of [³⁵S]methionine, and the radiolabeled product was analyzed by SDS–10% PAGE (lane 2, shown by arrowhead). In mock translation, the RNA template is omitted (lane 1). (C) In vitro-translated (unlabeled) ribin was tested directly in a cell-free transcription assay. pB7-2.0/HindIII-cut rat rDNA template (50 ng) was transcribed with 4 μl of DEAE-175 fraction alone (lane 1) or supplemented with 4 μl of mock translation reaction (lane 2), 4 μl of ribin translation (txn) reaction (lane 4), or 2 μl of each (lane 3). The specific transcript of 124 nt is marked by an arrow. (D) Nuclear extract from BHK cells was incubated with GST-ribin fusion protein (lanes 4 to 6) or GST alone (lanes 1 to 3), followed by sedimentation of the products formed on glutathione-Sepharose bead complexes. Aliquots taken from the total reaction mix before precipitation (T), from the supernatants (unbound fraction, U), and from the resuspended pellets (bound fraction, B) were subjected to immunoblotting with an anti-UBF antibody.

FIG. 3

Ribin expression levels upon serum depletion and after transfection. (A) Total cell extracts from hamster BHK cells and mouse N2a cells were probed in Western blotting with antiribin serum (lanes 1 and 2) or preimmune serum (lanes 3 and 4). In both types of cells, endogenous ribin (approximately 32 kDa) was detected. The positions of protein standards are shown (in kilodaltons) on the left. (B) BHK cells were cultured for 22 h with 10, 0, or 1% serum (lanes 1 to 3). Portions of the last two (serum deficient) samples were cultured for an additional 18 h with (lanes 4 and 5) or without (lanes 6 and 7) addition of serum back to 10%. Lane 8, control cells incubated continuously with 10% serum. Equal amounts of total cell protein from each variant were used for immunoblotting with antiribin antibody. The same cell extracts were also immunoprobed for actin as a reference control. (C) BHK and Vero cells stably transfected with pSinRep21 expression vector carrying ribin cDNA (lanes 2 and 4) or with blank vector (lanes 1 and 3) were probed for ribin by immunoblotting or Southwestern technique. In the latter, a mouse rDNA core promoter fragment, mCPr, was used as a binding probe.

FIG. 4

Effect of ribin overexpression on rRNA synthesis and cell proliferation. (A) Nuclei isolated from Vero cells stably transfected with pSinRep21 expression vector harboring ribin cDNA in the sense (S) or antisense (AS) orientation, or with blank vector (V), were used for a run-on transcription assay in the absence or presence of α-amanitin (150 μg/ml, lower panel). The samples in lanes V and S are the same cell lines shown in Fig. 3C, lanes 3 and 4, respectively. Radiolabeled nuclear RNA was then isolated and used in dot hybridization. Dot samples: D1 to D3, 1, 2, and 3 μg, respectively, of pBS-28SBE plasmid DNA, containing a 2,029-bp fragment of mouse 28S rDNA; R, 0.1 μg of sense RNA transcript of ribin cDNA; P, 3 μg of pBS vector DNA; GAP, 1 μg of plasmid pTRI-GAPDH, containing a 316-bp fragment of the GAPDH gene (note that P and GAP dots were exposed eight times longer than rDNA dots). SA N-RNA, specific activity of the radiolabeled nuclear RNA obtained after run-on transcription. Reactions were normalized by using equal amounts of nuclei for transcription and of nuclear RNA for hybridization. The panels on the right show a densitometry quantitation of the hybridization signals and the relative effect of ribin overexpression on Pol I and Pol II RNA transcription. (B) Growth rate of the cell transfectants. Equal starting amounts of the three cell variants were grown for 68 h (still subconfluent), and cell number was counted in the time intervals.

FIG. 5

Effect of serum starvation on rRNA synthesis in cells overexpressing ribin. BHK cells stably transfected with the pSinRep21 expression vector carrying ribin cDNA (S) or with the blank vector (V) were cultured with a control amount (10%, SER+) or a reduced amount (SER−) of serum (0.5% for 12 h). Nuclei isolated from these cells were used for run-on transcription in the presence of 150 μg of α-amanitin, followed by dot hybridization, as in Fig. 2. The dot samples D1, D2, P, and GAP are also as in Fig. 2. The specific activity of the radiolabeled nuclear RNA is given as in Fig. 4 (SA N-RNA). A densitometric quantitation of the average transcriptional signals is shown on the right. The values for the control (V, SER+) cells were taken as 100%, and the SER− values are presented by open bars.

FIG. 6

Cellular localization of ribin. BHK cells were transfected with GFP (A and C) or ribin-GFP fusion constructs (B and D). Identical accumulation of ribin in the nucleus was observed whether the GFP moiety was N or C terminal to the ribin ORF, as illustrated with the upper and lower images in each panel. Fluorescent images were taken by confocal microscope 42 h (A and B) or 7 days (C and D) after transfection.