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Isolation and Proteomic Characterization of Human Parvulin-associating Preribosomal Ribonucleoprotein Complexes* 210

Human parvulin (hParvulin; Par14/EPVH) belongs to the third family of peptidylprolyl cis-trans isomerases that exhibit an enzymatic activity of interconverting the cis-trans conformation of the prolyl peptide bond, and shows sequence similarity to the regulator enzyme for cell cycle transitions, human Pin1. However, the cellular function of hParvulin is entirely unknown. Here, we demonstrate that hParvulin associates with the preribosomal ribonucleoprotein (pre-rRNP) complexes, which contain preribosomal RNAs, at least 26 ribosomal proteins, and 26 trans-acting factors involved in rRNA processing and assembly at an early stage of ribosome biogenesis. Since an amino-terminal domain of hParvulin, which is proposed to be a putative DNA-binding domain, was alone sufficient to associate in principle with the pre-rRNP complexes, the association is probably through protein-RNA interaction. In addition, hParvulin co-precipitated at least 10 proteins not previously known to be involved in ribosome biogenesis. Coincidentally, most of these proteins are implicated in regulation of microtubule assembly or nucleolar reformation during the mitotic phase of the cell. Thus, these results, coupled with the preferential nuclear localization of hParvulin, suggest that hParvulin may be involved in ribosome biogenesis and/or nucleolar re-assembly of mammalian cells.


Supplementary Table 3. Identification of hParvulin-associating non-ribosomal proteins of previously unknown function in ribosome biogenesis.
Each entry contains the identified protein name with NCBI accession No. in parenthesis, the theoretical molecular weight (MW) and pI, the number (and the sequence coverage) of matching peptides of the PMF match, the average deviation in mass between measured and calculated values 33, 34). Origin of the proteins identified is indicated in the second colum (M, mouse; H, human). SDS staining band No. corresponding to FIG. 5 is also shown. For putative proteins, the best matching protein after TFAST searching is also indicated with % identity and the sequence length of the match. When yeast homologues are known or identified by TFAST search, protein names and their corresponding ORF names are given. If data are available, cellular localization and function are also indicated. As a control experiment, we identified the proteins present in GST-pulled down fraction, they include Bip and Hsp73 as indicated in Fig 4. Although these proteins were also detected in the hParvulin-associating complexes isolated, they are excluded from Supplementary

Fibroblast L929 Cells
To isolate hParvulin-associating proteins, we prepared the nuclear and cytosolic extracts from mouse fibroblast L929 cells. Initially, we tried to isolate hParvulinassociating proteins by eluting with a reduced glutathione from glutathione-Sepharose beads bound to GST-hParvulin after incubating with the nuclear and cytosolic extracts, respectively. While some potential hParvulin-associating proteins were detected following a reduced glutathione elution, the protein pattern was obscured by a high level of contaminating proteins due to nonspecific protein interaction to glutathione-Sepharose beads and GST for both nuclear and cytosolic extracts (data not shown). In order to reduce the contaminating proteins we attempted thrombin-mediated release of hParvulin-associating proteins from the glutathione-Sepharose beads bound to GST-hParvulin. Since the release of hParvulin from glutathione-Sepharose beads bound to GST-hParvulin was nearly complete-without any significant degradation of hParvulin after incubation with thrombin, we adopted the thrombin-mediated release of hParvulinassociating proteins after incubating with the nuclear and cytosolic extracts, respectively ( Fig. 3). In a control experiment, although GST and several contaminating proteins were eluted upon thrombin treatment from the glutathione-Sepharose beads bound to GST after incubation with the cell extracts, the number of the contaminating proteins was drastically reduced in the thrombin-released eluate (Fig. 3A lanes 1 and 3). We detected a number of hParvulin-associating proteins upon thrombin treatment of the GST-hParvulin-bound glutathione-Sepharose beads that were incubated with the nuclear extract ( Fig. 3A lane 4). In contrast, only a trace of proteins was detected in the cytoplasmic fraction (Fig. 3A lane 2). Similarly, when human HeLa and 293EBNA cells were used, a number of hParvulin-associating proteins were also detected in their nuclear extracts (Fig. 3B lane 2 and 3C lane 2). Thus, the hParvulin-associating proteins were present only in the nuclear extract. Those results suggest that the proteins isolated by thrombin-mediated release are the nuclear components associated specifically with hParvulin.

Cellular Localization of hParvulin
In order to analyze its cellular localization, we constructed two expression vectors; one contains a DNA sequence encoding hParvulin with the N-terminally tagged Flag epitope (Flag-N-hParvulin) (Fig. 1) and another with the C-terminally tagged Flag epitope (hParvulin-C-Flag). Each of the expression vectors was transfected into human 293EBNA cells transiently by use of LipofectAMINE. However, we observed that the efficiency was very low for both Flag-tagged hParvulins if compared with that of Flag-nucleolin or Flagnucleophosmin observed in separate experiments with the same expression vector and host cell (33). We estimate that the number of the cells transfected with the expression vector for Flag-tagged hParvulins is no more than one fifth of that for Flag-tagged nucleolin or nucleophosmin (data not shown). Although we do not yet know the reasons for the observed inefficiency of the transfection, it is likely that the overexpression of hParvulin causes some toxic effects on the cell. Despite the inefficiency of the transfection, we observed that hParvulin was dispersed throughout the cell, but it was present more in the nucleus including the nucleolus than in the cytoplasm in the 293EBNA cells that were transfected with both expression vectors for Flag-N-hParvulin and hParvulin-C-Flag (only Flag-N-hParvulin is shown in Fig. 4).

Identification of Protein Components in the hParvulin-Associating Complexes
For identification of hParvulin-associating proteins isolated by the thrombin-mediated release, individual protein bands were excised and digested with trypsin in the SDS-PAGE gel. The mass-to-charge ratio of the peptides released from the gel was measured by MALDI-TOF mass spectrometry with high-mass accuracy. The complete set of peptide masses from each protein band was compared with the tryptic peptide masses predicted for each protein in a comprehensive nonredundant database. A protein in the eluate released by thrombin-cleavage of GST-hParvulin was considered identified when the spectrum of its measured peptide masses met the previously established criteria for positive identification of proteins using MALDI-TOF mass spectrometry and automated database analysis (33). For this study, we searched the database for proteins with a mass range of 0 to 30kDa or 10 to 100kDa for smaller proteins (apparent Mr >20kDa by SDS-PAGE), and 50 to 200kDa or 100 to 300kDa for larger proteins, with constraint on mouse and human origins. Incomplete tryptic cleavage and peptide modifications that may alter the peptide masses, such as oxidized methionine or carbamidomethyl cysteine, were calculated for the putatively identified protein and compared with the measured masses.
The modified peptides identified in the search were added to the list to increase the number of matching peptides and sequence coverage. Protein that revealed the highest scored PMF matching by database search was retrieved as the identified protein.
Supplementary Tables I, II and III show the proteins identified as the hParvulinassociating proteins by applying the criteria described (33). The identification by the in-gel digestion-MALDI-TOF analysis was done for an individual protein band usually on silverstained 10% polyacrylamide gels. We also used 11% and 15% polyacrylamide gels to  Table 1). In addition to the ribosomal proteins, we also identified 23 non-ribosomal proteins as well as 24 unnamed or putative proteins (Supplementary Table 2 and 3).
Many of the identified non-ribosomal proteins are trans-acting factors that are known to be involved in ribosome biogenesis, including those such as p68 RNA helicase TNZ2, Bop1, nucleolin, Nop5/58, and fibrillarin. We also identified many ribosomal proteins and protein trans-acting factors for ribosome biogenesis as the hParvulin-associating proteins in the nuclear extracts obtained from human HeLa and 293E cells, respectively (Supplementary Tables 1 and 2). Thus, the presence of ribosomal proteins and transacting factors for ribosome biogenesis as the hParvulin-associating proteins in the three different cell strains analyzed, suggested that hParvulin was associated with preribosomal ribonucleoprotein (pre-rRNP) complexes from the nuclear extracts of mammalian cells. In order to ascertain these protein identification based on PMF data, we also analyzed a lysyl endopeptidase digest of the entire hParvulin-associating complexes by liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) 6 in a data-dependent manner followed by MS/MS spectra searching against NCBInr using batch processes of Mascot algorithm (manuscript in preparation). By a single operation of the LC-MS/MS, we confirmed the presence of the15 ribosomal proteins and the 18 transacting factors that were shown to be present in the isolated hParvulin-associating complexes by PMF method (supplementary Table I and II). Two examples of the protein identification with MS/MS spectra data are shown in supplementary Fig. 1. Thus, we believe that all of the proteins identified by PMF method are the reliable components of the hParvulin-associating protein complexes.

RNA Integrity of the hParvulin-Associating Complex
Since hParvulin was suggested to bind to the pre-rRNP complex, we addressed questions whether the association of the identified proteins with hParvulin is by direct interactions among these proteins, or by their association to common RNA molecules, or both. In order to clarify among those possibilities, we treated the nuclear extract with RNase A or ethidium bromide before incubating it with the GST-hParvulin-binding glutathione-Sepharose beads. Only a trace of the protein bands was detected as the hParvulin-associating proteins after treating the nuclear extract with RNase A or ethidium bromide (Fig. 6A). In addition, we also treated the hParvulin-associating complex on glutathione-Sepharose beads with RNase A and confirmed that RNase A dissociated most of the proteins associated with hParvulin (Fig. 6B). These results indicate that when RNA was degraded or precipitated before or after mixing with hParvulin, most of the proteins could no longer be associated with hParvulin. These results indicate that almost all protein constituents of the hParvulin-associating complex isolated are associated through a common RNA molecule. Thus, RNA integrity is necessary for the association of almost all of the proteins present in the hParvulin-associating complex we isolated. These results also suggest that the isolated hParvulin-associating complex is the pre-rRNP complex.

Presence of Pre-rRNA in the hParvulin-Associating RNP Complexes
Because all results we obtained suggest that the hParvulin-associating complexes are pre-rRNP complexes, we examined whether the complexes contain pre-rRNA. The identification of pre-rRNA in the isolated hParvulin-associating complexes was achieved by Northern-hybridization analyses with the different probes (Fig. 2). The hParvulinassociating complexes contained almost all rRNA species expected to be produced during rRNA processing in the nucleolus of mammalian cells. Namely, in addition to the matured 18S, 5.8S and 28S rRNAs, we detected 12S, 32S, 34S, 37S, 41S and 45S pre-rRNA species (Fig. 7). Among the pre-rRNA species detected, 32S and 12S pre-rRNAs are concentrated mostly in the isolated hParvulin-associating complexes. 32S pre-rRNA, is identified as the common precursor to 28S and 5.8S rRNA, encompassing the ITS 2 segment, while it reacts negatively with 18S, and 5'ETS probes. 12S pre-rRNA was detected only with ITS2. Thus, RNA species contained in the hParvulin-associating complexes are definitively shown to be rRNAs and pre-rRNAs. These results indicate that the isolated hParvulin-associating complexes are the nuclear precursor of the cytoplasmic ribosomal particles.

hParvulin Domain that is Involved in Associating to RNP Complexes
Since the amino-terminal domain of the hParvulin molecule is proposed to be a putative nucleic acid-associating domain (31), we addressed questions whether the entire hParvulin molecule participates in associating to the RNP complex, or whether any specific domain of the hParvulin molecule is sufficient to bind it. Therefore, we constructed genes for two domain mutants, the amino-terminal domain (GST-C-Par) and the carboxyl-terminal domain (GST-N-Par) (Fig. 1), each of which has a DNA sequence encoding a GST and a short segment with the thrombin cleavage site (TC).
The two domain mutants were expressed in a BL21 (DE3) strain, respectively, and were purified as described in Materials and Methods. Each was confirmed to have the expected molecular weight by SDS-PAGE and mass spectrometry analysis (data not shown).
When pulled down from the nuclear extract of L929 cells, the C-Par-associating protein complexes showed a protein profile similar to that for the entire hParvulin on SDS-PAGE gel (Fig. 8A). We could not detect any significant proteins that bound to C-Par from the cytosolic extract as the case of the entire hParvulin molecule.
Identification of the protein bands on SDS-PAGE gels by MALDI-TOF analysis and MS-Fit database search showed that all of the protein components of the C-Parassociating complexes so far analyzed are consistent with the protein components identified in the hParvulin-associating complexes (Supplementary Tables 1, 2 and 3). In addition, when the nuclear exract was treated with RNase A before incubation with GST-C-Par, we could not obtain almost all proteins that were pulled down with the entire GST-hParvulin (Fig. 8B). These results indicate that the N-terminal domain of hParvulin is