We previously reported the FANoS method to purify nucleolar DNA and RNA (Pontvianne et al., 2013, 2016b; Durut et al., 2014). Here, we applied FANoS to investigate the nucleolus proteome of 3 weeks-old Arabidopsis thaliana plants. Two nucleoli purifications from leaves of independently grown plants were performed (exp-1 and exp-2). We obtained ~9.83 × 10⁵ and ~8.25 × 10⁵ nucleoli in exp-1 and in exp-2 respectively, and by nanoLC-MS/MS analysis, we identified 1,001 (exp-1) and 778 (exp-2) different proteins (Figure 1A and Tables S1, S2). Comparative analysis revealed that 562 proteins were consistently identified in both experimental data sets (Figure 1A and Table S3). This subset of 562 common proteins identified in both biological replicates was considered in the subsequent analysis, if not otherwise specified. 99 and 409 proteins out of these 562 proteins have been previously identified by Pendle et al. (2005) and Palm et al. (2016) respectively (Figure S1A).

Based on functional characterization and cellular localization studies reported in the literature, we determined that ~35% of the proteins found in nucleolus fractions have been assigned as ribosome biogenesis factors (RBF); including 45S rRNA transcription and processing factors, 90S processome, 40S and 60S assembly factors and ribosomal proteins from large (RPL) and small (RPS) ribosome subunits. ~26% of the 562 proteins have been described as nucleolar proteins, but not yet characterized as RBFs while the remaining 39%, to our knowledge, have not been described as nucleolar and/or having a related nucleolar function (Figure 1B). Then, we assessed in which other subcellular compartments these (562) proteins could be also (transiently or not) localized. We therefore used the subcellular protein distribution report recently published in Palm et al. (2016). We determined that ~75.6% of these proteins have already been reported as nucleolar components with either nucleolar, nucleolar/nuclear or nucleolar/nuclear/cytoplasmic localization revealing the highly proteomic dynamic nature of the nucleolus. ~22.8% were reported as nuclear or nuclear/cytoplasmic and ~1.7% of them were only detected in the cytoplasmic fraction. None of the proteins are localized both in the nucleolus and in the cytosol fractions (Figure S1B and Table S4).

To distinguish particular extra ribosomal biogenesis functions of the nucleolus from Arabidopsis leaves, we assessed the enrichment of specific categories of proteins identified in the nucleolar fractions. For that, we compared the 562 protein accessions with a proteomic dataset we obtained from an Arabidopsis thaliana whole cell protein extract fraction (Table S5). For comparative purposes, the MS/MS spectrometry analysis of this fraction was performed in a similar manner to that with the nucleolar fractions. A Gene Ontology (GO) analysis revealed that in the nucleolar fractions there is an enrichment of ~3.8X of proteins linked to ribosome biogenesis, ~3.3X of RNA processing factors and ~3.5X of proteins related to Ribo Nucleo Protein (RNP) Complexes compared to whole cell protein extracts (Figure S2A). Additionally, we performed a Cluster of Orthologous Groups (COG) analysis and four major functional categories came up (Figure S2B): COG J (for ribosome biogenesis structure and translation) (17%), COG A (for RNA processing and modifications) (14%), COG O (for post-translational modification, protein turn over and chaperones) (13%) and COG R (for general function prediction only) (12%). These two analyses clearly show that nucleolus of Arabidopsis plants is enriched in proteins linked to ribosome biogenesis, to RNA processing and modifications and to RNP complexes. The COG analysis revealed also nucleolar enrichment of proteins linked to protein dynamics. More precisely, in the COG O category, 27% of the proteins correspond to 26S proteasome subunits while the 73% of the remaining proteins include redox activities (17%), HSP (10%), proteases (7%), chaperones (5%), and others (34%) (Figure 1C and Table S6).

All together these results demonstrate that FANoS methodology allows to obtain purified nucleoli from Arabidopsis leaves for proteasome analysis. This analysis indicates that in addition to ribosome biogenesis and RNA related factors the nucleolus is enriched in proteins/factors involved in enzymatic reactions and/or gene expression regulation. Because proteasome subunits are the most abundant proteins in the post-translational modification category, protein turn over and chaperones (COG O), we decided to study the functional relevance of the nucleolar localization of these subunits of the 26S proteasome.

The proteasome is a sophisticated complex that selectively degrades protein substrates marked by ubiquitin covalent linkage (Kurepa et al., 2009; Liepe et al., 2014; Bach and Hegde, 2016). The 26S proteasome complex is composed of the 19S regulatory and the 20S catalytic subunits. The 19S subunit is organized in two sub-complexes: the lid built of 8 Rpn proteins and the base composed of 3 Rpn proteins (Regulatory Particle Non-ATPase) as well as 6 Rpt (Regulatory Particle Triple-A or Regulatory Particle Triphosphatase) proteins. The 20S subunit is built of 7 alpha and 7 beta proteins (Vierstra, 2003; Kurepa and Smalle, 2008).

Among the 562 proteins identified in our nucleolar fractions, we found components of the lid (Rpn5, Rpn7), the base (Rpt1 and Rpt5), and the alpha (α1, α3-α6) and beta (β3, β5, β7) subunits (Figure S3A, blue labeled). We also detected additional proteins from the lid (Rpn6, Rpn8, Rpn9, and Rpn11), the base (Rpn1 and 2, Rpt2, 3, and 4) and the alpha (α2 and 7) and beta (β1, 2, 4, and 6) subunits in the individual exp-1 or exp-2 data sets (Figure S3A, orange labeled). Accordingly, several of these proteins were also reported in the nucleolus proteome of Arabidopsis cells (Palm et al., 2016; Table S7). To verify to which extend proteasome subunits are localized in the nucleolus, we analyzed the subcellular localization of 20S (Rpn5a and Rpt5b) and 19S (PBC1/β3 and PBG1/β7) proteins fused to GFP in A. thaliana protoplasts (Figure S3B). We noted that nucleolar localization of Rpn5a and PBG1/β7 is weak and dependent on the N- or C-terminal position of the GFP, while Rpt5b and PBC1/β3 do not show nucleolar localization with none of the constructs, suggesting either that only a small fraction of individual “tagged” proteins are assembled into proteasome and/or that they localized transiently in the nucleolus.

Therefore, to investigate a potential functional relationship between nucleolus and 26S proteasome, we determined proteasome localization and activity in nuc1.2 mutant plants which display a complete structural disorganization of the nucleolus (Pontvianne et al., 2007). A. thaliana contains two nucleolin protein genes NUC1 and NUC2, previously named AtNUC-L1 and AtNUC-L2 (Pontvianne et al., 2007, 2010). nuc1.2 plant corresponds to a T-DNA insertion mutant line described in Pontvianne et al. (2010) and Durut et al. (2014).

We first analyzed the cellular localization of proteasome subunits in nuc1.2 mutant plants (Figure 2). Immunolocalization experiments revealed that, Rpn1 and Rpn10 proteins localize in the cytoplasm and mostly in the nucleoplasm of root apex from WT plants (WT panels). However, Rpn10 might also localize in nucleolar subdomains (Figure S4), which are reminiscent of the nucleolar cavity also called nucleolar vacuoles (Saez-Vasquez and Medina, 2008; Stepinski, 2014). Interestingly, in nuc1.2 mutants, Rpn1 and Rpn10 proteins form discrete foci (Figure 2, white arrows) in the nucleolus suggesting that localization of proteasome subunits is closely linked to the nucleolus structure.

Secondly, we performed an in vitro 20S proteasome activity assay using total protein extracts from WT and nuc1.2 plants (Figures 3A,B). The results show that proteasome activity in nuc1.2 (~26 RFU) is reduced compared to WT (~39 RFU) plants (Figure 3A). Proteasome activity measured in proteasome subunit mutant plants rpt2 and rpt5 does not show significant variations (~35 and ~40 RFU, respectively) when compared to WT plant extracts, in agreement with previous reports (Lee et al., 2011; Sakamoto et al., 2011). Then, we investigated 26S proteasome activity in protein extracts from WT and nuc1.2 plants treated or not with proteasome inhibitors MG132 and ALLN (Figure 3B). WT plants treated with MG132 or ALLN show proteasome activity decrease (~22 RFU for each inhibitor) compared with untreated plants (~37 RFU). Remarkably, in nuc1.2 mutants, treatment with either MG132 or ALLN inhibitors slightly reduces the proteasome activity (~21 and ~20 RFU, respectively) compared to untreated conditions (~27 RFU). Interestingly, similar proteasome activity is observed between nuc1.2 (treated or not) and WT treated plants. These results show that nuc1.2 is hyposensitive to proteasome inhibitors.

To verify that lower proteasome activity in nuc1.2 protein extracts is not due to a reduced amount of proteasome protein in nuc1.2 plants, we determined the amount of two proteasome subunits (Rpn1 and Rpn10) in WT and nuc1.2 plants, treated or not with the MG132 inhibitor (Figure 3C). Western blot analysis does not reveal significant changes in protein level in WT and nuc1.2 plants treated or not with MG132, suggesting that proteasome complex amount is not affected in nuc1.2 plants. Similarly, we checked if NUC1 protein level could be affected by MG132 treatment. However, we do not observe detectable variations of NUC1 protein level in WT plants, suggesting that lower proteasome activity in these plants is not due to changes in the NUC1 protein level after MG132 treatment. If altered post-translational protein modifications of the 26S proteasome complex occur in nuc1.2 plants, remains to be determined.

Because proteasome activity can be detected both in the nucleus and cytoplasm (Kurepa and Smalle, 2008), we decided to determine proteasome activity in the nuclear and cytoplasmic fractions of nuc1.2 mutant plants (Figure 3D). The results show that the proteasome activity is reduced in nuc1.2 (~10.8 RFU) compared to WT (~15.2) plants in the nuclear fractions, while in cytosolic fractions, proteasome activity is higher in nuc1.2 (~16.6 RFU) compared to WT (~9.7) plants. Coomassie blue staining shows similar amount of nuclear and cytosolic proteins from nuc1.2 and WT protein fractions. Detection of H3 histone protein and absence of the cytosolic protein PRXII validates the purity of nuclear fractions (Figure S5).

Altogether these results suggest that functionally structured nucleolus and/or nucleolin protein is required for optimal proteasome dynamics and activity in plants.

It is not known if it is the absence of NUC1 protein or the nucleolar disorganization phenotype observed which is responsible of proteasome localization and/or activity previously observed in nuc1.2 mutant plants. Thus, we analyzed if inhibition of proteasome activity could have an impact on nucleolus functional organization. Three major structures are visualized in the nucleolus: the Fibrillar Centers (FC), the Dense Fibrillary Component (DFC) and the Granular Component (GC). rDNA transcription localizes to the periphery of the FC, pre-rRNA processing initiates in the DFC and later pre-rRNA processing and ribosome assembly occurs in the GC (Raska et al., 2006; Saez-Vasquez and Medina, 2008).

We investigated nucleolar structure in response to MG132 in WT and nuc1.2 plants expressing the Fib2:YFP nucleolar marker construct (Fibrillarin2:Yellow Fluorescent Protein) which allows to visualize nucleolus organization through the fluorescence of the YFP (Figure 4 and Picart and Pontvianne, 2017). The green signal of the Fib2:YFP protein reveals 3 distinct states of the nucleolus in WT plants: Structured, in which the FC and DFC are clearly recognized, Unstructured, in which the FC and the DFC are practically undetectable, and Intermediate, where nucleoli cannot be classed in the two previous categories. These three different states are observed in both WT and nuc1.2 plants treated or not with MG132, although the ratios are clearly different (Figures 4A,B). In untreated WT plants (DMSO only), ~54% of nucleoli appeared to be structured, ~17% are unstructured and ~29% are in an intermediate state (Figure 4B). In contrast, MG132 treatment increases the proportion of unstructured (~22%) and intermediate (~48%) nucleolus states, concomitant with a decreased proportion of structured nucleoli (~30%). In untreated nuc1.2 mutants (DMSO only), ~9% of the nucleoli are structured, while the others present unstructured (~32%) or intermediate states (~59%), which is in agreement with previous observations (Pontvianne et al., 2007; Picart and Pontvianne, 2017). Remarkably, this analysis shows that MG132 treatment does not result in further unstructured nucleoli in nuc1.2, in contrast to WT plants. The fraction of structured, unstructured and intermediate states in nuc1.2 plants treated with MG132 (~5, ~33, ~62%) remains similar to those observed in untreated plants (~9, ~32, ~59%). This result is also reminiscent to the analysis of proteasome activity in nuc1.2 showing minimal threshold to MG132 and ALLN (Figure 3B).

Altogether, these data show that inhibition of proteasome activity affects nucleolus structure/organization which might impact on rRNA transcription and processing.

In all eukaryotic cells, nucleolus formation and structure depend essentially on 45S rRNA synthesis and ribosome assembly (Hannan et al., 1998; Grummt, 2003; Sáez-Vásquez and Gadal, 2010). The 45S rRNA genes (encoding 18S, 5.8S, and 25S rRNAs) are transcribed in the nucleolus by RNA polymerase I (RNA pol I) as a single precursor (or pre-rRNA) containing internal (ITS1 and ITS2) and external transcribed spacers (5′ETS and 3′ETS). Pre-rRNA processing depends on the conserved U3 small nucleolar ribonucleoprotein particle (snoRNP) containing fibrillarin and on other transiently associated proteins such as nucleolin (Turner et al., 2009; Phipps et al., 2011; Henras et al., 2015). In Brassicaceae, we have shown that the nucleolin-U3 snoRNP complex binds both 5′ETS rDNA and the 5′end of nascent pre-RNA, suggesting coupling of transcription and processing of pre-rRNA (Sáez-Vasquez et al., 2004a,b). Furthermore, we demonstrated that 26S RPN subunits co-purified with the nucleolin-U3snoRNP complex suggesting that 26S proteasome might affect 45S rRNA gene expression (Samaha et al., 2010).

To investigate if 26S proteasomal activity can affect rRNA transcription and or processing, we measured the accumulation of (1) primary pre-rRNA precursor produced by RNA Pol I and (2) processed pre-rRNA at the primary cleavage site (P) in WT and/or nuc1.2 mutant plants treated or not with MG132 (Figure 5). Primer tis maps the transcription initiation site (TIS) (Saez-Vasquez and Pikaard, 1997) while primer p maps the P primary cleavage site (Sáez-Vasquez et al., 2004b). To compare the ratio between primary and cleaved pre-rRNA (TIS/P) in WT and nuc1.2 plants (treated or not with MG132), tis and p primers were added simultaneously to the same primer extension reactions (lanes 1-4). As previously reported, we observed an accumulation of pre-rRNA precursors in the nuc1.2 mutant compared to WT plants (Figure 5A, lanes 2 and 4), suggesting a defect in primary pre-rRNA processing in mutant plants (Pontvianne et al., 2007). Interestingly, while P/TIS ratio remains similar in WT plants treated or not with MG132, in nuc1.2 mutants, P/TIS ratio decreases after MG132 treatment (~5) compared to untreated conditions (~9); signifying an increase of ~2-fold of pre-rRNA cleaved at the P site, or alternatively a decrease of pre-RNA initiated at the TIS, in the nuc1.2 plants.

Altogether these observations indicate that proteasome inhibition does not affect RNA Pol I transcription or co-transcriptional cleavages of pre-rRNA, but rather later cleavages events taking place in the nucleolus. The data suggest also a role of NUC1 in proteasome activity or complex organization.

All lines were derived from Arabidopsis thaliana Columbia (Col 0) ecotype. Plants expressing Fib2:YFP nucleolar marker construct were described in Pontvianne et al. (2013) and Picart and Pontvianne (2017). The nuc1.2, rpt2a-1, and rpt5a-4 T-DNA insertion mutant lines were reported previously in Pontvianne et al. (2007, 2010), Wang et al. (2009), Sakamoto et al. (2011). Seeds were sown either on soil or on 1X Murashige and Skoog medium (MS containing 1% sucrose) and left for 2 days at 4°C to synchronize. Plants were then grown in controlled growth chambers under a 16 h light/8 h dark cycle at 21°C for 3 weeks (FANoS) or 2 weeks (in vitro activity assay) or under continuous light for 14 days (Western blot). For treatment with proteasome inhibitors MG132 and ALLN (Sigma), 15-days-old plant seedlings were transferred to petri dishes containing 6 mL of liquid MS medium complemented with 50 μM MG132 and/or 50 μM ALLN for 24 h before harvesting.

Leaves (without petiole) from 3-weeks-old Fib2-YFP plants, were fixed for 20 min in 4% formaldehyde in cold Tris buffer (10 mM Tris-HCl pH7,5, 10 mM EDTA, 100 mM NaCl), and washed twice for 10 min with Tris buffer. Then, leaves were chopped with a razor blade in FACS buffer (45 mM MgCl2, 20 mM MOPS pH7, 30 mM Sodium citrate, 0.1% TritonX-100) containing protease inhibitors (cOmplete, Mini Protease Inhibitor Cocktail from Roche) and filtered through a 30 μM PARTEC CellTrics membrane. Filtrates were sonicated using a Bioruptor (Diagenode) with parameters setted up as follows: three 5-min pulse ON/30-s OFF at Medium intensity. Samples were kept on ice and protected from light until sorting experiment was performed using BD FACS ARIA II (Biosciences), at the IGMM institute, Montpellier (MRI platform), and with parameters described previously in Pontvianne et al. (2016a).

Fifteen-days-old plant seedlings were collected, shock-frozen in liquid nitrogen and grinded in fine powder. Samples were homogenized in three volumes of Extraction buffer (20 mM HEPES/KOH, 10 mM MgCL2, 0.5M Hexylene glycol), filtrated through a Miracloth (EMD Millipore Corporation) and a bolting cloth (Sefar AG, 31 μm). Then, Triton X-100 was added (0.5% final) and samples incubated on a rotor for 15 min at 4°C. To obtain cytosolic and nuclear fractions, whole cell extract samples were centrifuged at 1,000 g during 10 min at 4°C. The supernatant corresponds to the cytosolic fraction. The pellet was washed with 1 mL of Extraction buffer containing 0.5% Triton X-100, centrifuged at 1,000 g for 10 min at 4°C and finally resuspended in 150 μL of Extraction buffer (supplemented with 0.5% Triton X-100). This corresponds to the nuclear fraction. For each proteasome activity assay, 1 μg of protein of each fraction was used.

Subcellular localization was performed as described in Sommer et al. (2011) and Palm et al. (2016). In brief, the coding sequence of Rpn5a (At5g09900), Rpt5b (At1g09100), PBC1/β3 (At1g21720), and PBG1/β7 (At1g56450) genes was amplified using Arabidopsis thaliana cDNA and specific oligonucleotides (Table S8). Then, amplified fragments were cloned in the pRTds vector to generate C- and N-terminal GFP fusion constructs. As a nucleolar localization control, atFIB2 (At4g25630) was cloned in front of mCherry into the same vector and co-transformed with the GFP-fusion constructs (Missbach et al., 2013).

Leaves of 4-weeks-old Arabidopsis thaliana plants were rubbed on K240 sandpaper and then incubated in 25 mL of extraction buffer [1% (w/v) cellulase R10, 0.3% (w/v) macerozyme in MCP (29 mM MES-KOH pH 5.6, 500 mM sorbitol, 1 mM CaCl2)] for 2 h at 30°C to isolate protoplasts from mesophyll cells. After incubation, the released protoplasts were filtered through a 75 μm nylon mesh and underlayed with 2.5 mL of 100% (v/v) Percoll MCP (pH 5.6 containing 5 mM MES, 500 mM sorbitol, 1 mM CaCl2). After centrifugation at 405 g for 8 min, the clear supernatant of around 20 mL was removed and the remaining protoplast fraction was mixed with the Percoll cushion, followed by overlaying with 7.5 mL 25% (v/v) Percoll in MCP and 5 mL MCP. The mixture was centrifuged at 270 g for 8 min and the green protoplast fraction between MCP and 25% (v/v) Percoll was collected in a new tube. After centrifugation at 100 g for 5 min, the protoplast pellet was diluted in MMg (5 mM MES-KOH pH 5.6, 400 mM sorbitol, 15 mM MgCl2) to a cell number of 106 cells per mL. For transfection, 100 μL protoplasts were mixed with 10 μg pDNA per construct. 100 μL PEG-solution [40% (w/v) PEG-4000, 100 mM Ca(NO3)2, 400 mM sorbitol] was added to the protoplasts. After incubation for 20 min at room temperature, the reaction was stopped with K3-solution (20 mM MES-KOH pH 5.6, 400 mM sucrose, 1 mM CaCl2, MS salts). The protoplasts were incubated over night at room temperature and under constant light condition. The expression analysis was done by confocal laser scanning microscopy (CLSM) using a HCX PL APO CS 40 × 1.25 NA 1.25 oil objective. Transformed protoplasts (around 10 μL) were spotted on an object slide. Fluorescence was excited and detected as follows: GFP 488 nm/505–525 nm, mCherry 568 nm/580–610 nm, chlorophyll fluorescence 514 nm/650–750 nm.

Fifteen-days-old plant seedlings treated or not with MG132 or ALLN were collected, shock-frozen in liquid nitrogen and grinded in fine powder. Samples were incubated on ice in Extraction buffer (50 mM HEPES/KOH, 2 mM MgCl2, 150 mM NaCl, 10% Glycerol, 1% Triton X-100) for 30 min with vortexing steps every 10 min. Then samples were centrifuged at 22,000 g for 20 min at 4°C and supernatant recovered. Activity assay was performed using the kit “20S Proteasome Activity Assay Kit” (Chemicon® International) according to the manufacturer's instructions. For each assay, 2 μg of protein extract was used. Fluorescence was determined using “Fluoroskan Ascent FL” (Thermo Scientific), with light excitation at 355 nm and emission at 460 nm.

Plant material (100 mg) treated or not with MG132, was homogenized and extracted in protein extraction buffer [50 mM Tris-HCl pH8, 150 mM NaCl, 10 mM EDTA, 50 mM NaFluoride, 1% NP40, 0.5% Deoxycholate, 0.1% SDS and protease inhibitors (cOmplete, Mini Protease Inhibitor Cocktail from Roche)]. Samples were cleared by centrifugation at 13,000 g for 20 min at 4°C and proteins extracted in 1X SDS-Laemmli buffer. Western blot was performed as described previously (Durut et al., 2014) using, α-H3 (CT, pan from Millipore) α-NUC1 (Pontvianne et al., 2010), α-RPN1a (Wang et al., 2009), α-RPN10 (Lin et al., 2011), and α-PRXII (Bréhélin et al., 2003) antibodies.

Immunofluorescence was performed on roots apex from 8 day –old seedlings as previously described in Durut et al. (2014). Briefly, treated roots were incubated overnight at 4°C with α-RPN1 (1:1,000) and α-RPN10 (1:1,000) and then with anti-rabbit coupled with Alexa 488 (1:1,000, Invitrogen), for 3 0h at room temperature. Slides were then mounted in Vectashield medium containing DAPI solution. For nucleolus structural studies, 2 week-old Fib2-YFP plants (WT and nuc1.2, treated or not with MG132), grown on MS medium, were fixed for 20 min in 4% formaldehyde in cold Tris buffer (10 mM Tris-HCl pH7,5, 10 mM EDTA, 100 mM NaCl) and washed twice for 10 min with Tris buffer. Then plants were chopped with a razor blade in LB01 buffer (15 mM Tris-HCl pH 7,5, 2 mM NaEDTA, 0.5 mM spermine, 80 mM KCl, 20 mM NaCl, 0.1% Triton X-100) and filtered through a 30 μM PARTEC CellTrics membrane. Filtrates were completed with an equal volume of sorting buffer (100 mM Tris-HCl pH7,5, 50 mM KCl, 2 mM MgCl2, 0.05% Tween-20, 5% sucrose, filtered through 0.45 μm filter) before spreading on a polysine slide. After air-drying, slides were post-fixed in 2% formaldehyde in phosphate buffer (PBS) for 5 min and washed twice with 1X PBS. Slides were mounted in Vectashield medium containing DAPI solution. Observations and imaging were performed using a confocal microscope LSM 700 from Zeiss.

Total RNAs from A. thaliana WT and nuc1.2 plant mutants were extracted using Trizol reagent (Invitrogen), according to manufacturer's instructions. Then, all samples were then treated with RQ-DNase (Promega) to eliminate contaminant genomic DNA. Primer extension analysis to detect TIS and P sites was done using 5–10 μg of RNAs and specific 5′end labeled primers, as previously described (Sáez-Vasquez et al., 2004b; Pontvianne et al., 2007). Products of the reaction were analyzed on 8% polyacrylamide/ 7 M urea sequencing gel.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors JS, and states that the process met the standards of a fair and objective review.