Nop19p is a nucleolar protein associated with preribosomes.
A large number of ribosomal assembly factors have been identified by combining genetic studies and pre-ribosomal analysis using mass-spectrometry. To unveil protein that might have been overlooked in previous study, we focus our analysis on nucleolar proteins of unknown function. Previous global analyses of yeast proteins localization have shown that the protein Nop19p, encoded by the open reading frame YGR251W
, localizes to the nucleolus. In order to confirm the subcellular localization of Nop19p23
, a construct expressing a C-terminal YFP fusion was integrated at the endogenous locus using a one step PCR strategy. Clones were verified by PCR and transformed with plasmid pUN100-mCherry-NOP125
expressing a mCherry-fused version of Nop1p (mCherry-Nop1p), a core component of box C/D snoRNPs involved in early, nucleolar stages of the pre-rRNA maturation pathway, to visualize the nucleolus. The strain was grown exponentially and living cells were analyzed by fluorescence microscopy (). The YFP signal observed in these conditions is highly enriched within a crescent-shaped region also containing mCherry-Nop1p, consistent with a nucleolar localization. Nop19p-YFP is also present at low levels in a region immediately adjacent to the nucleolus corresponding to the nucleoplasm. We concluded that Nop19p accumulates predominantly in yeast cell nucleoli suggesting that Nop19p might be a component of preribosomes. To get insights into the nature of macromolecular complexes containing Nop19p, we analyzed the sedimentation profile of Nop19p with that of (pre)ribosomal particles on density gradients. We used a strain expressing a C-terminal fusion between NOP19 and the TAP tag from the genomic locus (Open Biosystems). Expression of this fusion protein remained under the control of the endogenous promoter. Growth of the tagged strain was indistinguishable from wild-type strain (data not shown), indicating that the fusion protein is functional. Sedimentation profile of Nop19-TAP was tested by fractionation of a cell lysate on a 4.5%–45% sucrose gradient (). Western blot analysis of gradient fractions showed that Nop19p concentrates in two broad peaks. The dense fraction containing Nop19p-TAP (fractions 11–13) likely corresponds to the SSU processome/90S pre-ribosome whereas the light sedimenting fractions (fractions 1–3) probably represent the soluble protein plus UTP subcomplex(es). Signal was also observed in fractions 7–10 suggesting that few amounts of Nop19p are also present in pre-40S and/or pre-60S particles. Thus, a large part of Nop19p-TAP is engaged within large complexes, the density of which is consistent with that of various preribosomal particles. We next carried out immunoprecipitation experiments to assess whether Nop19p interacts physically with preribosomal particles. The NOP19::TAP
strain was grown in the presence of glucose, and Nop19p-TAP was precipitated in non-denaturing conditions from a total cellular extract by the use of IgG-conjugated Sepharose. Following immunoprecipitations, bound RNAs were analyzed by northern hybridization () and compared with RNAs recovered in parallel from the non-tagged control strain (no TAG lanes). We found that the RNA component of 90S preribosomal particles, the 35S pre-rRNA is efficiently precipitated (~20%) as well as the 23S pre-rRNA (~15%), compared to the isogenic non tagged strain ( and lanes 1–4). The tight association of Nop19p with 23S pre-rRNA might suggests that A0
cleavages are required for the release of the protein from pre-ribosomal particles. Late 20S and 27S pre-rRNAs were also co-precipitated with Nop19p-TAP clearly above background levels but appeared weaker than for 35S or 23S pre-rRNAs (respectively ~4% and ~2%), suggesting Nop19p is quickly released from these particles after A0
cleavages. Our results show that, Nop19p is tightly associated with early pre-rRNA, component of the 90S preribosomal particles, a conclusion further supported by the presence, above the background, in the Nop19p-TAP immunoprecipitate, of various C/D and H/ACA snoRNAs ( and lanes 5–8).
Figure 2 Nop19p is a nucleolar protein associated with preribosomes. (A) Subcellular localization of Nop19p. Yeast strain expressing Nop19p-YFP and mCherry-Nop1p were grown exponentially and cell samples were used for fluorescence microscopy analysis. (B) Sedimentation (more ...)
Nop19p is required for cleavages of the pre-rRNA at sites A0, A1 and A2.
ORF was initially reported to be essential for cell viability.26
In order to investigate the function of Nop19p in ribosome biogenesis, we constructed a yeast strain that conditionally expresses the Nop19p protein fused to the 3HA tag at the N-terminus (3HA-Nop19p protein), allowing its easy detection. The NOP19
open reading frame was tagged by the HA-encoding sequence and placed under the control of the regulated GAL1-10
promoter by homologous recombination,27
creating strain GAL1::3HA::NOP19
. This strain was propagated in a medium containing galactose as carbon source and was then shifted to a glucose-containing medium to allow depletion of Nop19p.
On galactose containing medium, the growth rate of GAL1::3HA::NOP19 strain and otherwise isogenic wild type were almost identical. Two hours after transfer to the non-permissive glucose medium, the growth rate of GAL1::3HA::NOP19 strain was already substantially reduced compared to wild-type, with a doubling time of ~5 h. Growth essentially ceased by 25 h after transfer ().
Figure 3 Nop19p depletion affects 40S ribosomal subunit accumulation in yeast cells. (A) Growth rate of wild-type and Gal::3HA::NOP19 strains following a transfer from permissive galactose medium to glucose medium for the times indicated. Cells were maintained (more ...)
Aliquots of GAL1::3HA::NOP19 cells grown in galactose-containing medium or grown for 1, 3, 6, 12, 24 and 48 hours in glucose-containing medium were harvested. From these aliquots, total proteins and RNAs were extracted and the kinetics of Nop19p depletion was assessed by western-blotting () analyses. In these conditions, the abundance of 3HA-Nop19p was strongly reduced after transfer to glucose medium and became undetectable after 1 h.
As a first step in assessing whether Nop19p is involved in the production of ribosomal subunits, we compared the ribosome profiles in the presence and absence of Nop19p on sucrose gradient (). GAL1::3HA::NOP19 and otherwise isogenic wild type cells were grown for 6 h in glucose-containing medium and sedimentation profiles were compared by 4.5% to 45% sucrose gradient analysis. Depletion of 3HA-Nop19p resulted in a clear reduction of the sedimentation peak corresponding to the free 40S subunits (fractions 7 and 8), a correlated strong increase of the 60S subunit sedimentation peak (fraction 10) and a concomitant polysome decrease (fractions 13–18). This phenotype clearly suggests a putative role of Nop19p in the production of the small ribosomal subunit.
To investigate the origin of this phenotype, we compared pre-rRNA processing in WT and Nop19p-depleted cells by northern-blot analysis (). Strains depleted of Nop19p showed defects in the pathway of 40S subunit synthesis. As early as 1 h after the nutritional shift, levels of the 35S pre-rRNA increased, while the 27SA2 and 20S pre-rRNAs were reduced ( and lanes 7–12). This suggests that early cleavages of 35S pre-rRNA at sites A0, A1 and A2, necessary for the production of 20S pre-rRNA (the immediate precursor to 18S rRNA) are somewhat impaired (for a cartoon of the pre-ribosomal RNA processing pathway, see ). This conclusion was supported by a strong accumulation of the aberrant 23S RNA, which is produced by direct cleavage of the 35S pre-rRNA at site A3 in the absence of cleavage at sites A0, A1 and A2. At later time points, the mature 18S rRNA was depleted in the strains lacking Nop19p, whereas levels of the 25S and 5.8S rRNAs were stable. According to phosphorimager quantifications, the level of 18S rRNA, relative to that of 25S rRNA, is diminished by 90% after 24 hours of growth in glucose-containing medium. The levels of the 27SB and 7S pre-rRNA were also lower in Nop19p-depleted cells after 12 h of depletion, but this may largely reflect reduced synthesis as a consequence of growth inhibition. The ratio between the long and short forms of 5.8S rRNA was unaltered, indicating that the alternative pre-rRNA processing pathways that generate these rRNAs both remain active. Together these observations indicate that depletion of Nop19p leads to the inhibition of cleavage at sites A0, A1 and A2.
Figure 4 Nop19p depletion leads to a defect in A0, A1 and A2 cleavages. (A) WT BY4741 and Gal::3HA::NOP19 strains were shifted from a galactose to a glucose medium. Samples were collected before and at different times after the nutritional shift. Total RNAs were (more ...)
The effects of reduced 3HA-Nop19p level on pre-rRNA processing were also assessed by pulse-chase labeling, performed 3 h after transfer of the GAL1::3HA::NOP19
strain to glucose medium. Consistent with the northern data, processing of the 35S pre-rRNA was delayed, whereas synthesis of 27SA and 20S pre-rRNA was greatly reduced in the strains depleted of Nop19p ( and lanes 8–14) compared to the wild type ( and lanes 1–7). Maturation of the 27SB pre-rRNA appeared to be slowed, but accumulation of mature 25S rRNA was not clearly reduced. Similar result was previously observed during Has1p depletion.28
In contrast, 18S rRNA synthesis was strongly inhibited, most probably as a direct result of reduced 20S pre-rRNA production detected by this approach. These results confirmed that cleavages at A0
are strongly impaired in cells lacking Nop19p.
Nop19p is not required for proper assembly of U3 processome.
As previously shown, Nop19p is a novel component of the 90S preribosome and is required for early cleavages of the 35S pre-rRNA. In order to get insight into the function of Nop19p in the hierarchical assembly of the 90S preribosome, we first determined whether Nop19p is required for proper accumulation of components of this particle. For this purpose, we individually expressed TAP-tagged versions of UTP proteins Utp17p (UTP-A), Pwp2p (UTP-B), Utp22p (UTP-C) as well as Rrp5p in the GAL1::3HA::NOP19 strain. Depletion of 3HA-Nop19p clearly did not result in reduced levels of any of these factors (data not shown), suggesting Nop19p is not required for their accumulation.
We next determined whether Nop19p is required for the incorporation of the UTP-A, UTP-B, UTP-C modules as well as Rrp5p within the SSU processome. To study the importance of Nop19p in the assembly of the 90S preribosome, we compared the sedimentation profiles of components of the UTP-A, UTP-B or UTP-C subcomplexes in WT and Nop19p-depleted cells (). For this experiment, we choose to deplete GAL1::3HA::NOP19 strains for 3 h. At this time point indeed, 3HA-Nop19p levels are already undetectable but ribosome biogenesis is only slightly affected, arguing against indirect effect. The A254 absorbance profiles and notably the large versus small ribosomal subunit ratio presented in the parts in confirmed that depletion of Nop19p induced a berely detectable defect in the production of the mature 40S ribosomal subunit. The Utp17p-TAP, Pwp2p-TAP, Utp22p-TAP or Rrp5-TAP proteins were still detected in fractions of the gradient containing high-molecular-weight complexes and none of them was mainly detected in the top fractions containing small particles and free proteins. We concluded that the preribosomal particles lacking Nop19p contain the tested components of the different UTP modules and that Nop19p is therefore probably not required for their incorporation.
Figure 5 Nop19p and U3 processome assembly. (A) Depletion of Nop19p in yeast does not affect the incorporation of components of the UTP-A, UTP-B or UTP-C modules within preribosomes. Strains expressing TAP-tagged versions of Utp17p, Pwp2p, Utp22p or Rrp5p that (more ...)
We next studied whether Utp17p (UTP-A complex), Pwp2p (UTP-B complex), or Rrp5p are required for Nop19p recruitment to the nascent preribosomal particles. We replaced the endogenous promoters of the UTP17, PWP2 or RRP5 genes with the conditional GAL1 promoter in the previously described NOP19::TAP strain. These strains were shifted from a galactose- to a glucose-containing medium and grown to deplete Utp17p, Pwp2p or Rrp5p. Depletion of these factors did not result in a reduction of 3HA-Nop19p (data not shown), suggesting they are not required for Nop19p-TAP accumulation.
Extracts prepared from the depleted cells were analyzed by sedimentation on sucrose gradients. The absorbance profiles observed in Utp17p-, Pwp2p- and Rrp5p-depleted cells () are consistent with those reported previously by others in reference 15, 19
and very similar to those observed in the absence of Nop19p. In absence of Utp17p, Pwp2p and Rrp5p, Nop19p was still detected in the fractions of the gradient containing preribosomal particles. However, the sedimentation profiles of Nop19-TAP in Utp17p-, Pwp2p- and Rrp5p-depleted cells appeared significantly different from that observed with WT cells. The protein is indeed more concentrated in fractions 11 and 12 in WT cells whereas it appeared shifted to fractions 8 to 10 in depleted cells and appeared less concentrated in the top fractions containing free proteins and other low-molecular-weight complexes. These data indicate that the incorporation of Nop19p into preribosomes is not prevented in the absence of UTP-A or UTP-B components or Rrp5p. The observed shift in sedimentation may reflect that Nop19p remains associated with stalled preribosomes containing the aberrant 23S pre-rRNA. Therefore, Nop19p is essential for rRNA processing, but has no detectable function in hierarchical assembly of the 90S preribosome.
Dhr2p and Utp25p are in tight association with Nop19p.
In order to get insights into the function of this novel factor, we next attempted to identify “preferential” Nop19p-associated factors. The aim was to identify tight interactions between Nop19p and 90S preribosomal proteins using stringent conditions. Hence, Nop19p fused to the TAP tag (Nop19p-TAP) was purified over an IgG-sepharose column in native conditions. Nop19p-TAP-containing complexes bound to the column were then submitted to a “disruption treatment” consisting in extensive washes with buffers containing 200 mM, 1 M or 2 M salt (KCl) concentration (see Materials and Methods). The protein A-IgG interaction is resistant to 1 M and 2 M salt treatment,30
but most of the proteins component of the 90S preribosome are expected to be released following these stringent washes. Following TEV elution, a second calmodulin affinity column was employed as described in reference 31
. The polypeptides copurified with Nop19p following this modified TAP tag protocol were resolved by SDS-PAGE and stained with coomassie blue (Fig. S1
). Coomassie-stained bands were excised and polypeptides contained in these bands were subjected to in-gel trypsin digestion. The resulting peptides were analyzed by on-line capillary liquid chromatography/nanospray ion trap tandem mass spectrometry, allowing identification of the proteins from which they were derived.
Among the identified factors, we noticed the presence of two 90S components, Dhr2p and Utp25p. Dhr2p is a DEAH-box RNA helicase essential for cell viability. Dhr2p is required for A0
cleavages and so on for 18S rRNA production.32
Dhr2p presents a strong conservation in its core domain with Dhr1p, one other DEAH-box RNA helicase also required for early 35S pre-rRNA cleavages. Moreover, Dhr2p has been shown to be associated in vivo with U3 processome components including U3 snoRNA, Mpp10p,33
Utp25p is an essential factor, also required for A0
cleavages (reviewed in ref. 36
and Fig. S2
). As Dhr2p, Utp25p coimmunoprecipitated SSU processome factors such as U3 snoRNA, Mpp10p, Utp3p/Sas10p, Utp8p, Utp18p and Utp21p.34,36,37
All together, these results suggested that Nop19p is a component of the SSU processome interacting “preferentially” with Dhr2p and Utp25p.
In order to independently verify the interactions seen using the modified TAP protocol, we individually expressed in the NOP19::TAP strain, HA-tagged versions of Dhr2p, Dhr1p and Utp25p as well as various UTP proteins including Utp17p (UTP-A), Pwp2p (UTP-B), Utp22p (UTP-C) and Rrp5p. Strains were exponentially grown in YPD medium at 30°C and cell pellets were broken in liquid nitrogen. Nop19p-TAPcontaining complexes bound to the IgG sepharose column were extensively washed with buffers containing 0.2 M, 1 M or 2 M KCl. Protein complexes were next eluted using a denaturing buffer. The released peptides were analyzed by western-blot using anti-HA antibodies (). As expected, all tested factors were found associated with Nop19p-TAP at low salt concentration (0.2 M). In accordance with our previous observations, Dhr2p-HA and Utp25p-HA interactions with Nop19p-TAP were resistant to high salt concentrations (1 M and 2 M) (). In contrast, Dhr1p-HA is totally released from the column after 1 M KCl washes. These results confirm that Nop19p stably interacts with Dhr2p-HA and Utp25p-HA when compared with other 90S preribosomal proteins. Moreover, little amounts of Utp17p-HA (UTP-A) and Pwp2p-HA (UTP-B) remain associated with Nop19p after high salt disruption, suggesting stable association of these two factors with Nop19p-TAP, althought to a lesser extend as compared with Dhr2p and Utp25p. UTP-C factor (Utp22p-HA), as well as Rrp5p-HA are still associated to Nop19p-TAP at 1 M salt concentration but are released from the column during 2 M KCl washes. All together, these results suggest that Nop19p is specifically and stably associated to Dhr2p and Utp25p. Moreover, stable association between Nop19p and UTP-A and UTP-B components were identified. Nop19p also appeared associated with UTP-C subcomplex and Rrp5p, to a lower extent however.
Figure 6 TAP purification of Nop19p-TAP under stringent conditions. (A) Nop19p-TAP was affinity purified under native conditions (see Materials and Methods). Nop19p-TAP-containing complexes bound to IgG were extensively washed with buffers containing 200 mM, 1 (more ...)
We next determined whether Nop19p is required for the incorporation of Dhr2p and Utp25p within the SSU processome. For this purpose, we compared the sedimentation profiles of Dhr2p-TAP and Utp25p-TAP in WT and Nop19p-depleted cells (). Western blot analysis of gradient fractions showed that Dhr2p concentrates in two broad peaks in WT cells. The lower peak of Dhr2p-TAP (fractions 11–14) may correspond to the SSU processome/90S preribosome whereas the slower sedimenting fractions (fractions 1–3) probably represents the free protein plus small protein complex(es). Signal is also observed in fractions 8–10 suggesting few amounts of Dhr2p are present in pre-40S and/or pre-60S particles. In Nop19p-depleted cells, Dhr2p was still present in heavy fractions, suggesting Nop19p is not required for its incorporation into the preribosomes. Interestingly, Dhr2p was no more detected in top fractions while it concomitantly accumulated in fractions 7–9. These results probably reflect that in absence of Nop19p, Dhr2p is trapped within stalled preribosomes containing the aberrant 23S pre-rRNA avoiding its recycling.
Figure 7 Depletion of Nop19p affects the sedimentation profile of Dhr2p and Utp25p. Strains expressing TAP-tagged versions of Dhr2p (A) and Utp25p (B) that were otherwise WT or expressing 3HA-Nop19p under the control of the GAL1 promoter were transferred from (more ...)
In accordance with observations by other,37
Utp25p is present in high molecular mass fractions probably corresponding to SSU processome/90S and pre-40S preribosomes (). These results were also assessed by co-immunoprecipitation experiments which confirmed that Utp25p is associated with both the 35S and 20S pre-rRNAs (Fig. S2
) data not shown. In absence of Nop19p, very weak signal was observed in high molecular complexes whereas signal strongly increased in top fractions. This data suggest that the incorporation of Utp25p into preribosomes is affected in absence of Nop19p. In these conditions, Utp25p accumulates in the pool of free proteins and/or small protein complexes.