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. 2001 Nov;12(11):3680-9.
doi: 10.1091/mbc.12.11.3680.

Basic Domains Target Protein Subunits of the RNase MRP Complex to the Nucleolus Independently of Complex Association

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Free PMC article

Basic Domains Target Protein Subunits of the RNase MRP Complex to the Nucleolus Independently of Complex Association

H van Eenennaam et al. Mol Biol Cell. .
Free PMC article

Abstract

The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both RNase MRP and RNase P accumulate in the nucleolus of eukaryotic cells. In this report we show that for three protein subunits of the RNase MRP complex (hPop1, hPop4, and Rpp38) basic domains are responsible for their nucleolar accumulation and that they are able to accumulate in the nucleolus independently of their association with the RNase MRP and RNase P complexes. We also show that certain mutants of hPop4 accumulate in the Cajal bodies, suggesting that hPop4 traverses through these bodies to the nucleolus. Furthermore, we characterized a deletion mutant of Rpp38 that preferentially associates with the RNase MRP complex, giving a first clue about the difference in protein composition of the human RNase MRP and RNase P complexes. On the basis of all available data on nucleolar localization sequences, we hypothesize that nucleolar accumulation of proteins containing basic domains proceeds by diffusion and retention rather than by an active transport process. The existence of nucleolar localization sequences is discussed.

Figures

Figure 1
Figure 1
Schematic representation of the hPop1, Rpp38, and hPop4 proteins. The length, putative nuclear localization signals (NLS) and nucleolar localization signals (NoLSs) as established by Jarrous and coworkers (Jarrous et al., 1999b) for the hPop1 (A), Rpp38 (B), and hPop4 (C) protein subunits are depicted in the upper parts. The lower parts represent charge plots of these proteins. Values on the vertical axis represent positively charged and negatively charged regions, respectively. These values were calculated with the use of a window of nine amino acids. Gray regions in the charge plots represent the regions of the proteins important for their nucleolar localization. For the hPop1 protein (A) the charge plot of only amino acids 100–400 is depicted (hatched area in upper part). The R-, W- and G-box refer to conserved sequence elements observed in the hPop1 protein (Lygerou et al., 1996b).
Figure 2
Figure 2
Subcellular localization of deletion mutants of hPop1. (A) VSV-hPop1 constructs were transiently transfected into HEp-2 cells. Cells were fixed with paraformaldehyde, and the expressed proteins were visualized with the use of anti-VSV antibodies (panels 1, 3, and 5). A phase-contrast image of the same cells is shown in panels 2, 4, and 6. Panels 1 and 2: VSV-hPop1 construct; panels 3 and 4: VSV-hPop1 Δ(1–318); panels 5 and 6: VSV-hPop1 Δ(129–1024). (B) EGFP-hPop1 constructs were transiently transfected into HEp-2 cells. Cells were fixed with methanol/acetone, and the fluorescent proteins were visualized by direct fluorescence microscopy (panels 1, 3, and 5). Phase-contrast images of the same cells are shown in panels 2, 4, and 6. Panels 1 and 2: EGFP-hPop1 128–319; panels 3 and 4: EGFP-hPop1 128–167; panels 5 and 6: EGFP-hPop1 168–245.
Figure 3
Figure 3
Subcellular localization of deletion mutants of Rpp38. ECFP-Rpp38 constructs were transiently transfected into HEp-2 cells. Cells were fixed with methanol/acetone, and the fluorescent proteins were visualized by direct fluorescence microscopy (panels 1, 3, 5, and 7). Phase-contrast images of the same cells are shown in panels 2, 4, 6, and 8. Panels 1 and 2: ECFP-Rpp38; panels 3 and 4: ECFP-Rpp38 Δ(1–98); panels 5 and 6: ECFP-Rpp38 Δ(1–141); and panels 7 and 8: ECFP-Rpp38 Δ(181–283).
Figure 4
Figure 4
Association of ECFP-Rpp38 (mutants) with RNase MRP and RNase P ribonucleoprotein particles and RNase P activity associated with ECFP-Rpp38 deletion mutants. (A) Constructs encoding (deletion mutants of) ECFP-Rpp38 were transiently transfected in HEp-2 cells. Extracts from these cells used for immunoprecipitations with anti-ECFP antibodies. RNA isolated from total cell extracts (lanes 1–7) and immunoprecipitates (lanes 8–14) was analyzed by Northern blot hybridization with the use of riboprobes specific for RNase MRP and RNase P RNA. Lanes 1 and 8: material from control cells expressing ECFP alone; lanes 2 and 9: ECFP-Rpp38; lanes 3 and 10: ECFP-Rpp38 Δ(1–40); lanes 4 and 11: ECFP-Rpp38 Δ(1–98); lanes 5 and 12: ECFP-Rpp38 Δ(1–141); lanes 6 and 13: ECFP-Rpp38 Δ(181–283) and lanes 7 and 14, ECFP-Rpp38 Δ(246–283). The positions of RNase P and RNase MRP RNA are indicated. (B) RNase P activity assay associated with anti-ECFP immunoprecipitates from extracts of cells transiently transfected with ECFP-Rpp38 (deletion mutants). Lane 1: material from control cells expressing ECFP alone; lane 2: ECFP-Rpp38; lane 3: ECFP-Rpp38 Δ(1–40); lane 4: ECFP-Rpp38 Δ(1–98); lane 5: ECFP-Rpp38 Δ(1–141); lane 6: ECFP-Rpp38 Δ(181–283); lane 7: ECFP-Rpp38 Δ(246–283); lane 8: beads alone; and lane 9, substrate RNA. On the right, the positions of the pre-tRNA, the mature tRNA and the 5′-leader are indicated.
Figure 5
Figure 5
Subcellular localization of deletion mutants of hPop4. VSV-hPop4 constructs were transiently transfected into HEp-2 cells. Cells were fixed with methanol/acetone, and the expressed proteins were visualized by indirect fluorescence microscopy with the use of anti-VSV antibodies (panels 1, 3, 5, and 7). Phase-contrast images of the same cells are shown in panels 2, 4, 6, and 8. Panels 1 and 2: VSV-hPop4; panels 3 and 4: VSV-hPop4 Δ(61–109), nucleolar accumulation; panels 5 and 6: VSV-hPop4 Δ(61–109); accumulation in the Cajal bodies; and panels 7 and 8: VSV-hPop4 Δ(162–220), nucleolar pattern.
Figure 6
Figure 6
Association of VSV-hPop4 (mutants) with RNase MRP and RNase P ribonucleoprotein particles and RNase P activity associated with VSV-hPop4 deletion mutants. (A) Constructs encoding (deletion mutants) of VSV-hPop4 were transiently transfected into HEp-2 cells. Extracts from these cells were used for immunoprecipitation with anti-ECFP antibodies. RNA isolated from total cell extracts (lanes 1–8) and immunoprecipitates (lanes 9–16) was analyzed by Northern blot hybridizations with the use of riboprobes specific for RNase MRP and RNase P RNA. Lanes 1 and 9: material from cells transfected with empty vector alone; lanes 2 and 10: VSV-hPop4; lanes 3 and 11: VSV-hPop4 Δ(1–10); lanes 4 and 12: VSV-hPop4 Δ(1–47); lanes 5 and 13: VSV-hPop4 Δ(61–109); lanes 6 and 14: VSV-hPop4 Δ(210–220); lanes 7 and 15: VSV-hPop4 Δ(181–220); and lanes 8 and 16, VSV-hPpop4 Δ(162–220). The positions of RNase MRP and RNase P RNA are indicated on the right. (B) RNase P activity assay associated with immunoprecipitates from extracts of cells transiently transfected with VSV-hPop4 (deletion mutants). Lane 1: material from cells transfected with empty vector alone; lane 2: material from cells expressing VSV-hPop4; lane 3: VSV-hPop4 Δ(1–10); lane 4: VSV-hPpop4 Δ(1–47); lane 5: VSV-hPop4 Δ(61–109); lane 6: VSV-hPop4 Δ(162–220); lane 7: VSV-hPop4 Δ(181–220); lane 8: VSV-hPop4 Δ(210–220); and lane 9: input. On the right, the positions of the pre-tRNA, the mature tRNA and the 5′-leader are indicated.

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