Localization in the Nucleolus and Coiled Bodies of Protein Subunits of the Ribonucleoprotein Ribonuclease P

The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors.

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

Molecular Biology of the Cell ◽

10.1091/mbc.12.11.3680 ◽

2001 ◽

Vol 12 (11) ◽

pp. 3680-3689 ◽

Cited By ~ 15

Author(s):

Hans van Eenennaam ◽

Annemarie van der Heijden ◽

Rolf J. R. J. Janssen ◽

Walther J. van Venrooij ◽

Ger J. M. Pruijn

Keyword(s):

Protein Composition ◽

Rnase P ◽

Nucleolar Localization ◽

Rrna Processing ◽

Protein Subunits ◽

Trna Processing ◽

Rnase Mrp ◽

Human Rnase ◽

The Difference ◽

Active Transport Process

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.

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The organization of ribosomal RNA processing correlates with the distribution of nucleolar snRNAs

Journal of Cell Science ◽

10.1242/jcs.109.6.1241 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1241-1251 ◽

Cited By ~ 4

Author(s):

A.F. Beven ◽

R. Lee ◽

M. Razaz ◽

D.J. Leader ◽

J.W. Brown ◽

...

Keyword(s):

Small Bodies ◽

Transcript Processing ◽

Coiled Bodies ◽

External Transcribed Spacer ◽

Immunofluorescence Labelling ◽

Late Telophase ◽

Ribosomal Rna Processing ◽

Fibrillar Component ◽

High Degree

We have analyzed the organization of pre-rRNA processing by confocal microscopy in pea root cell nucleoli using a variety of probes for fluorescence in situ hybridization and immunofluorescence. Our results show that transcript processing within the nucleolus is spatially highly organized. Probes to the 5′ external transcribed spacer (ETS) and first internal transcribed spacer (ITS1) showed that the excision of the ETS occurred in a sub-region of the dense fibrillar component (DFC), whereas the excision of ITS1 occurred in the surrounding region, broadly corresponding to the granular component. In situ labelling with probes to the snoRNAs U3 and U14, and immunofluorescence labelling with antibodies to fibrillarin and SSB1 showed a high degree of coincidence with the ETS pattern, confirming that ETS cleavage and 18 S rRNA production occur in the DFC. ETS, U14, fibrillarin and SSB1 showed a fine substructure within the DFC comprising closely packed small foci, whereas U3 appeared more diffuse throughout the DFC. A third snoRNA, 7–2/MRP, was localised to the region surrounding the ETS, in agreement with its suggested role in ITS1 cleavage. All three snoRNAs were also frequently observed in numerous small foci in the nucleolar vacuoles, but none was detectable in coiled bodies. Antibodies to fibrillarin and SSB1 labelled coiled bodies strongly, though neither protein was detected in the nucleolar vacuoles. During mitosis, all the components analyzed, including pre-rRNA, were dispersed through the cell at metaphase, then became concentrated around the periphery of all the chromosomes at anaphase, before being localized to the developing nucleoli at late telophase. Pre-rRNA (ETS and ITS1 probes), U3 and U14 were also concentrated into small bodies, presumed to be pre-nucleolar bodies at anaphase.

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Nuclear domains of the RNA subunit of RNase P

Journal of Cell Science ◽

10.1242/jcs.110.7.829 ◽

1997 ◽

Vol 110 (7) ◽

pp. 829-837

Author(s):

M.R. Jacobson ◽

L.G. Cao ◽

K. Taneja ◽

R.H. Singer ◽

Y.L. Wang ◽

...

Keyword(s):

Ribosomal Rna ◽

Rna Processing ◽

Mammalian Cells ◽

Rat Kidney ◽

Rnase P ◽

Transfer Rna ◽

Binding Domain ◽

Ribosomal Rna Processing ◽

Nuclear Domains ◽

Fibrillar Component

The ribonucleoprotein enzyme RNase P catalyzes the 5′ processing of pre-transfer RNA, and has also recently been implicated in pre-ribosomal RNA processing. In the present investigation, in situ hybridization revealed that RNase P RNA is present throughout the nucleus of mammalian cells. However, rhodamine-labeled human RNase P RNA microinjected into the nucleus of rat kidney (NRK) epithelial cells or human (HeLa) cells initially localized in nucleoli, and subsequently became more evenly distributed throughout the nucleus, similar to the steadystate distribution of endogenous RNase P RNA. Parallel microinjection and immunocytochemical experiments revealed that initially nucleus-microinjected RNase P RNA localized specifically in the dense fibrillar component of the nucleolus, the site of pre-rRNA processing. A mutant RNase P RNA lacking the To antigen binding domain (nucleotides 25–75) did not localize in nucleoli after nuclear microinjection. In contrast, a truncated RNase P RNA containing the To binding domain but lacking nucleotides 89–341 became rapidly localized in nucleoli following nuclear microinjection. However, unlike the full-length RNase P RNA, this 3′ truncated RNA remained stably associated with the nucleoli and did not translocate to the nucleoplasm. These results suggest a nucleolar phase in the maturation, ribonucleoprotein assembly or function of RNase P RNA, mediated at least in part by the nucleolar To antigen. These and other recent findings raise the intriguing possibility of a bifunctional role of RNase P in the nucleus: catalyzing pre-ribosomal RNA processing in the nucleolus and pre-transfer RNA processing in the nucleoplasm.

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Folding of a universal ribozyme: the ribonuclease P RNA

Quarterly Reviews of Biophysics ◽

10.1017/s0033583507004623 ◽

2007 ◽

Vol 40 (2) ◽

pp. 113-161 ◽

Cited By ~ 17

Author(s):

Nathan J. Baird ◽

Xing-Wang Fang ◽

Narayanan Srividya ◽

Tao Pan ◽

Tobin R. Sosnick

Keyword(s):

Rnase P ◽

Model System ◽

Ribonuclease P ◽

Rna Catalysis ◽

Protein Subunit ◽

Catalytically Active ◽

Ribonuclease P Rna

AbstractRibonuclease P is among the first ribozymes discovered, and is the only ubiquitously occurring ribozyme besides the ribosome. The bacterial RNase P RNA is catalytically active without its protein subunit and has been studied for over two decades as a model system for RNA catalysis, structure and folding. This review focuses on the thermodynamic, kinetic and structural frameworks derived from the folding studies of bacterial RNase P RNA.

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Crystal structures of the archaeal RNase P protein Rpp38 in complex with RNA fragments containing a K-turn motif

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17018039 ◽

2018 ◽

Vol 74 (1) ◽

pp. 57-64 ◽

Cited By ~ 5

Author(s):

Kosuke Oshima ◽

Xuzhu Gao ◽

Seiichiro Hayashi ◽

Toshifumi Ueda ◽

Takashi Nakashima ◽

...

Keyword(s):

Crystal Structure ◽

Rnase P ◽

Ribonuclease P ◽

Stem Loop ◽

P Protein ◽

Human Rnase ◽

Human Proteins ◽

Rna Fragments ◽

Specificity Domain

A characteristic feature of archaeal ribonuclease P (RNase P) RNAs is that they have extended helices P12.1 and P12.2 containing kink-turn (K-turn) motifs to which the archaeal RNase P protein Rpp38, a homologue of the human RNase P protein Rpp38, specifically binds. PhoRpp38 from the hyperthermophilic archaeon Pyrococcus horikoshii is involved in the elevation of the optimum temperature of the reconstituted RNase P by binding the K-turns in P12.1 and P12.2. Previously, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was determined at 3.4 Å resolution. In this study, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was improved to 2.1 Å resolution and the structure of PhoRpp38 in complex with the K-turn in P12.1 was also determined at a resolution of 3.1 Å. Both structures revealed that Lys35, Asn38 and Glu39 in PhoRpp38 interact with characteristic G·A and A·G pairs in the K-turn, while Thr37, Asp59, Lys84, Glu94, Ala96 and Ala98 in PhoRpp38 interact with the three-nucleotide bulge in the K-turn. Moreover, an extended stem-loop containing P10–P12.2 in complex with PhoRpp38, as well as PhoRpp21 and PhoRpp29, which are the archaeal homologues of the human proteins Rpp21 and Rpp29, respectively, was affinity-purified and crystallized. The crystals thus grown diffracted to a resolution of 6.35 Å. Structure determination of the crystals will demonstrate the previously proposed secondary structure of stem-loops including helices P12.1 and P12.2 and will also provide insight into the structural organization of the specificity domain in P. horikoshii RNase P RNA.

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Inhibition of the Expression of the Human RNase P Protein Subunits Rpp21, Rpp25, Rpp29 by External Guide Sequences (EGSs) and siRNA

Journal of Molecular Biology ◽

10.1016/j.jmb.2004.06.006 ◽

2004 ◽

Vol 342 (4) ◽

pp. 1077-1083 ◽

Cited By ~ 15

Author(s):

Haifeng Zhang ◽

Sidney Altman

Keyword(s):

Rnase P ◽

Protein Subunits ◽

P Protein ◽

Human Rnase

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Interactions between RNase P protein subunits in Archaea

Archaea ◽

10.1155/2004/743956 ◽

2004 ◽

Vol 1 (4) ◽

pp. 247-254 ◽

Cited By ~ 25

Author(s):

Thomas A. Hall ◽

James W. Brown

Keyword(s):

Protein Interactions ◽

Rnase P ◽

Ribonuclease P ◽

Protein Protein Interactions ◽

Yeast Two Hybrid ◽

Protein Subunits ◽

P Protein ◽

Yeast Two Hybrid System ◽

Methanothermobacter Thermoautotrophicus ◽

Two Hybrid

A yeast two-hybrid system was used to identify protein–protein interactions between the ribonuclease P (RNase P) protein subunits Mth11p, Mth687p, Mth688p and Mth1618p from the archaeonMethanothermobacter thermoautotrophicus. Clear interactions between Mth688p and Mth687p, and between Mth1618p and Mth11p, were confirmed byHIS3andLacZreporter expression. Weaker interactions of Mth687p and Mth688p with Mth11p, and Mth11p with itself, are also suggested. These interactions resemble, and confirm, those previously seen among the homologs of these proteins in the more complex yeast RNase P holoenzyme.

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The Dynamic Network of RNP RNase P Subunits

International Journal of Molecular Sciences ◽

10.3390/ijms221910307 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10307

Author(s):

Athanasios-Nasir Shaukat ◽

Eleni G. Kaliatsi ◽

Ilias Skeparnias ◽

Constantinos Stathopoulos

Keyword(s):

Dynamic Network ◽

Rnase P ◽

Ribonuclease P ◽

Protein Subunit ◽

Protein Subunits ◽

Phosphodiester Bond ◽

Cellular Processes ◽

Protein Partners

Ribonuclease P (RNase P) is an important ribonucleoprotein (RNP), responsible for the maturation of the 5′ end of precursor tRNAs (pre-tRNAs). In all organisms, the cleavage activity of a single phosphodiester bond adjacent to the first nucleotide of the acceptor stem is indispensable for cell viability and lies within an essential catalytic RNA subunit. Although RNase P is a ribozyme, its kinetic efficiency in vivo, as well as its structural variability and complexity throughout evolution, requires the presence of one protein subunit in bacteria to several protein partners in archaea and eukaryotes. Moreover, the existence of protein-only RNase P (PRORP) enzymes in several organisms and organelles suggests a more complex evolutionary timeline than previously thought. Recent detailed structures of bacterial, archaeal, human and mitochondrial RNase P complexes suggest that, although apparently dissimilar enzymes, they all recognize pre-tRNAs through conserved interactions. Interestingly, individual protein subunits of the human nuclear and mitochondrial holoenzymes have additional functions and contribute to a dynamic network of elaborate interactions and cellular processes. Herein, we summarize the role of each RNase P subunit with a focus on the human nuclear RNP and its putative role in flawless gene expression in light of recent structural studies.

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Kinetic mechanism of human mitochondrial RNase P

10.1101/666792 ◽

2019 ◽

Cited By ~ 1

Author(s):

Xin Liu ◽

Nancy Wu ◽

Aranganathan Shanmuganathan ◽

Bradley P. Klemm ◽

Michael J. Howard ◽

...

Keyword(s):

Conformational Changes ◽

Rnase P ◽

Kinetic Mechanism ◽

Ribonuclease P ◽

Substrate Affinity ◽

P Protein ◽

Binding Data ◽

Catalytically Active

ABSTRACTA first step in processing mitochondrial precursor tRNA (pre-tRNA) is cleavage of the 5’ leader catalyzed by ribonuclease P (RNase P). Human mitochondrial RNase P (mtRNase P) is composed of three protein subunits: mitochondrial RNase P protein (MRPP) 1, 2 and 3. Even though MRPP3 is the metallonuclease subunit responsible for catalysis, cleavage is observed only in the presence of the MRPP1/2 subcomplex. To understand the functional role of MRPP1/2, we reconstituted human mitochondrial RNase P in vitro and performed kinetic and thermodynamic analyses. MRPP1/2 significantly enhances both the catalytic activity and the apparent substrate affinity of mtRNase P. Additionally, pull-down and binding data demonstrate synergy between binding pre-tRNA and formation of a catalytically active MRPP1/2/3 complex. These data suggest that conformational changes in the MRPP1/2-pre-tRNA complex lead to protein-protein or protein-RNA interactions that increase both MRPP3 recognition and cleavage efficiency. This work presents the first kinetic model for human mtRNase P, providing a fundamental framework for the function of MRPP1/2 for recognition and processing of pre-tRNA.

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