Figure 1.
GRIM-1 is a novel growth suppressor.
A) GRIM-1 depletion confers a growth advantage during RA/IFN stimulation. Top: Growth of HeLa cells stably expressing pLKO1 (EV; ○) or scrambled shRNA (SC; ▪) or GRIM-1-specific (GS; ▴) shRNA were monitored for 4 days in the presence of a sub-lethal but growth-inhibitory concentration of hIFN-β (1000 U/ml) and RA (1 µM). GRIM-1-depleted cells continue to grow while the others are inhibited by RA/IFN. B) Depletion of GRIM-1 ascertained by WB analysis using GRIM-1-specific antibody and actin-specific antibody was used as loading control. GRIM-1 in GS cells represent ∼12% compared to levels in EV and/or SC when normalized to corresponding actin levels. C) GRIM-1 over-expression sensitizes cells to RA/IFN-induced cell death. HeLa cells transiently expressing GFP-tagged GRIM-1 isoforms (Open bars) were challenged with hIFN-β (2000 U/ml) and RA (2.5 µM) (Solid bars) for 12 h. Change in cell/nuclear appearance of GFP-positive cells was used as a measure of cells undergoing apoptosis. Numbers to the side in WB indicate positions of protein markers.
Figure 2.
GRIM-1 isoforms interact with endogenous NAF1 in mammalian cells.
HeLa cells transiently expressing Myc-tagged GRIM-1 isoforms were analyzed for interaction with NAF1 using the indicated antibodies. A) Expression levels of GRIM-1 isoforms (top), NAF1 (middle) and actin (bottom) in the indicated transfectants. GRIM-1γ levels are low compared to GRIM-1α/β when normalized to actin levels. No significant change in NAF1 levels are observed upon GRIM-1 expression. B & C) Total cell lysates (signal-normalized lysate amount) were subjected to IP with the indicated antibodies and subsequently analyzed by WB with the indicated antibodies. All GRIM-1 isoforms interact with NAF1. More NAF1 in GRIM-1γ-expressing cells is due to more lysate used for IP. D–E) GRIM-1 and NAF1 co-localize in situ. D) Confocal images of Myc-tagged GRIM-1 isoforms (green channel) and NAF1 (red channel). Nuclei (blue channel) were visualized using DAPI. GRIM-1 isoforms and NAF1 complexes (yellow) can be seen in the merged channel indicative of interaction in situ. Lobed nuclei appear with a high frequency in GRIM-1γ-expressing cells. E) GRIM-1 is necessary for delocalizing NAF1. Immunofluorescent images of GRIM-1 and NAF1 in the indicated cell lines at steady state and RA/IFN-stimulated state. In both cell lines, NAF1 is nuclear at steady state. Upon RA/IFN stimulation, NAF1 is delocalized in SC cells, but not in GS cells. F) Confocal images of endogenous GRIM-1. RA/IFN stimulation increases GRIM-1 that accumulates more in the cytoplasm. G) NAF1 levels are not influenced by IFN/RA treatment. Total cell lysates were prepared at the indicated time point after RA/IFN treatment and analyzed using the indicated antibodies in WB. Numbers to the side in WB indicate positions of protein markers.
Figure 3.
GRIM-1 regulates small non-coding RNA levels.
Relative abundance was calculated with respect to spliceosomal RNA (snRNA-U6) levels by real-time PCR. A) Levels of the indicated RNA in HeLa cells stably expressing Myc-tagged GRIM-1 isoforms. GRIM-1 isoforms differentially suppress box H/ACA-containing RNA. Top: snoRNA-73 (open), -74 (hatched), -50 (light gray); Middle: scaRNA-8 (stippled), -13 (brick), TERC (black); Bottom: spliceosomal RNA (snRNA-U2 (wavy)) and box C/D-containing RNA (snoRNA-U3 (checkered)) levels. A related box C/D snoRNA-U13 (dark gray) levels are not influenced by GRIM-1. B) Levels of the indicated RNA upon GRIM-1 depletion in HeLa cells. GRIM-1-depleted cells (GS) show increased box H/ACA-containing RNA. Top: snoRNA-73, -74 and -50; Middle: scaRNA-8, -13, TERC; Bottom: snRNA-U2 and snoRNA-U3 levels. A related box C/D snoRNA-U13 levels is not influenced by GRIM-1. Bar patterns same as in (A). **, p<0.01.
Figure 4.
GRIM-1 regulates small non-coding RNA levels.
A) Construction of shRNA-resistant GRIM-1 variant. Top: GRIM-1 shRNA-targeted region showing the wild-type sequence (WT Seq) and their corresponding amino acids (AA Seq) number (Codon No). Base changes coding for the same amino acids are underlined in the variant sequence (Var Seq). Bottom: GRIM-1 shRNA does not target the variant sequence for silencing. Expression of Myc-tagged GRIM-1Var isoforms in GRIM-1-depleted cells. Actin was used as a loading control. B) The repressive effect of GRIM-1 on small non-coding RNA reappears upon GRIM-1Var expression in GRIM-1-depleted cells (GS). Levels of the indicated RNA in HeLa cells stably expressing Myc-tagged GRIM-1Var isoforms. GRIM-1Var isoforms differentially suppress snoRNA-73, -74, -50, scaRNA-8, -13, TERC, snRNA-U2 and snoRNA-U3 levels. A related box C/D snoRNA-U13 levels are not influenced by GRIM-1. Relative abundance was calculated with respect to spliceosomal RNA (snRNA-U6) levels by real-time PCR. *, p<0.01; **, p<0.001. Numbers to the side in WB indicate positions of protein markers. Bar patterns same as in Figure 3.
Figure 5.
GRIM-1-mediated effects on rRNA processing.
A) A Modular representation of mammalian rRNA primary transcript. Top: Relative positions of regions absent (thin lines) and present in the mature rRNA (solid lines). Bottom: Relative positions of primers used to analyze rRNA processing. Primer (1 or A) is used for first-strand synthesis in RT. The Ct value obtained using primers 6/1 and F/A represent the total rRNA (mature and unprocessed) present in the preparation. The Ct value obtained using two different primer sets (4/5 and 2/3) for 5′ETS-18S and (D/E and B/C) for ITS2-28S represent unprocessed rRNA present in the preparation. Primers 2 and 5 cannot amplify when 18S rRNA has matured. Similarly, primers B and E cannot amplify when 28S rRNA has matured. B) GRIM-1 isoforms suppress rRNA processing. Isoform-specific inhibitory effects are evident on 5′ETS-18S region with GRIM-1γ being a potent inhibitor of processing and GRIM-1α is the least inhibitory. The values represent the averages of primer pairs 4/5 (unprocessed) over 6/1 (total) and primer pairs 2/3 (unprocessed) over 6/1 (total) for 18S rRNA. C) Efficient processing of 18S rRNA regions in GRIM-1-depleted cells (GS) compared to empty vector control (pLKO1; EV) and/or scrambled shRNA control (SC). Values were calculated as mentioned above. D) Reappearance of the inhibitory effect on rRNA processing upon GRIM-1Var expression in GRIM-1-depleted cells (GS). Isoform-specific effects are evident on 5′ETS-18S region. E) Efficient processing of 28S rRNA regions in GRIM-1-depleted cells (GS) compared to empty vector control (pLKO1; EV) and/or scrambled shRNA control (SC). The values represent the averages of primer pairs D/E (unprocessed) over F/A (total) and primer pairs B/C (unprocessed) over F/A (total) for 28S rRNA. F) GRIM-1var isoforms show inhibitory effect on ITS2-28S region. GRIM-1βvar and GRIM-1γvar exert similar inhibitory effect while GRIM-1αvar is less inhibitory on ITS2-28S rRNA processing. *, p<0.01; **, p<0.001.
Figure 6.
GRIM-1 isoforms interact with endogenous Dyskerin in mammalian cells.
HeLa cells transiently expressing Myc-tagged GRIM-1 isoforms were analyzed for interaction with Dyskerin using the indicated antibodies. A) Expression levels of Dyskerin (top) and actin (bottom) in the indicated transfectants. No significant change in Dyskerin levels are observed upon GRIM-1 expression. B & C) Total cell lysates (signal-normalized lysate amount) were subjected to IP with the indicated antibodies and subsequently probed with the indicated antibodies. All GRIM-1 isoforms interact with Dyskerin. D) Quantification of Dyskerin-bound RNA in the indicated cell lines. RNA extracted following RNP-IP of cross-linked cell lysates using Dyskerin-specific antibody, was used in RT and analyzed by real-time PCR using primers. Dyskerin-bound RNA levels are low in GRIM-1-expressing cells. Majority of snoRNA-74 fraction is bound by Dyskerin (a box H/ACA core protein) while only a minor fraction of snoRNA-U3 is bound by Dyskerin. Open bars represent RNA levels (total) in cells, Checkered bars represent Dyskerin-bound RNA levels in cells and Solid bars represent RNP-IP performed using an IgG control. *, p<0.01; **, p<0.001. Numbers to the side in WB indicate positions of protein markers.
Figure 7.
Yeast Shq1p protein exhibits GRIM-1α-like behavior in mammalian cells.
A) Expression of FLAG-tagged Shq1p in GRIM-1-depleted HeLa cells ascertained by WB. B) Shq1p interacts with endogenous NAF1. IP and WB analysis were performed as described previously with the indicated antibodies. C) Growth assay of empty vector control (VC-F; ♦), Shq1p-expressing (▪) and GRIM-1α-expressing (▴) cells. D) Box H/ACA RNA levels are suppressed by Shq1p and GRIM-1α to a similar extent. Bar patterns are: pCXN2-FLAG vector (Open; VC-F), FLAG-Shq1p (Hatched; Shq1p), pCXN2-Myc vector (Stippled; VC-M) and Myc-tagged GRIM-1α (Solid; 1α). E) Inhibition of 18S rRNA processing by Shq1p and GRIM-1α are similar. Bar patterns as in D. The values represent the averages of primer pairs 4/5 (unprocessed) over 6/1 (total) and primer pairs 2/3 (unprocessed) over 6/1 (total) for 18S rRNA. See Fig. 5 legend for additional information. F) Over-expressed Shq1p more accumulates in the cytoplasm. Confocal images showing more Shq1p in the cytoplasm compared to the nucleus. *, p<0.01. Numbers to the side in WB indicate positions of protein markers.
Figure 8.
GRIM-1 expression is lost in prostatic epithelial cells.
A) NCI-TARP array was analyzed by immunohistochemistry as per standard procedures. A representative patient-matched sample showing low expression of GRIM-1 (brown signals) in prostate tumors compared to normal prostate. See Table 1 for additional information. B) GRIM-1 mRNA levels are low in VCaP compared to RWPE-1 as analyzed by qPCR while the other indicated RNAs are high in VCaP compared to RWPE-1 cell line. GRIM-1 mRNA levels were analyzed using three different primer pairs (Table S3). *, p<0.01; **, p<0.001.
Table 1.
GRIM-1 expression levels in normal and tumor specimens analyzed by immunohistochemistry.