We compared the localization of stably expressed GFP-SENP3 and GFP-SENP5 with components that are characteristic of nucleolar subcompartments, including the fibrillar centers (anti-UBF), the dense fibrillar component (dsRed-fibrillarin), and the granular component (dsRed-B23/nucleophosmin). The signal from GFP-SENP3 and GFP-SENP5 overlapped poorly with UBF staining and only partially with dsRed-fibrillarin, suggesting that they were not concentrated in either the fibrillar centers or the dense fibrillar component (, top). In contrast, both proteins showed distributions that were very similar to B23/nucleophosmin, which could be observed either by immunostaining or by coexpression of B23/nucleophosmin as a fusion with the dsRed fluorescent protein (, bottom). We conclude that SENP3 and SENP5 are concentrated within the granular component.
Figure 1. SENP3 and SENP5 colocalize and associate with B23/nucleophosmin. (A) U2OS-derived cell lines expressing GFP-SENP3 and GFP-SENP5 were transfected with a plasmid for expression of dsRed-fibrillarin (top). After 48 h, the cells were fixed and immunostained (more ...)
The presence of SUMO-2/3–specific proteases within nucleoli suggested that the absence of these SUMO paralogues might be the result of their ongoing removal from nucleolar targets. To test this idea, we depleted SENP3 and SENP5 individually and together by oligonucleotide-mediated RNA interference (RNAi) from HeLa cells (). Depletion of either protease did not substantially alter the distribution of SUMO proteins. However, nucleolar SUMO-1 increased after codepletion of SENP3 and SENP5 ( and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200807185/DC1
). Significantly, SUMO-2/3 also became concentrated within nucleoli in the absence of both enzymes. The redistribution of SUMO-2/3 was striking because these paralogues are normally not evident within nucleoli. Depletion of nuclear pore–associated SENP/Ulps (SENP1 and SENP2) did not increase nucleolar levels of SUMO proteins (unpublished data), arguing that such accumulation is not a general result of SUMO pathway manipulation or of RNAi machinery activation.
Figure 2. SENP3 and SENP5 codepletion cause nucleolar SUMO protein accumulation. (A) HeLa cells were transfected with siRNAs directed against SENP3 and SENP5, either singly or in combination. 72 h after transfection, depletion was confirmed by Western blotting. (more ...)
SENP3 and SENP5 show robust enzymatic activities against SUMO-2/3–containing substrates (Di Bacco et al., 2006
; Gong and Yeh, 2006
; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200807185/DC1
), so they may directly control the half-lives of nucleolar SUMO-2/3–conjugated species. The functions of SENP3 and SENP5 appear to be at least partially redundant because high levels of SUMO-2/3 did not accumulate unless both proteins were depleted. Their role in controlling nucleolar SUMO-1 accumulation is somewhat harder to understand. It is possible that deconjugation of multiple SUMO moieties from some targets must occur in a particular sequence, so that failure to remove SUMO-2/3 in the absence of SENP3 and SENP5 might block subsequent removal of SUMO-1, and thus indirectly result in its nucleolar accumulation. Alternatively, SENP3 and SENP5 may be more active in vivo against SUMO-1–conjugated species than indicated by in vitro assays.
Budding yeast Ulp1p is required for 60S preribosome maturation and export (Panse et al., 2006
). To test whether SENP3 and SENP5 are similarly required, we examined rRNA synthesis and processing through pulse-chase analysis with [3
H]uridine (). Although 47S rRNA transcription was not substantially decreased after SENP3 depletion, the production of 28S rRNA from their 32S precursor RNA was inhibited. After a 2-h chase, control cells showed a ratio of labeled 32S versus 28S RNAs that was less than 0.5:1, whereas this ratio was over 2:1 in SENP3-depleted cells (). In contrast, loss of SENP5 did not have a strong effect on rRNA processing, but caused the levels of 47S RNA transcription to drop by >60% (). Cells depleted of both proteins showed both decreased 47S RNA precursor levels and increased 32S/28S ratios. Relocalization of ribosomal assembly factors further suggested disruption of ribosome biogenesis in the absence of SENP3 and SENP5 (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200807185/DC1
). Simultaneous depletion of SENP1 and SENP2 marginally reduced 47S RNA transcription but did not appreciably alter the rate of rRNA processing or alter assembly factor localization (unpublished data), arguing that defects in ribosome biogenesis were specifically caused by depletion of SENP3 and SENP5. Our results show that SENP3 and SENP5 individually play important roles in ribosome biogenesis (), but depletion of both proteases is required for elevation of SUMO-conjugated species within nucleoli (). Together, these results might suggest that although both SENP3 and SENP5 can act on the bulk of nucleolar substrates, they may each specifically regulate a subset of conjugates that are critical for particular events in the biogenesis pathway.
Figure 3. Depletion of SENP3 or SENP5 cause defects in ribosome biogenesis. (A) HeLa cells were transfected with control oligonucleotides (Control) or siRNAs directed against SENP3 (ΔSENP3) and SENP5 (ΔSENP5) or both SENP3 and SENP5 (ΔSENP3/5). (more ...)
We were unable to extract SENP3 and SENP5 from mammalian nucleoli under conditions that would allow analysis of protein–protein interactions. As an alternative, we used Xenopus laevis
egg extracts (XEEs) to look for binding partners. XEEs offer major advantages for these experiments because most nuclear and nucleolar proteins are stored in soluble forms (Powers et al., 2001
). Human SENP3 and Xenopus
SENP3 (xSENP3) proteins are ~85% identical within their catalytic domains and 50% identical throughout their entire sequences. Human SENP5 and xSENP5 proteins are ~87% identical within their catalytic domains and 43% identical throughout their entire sequences. There was little xSENP5 in Xenopus
eggs (unpublished data), which is perhaps consistent with the more specialized tissue distribution of mammalian SENP5 (Fig. S2 C). Antibodies against xSENP3 recognized a protein of the appropriate size in XEEs. Analysis using vinyl sulfone derivatives of different SUMO paralogues demonstrated that xSENP3 shared the same enzymatic specificity as mammalian SENP3 (Fig. S2 D).
We observed a protein that strongly and specifically coprecipitated with xSENP3, which mass spectrometric analysis revealed to be the Xenopus
homologue of B23/nucleophosmin (xB23/nucleophosmin). We confirmed this interaction by Western blotting of anti-xSENP3 immunoprecipitates (). Given the low levels of xSENP5 within XEEs, we used an alternative strategy to determine whether SENP5 might likewise associate with xB23/nucleophosmin. FLAG-tagged xSENP5 was translated within XEEs (Boyarchuk et al., 2007
), followed by precipitation with anti-FLAG or anti-xB23/nucleophosmin antibodies. Like xSENP3, we found that FLAG-tagged xSENP5 strongly bound with xB23/nucleophosmin in XEEs ().
Mammalian cells lacking B23/nucleophosmin show defects in ribosome biogenesis and particularly in 32S to 28S RNA processing (Itahana et al., 2003
). The association of B23/nucleophosmin and nucleolar SENPs, as well as their colocalization and phenotypic similarities, lead us to further examine their relationship. Both SENP3 and SENP5 levels decreased dramatically after RNAi-mediated depletion of B23/nucleophosmin (). This could be observed either by immunoblotting () or by examination of GFP fluorescence in U2OS cells stably expressing GFP-SENP3 or GFP-SENP5 (). SENP1 and SENP2 levels did not change after B23/nucleophosmin depletion (). As in experiments where SENP3 and SENP5 were codepleted (), depletion of B23/nucleophosmin caused a shift of SUMO protein distribution toward nucleoli (). Our findings suggest that B23/nucleophosmin physically interacts with SENP3 and SENP5 and regulates their abundance and that it thus plays a critical role in controlling the profile of SUMO conjugates within nucleoli through SENP3 and SENP5.
Figure 4. B23/nucleophosmin regulates SENP3 and SENP5 abundance. (A) HeLa cells were transfected with control oligonucleotides (left lane), siRNAs for codepletion of SENP1 and SENP2 (second lane), siRNAs for codepletion of SENP3 and SENP5 (third lane), or siRNAs (more ...)
Several observations indicated that SENP3 and SENP5 loss was caused by increased degradation rather than decreased expression. First, GFP-SENP3 and GFP-SENP5 were not transcribed from the native SENP3 and SENP5 promoters, so the drop in their levels upon B23/nucleophosmin depletion () argues that repression of those promoters cannot account for changes in protein levels. Second, the concentrations of SENP3 and SENP5 mRNAs were not substantially altered by B23/nucleophosmin depletion in HeLa cells, as measured by RT-PCR (unpublished data), further arguing against B23/nucleophosmin control of SENP3 and SENP5 transcription or mRNA stability. Third, B23/nucleophosmin-depleted cells did not show a substantial drop in overall protein translation, as measured by incorporation of [35
S]methionine (unpublished data), indicating that preexisting ribosomes were largely sufficient to maintain protein synthesis levels. Notably, depletion of a key ribosomal assembly factor, Rrp9 (Venema et al., 2000
), did not destabilize SENP3 and SENP5 or cause substantial nucleolar accumulation of SUMO proteins (unpublished data), demonstrating that their loss was not a simple consequence of ribosome synthesis inhibition but may be more specifically linked to the function of B23/nucleophosmin.
Finally, SENP3 and SENP5 levels in B23/nucleophosmin-depleted cells showed substantial, albeit incomplete, recovery upon treatment with the proteasome inhibitor MG132, which is consistent with the notion that they are subject to degradation in the absence of B23/nucleophosmin (). Stable cell lines expressing GFP-SENP3 and SENP5 showed a partial redistribution of GFP signals after B23/nucleophosmin depletion and MG132 treatment, with an increase in the ratio of nucleoplasmic to nucleolar distribution of both fusion proteins (unpublished data). This observation might indicate a role for B23/nucleophosmin in retention of these Ulp/SENPs within the nucleolus. However, we noted that the distribution of many other nucleolar proteins was rearranged after MG132 treatment, so it is also possible that relocalization of GFP-SENP3 and GFP-SENP5 may reflect alterations of nucleolar structure.
We hypothesized that failure to deconjugate some SUMO species might disrupt ribosome biogenesis in the absence of SENP3 and SENP5. We therefore examined the SUMOylation of mammalian homologues of ribosomal proteins or ribosome assembly factors that have been implicated as SUMO conjugation targets in yeast (Panse et al., 2004
; Wohlschlegel et al., 2004
; Denison et al., 2005
; Hannich et al., 2005
). Using cells that stably expressed His6
-tagged SUMO-1 (His-SUMO1F) or SUMO-2 (His-SUMO2F) proproteins, we examined changes in electrophoretic mobility of potential conjugation targets by Western blotting after codepletion of SENP3 and SENP5. We also purified the SUMO-conjugated fraction from each cell line by affinity chromatography and examined whether SUMOylated forms of the proteins could be found by Western analysis.
A subset of proteins showed clearly altered SUMOylation patterns under these circumstances: GNL2, the mammalian homologue of a 60S preribosomal export factor, showed a supershifted form upon depletion of SENP3/5 (), which was verified as a SUMOylated form through its preferential retention on nickel–nitrilotriacetic acid (Ni-NTA) agarose beads. We observed a supershifted form of the 60S ribosomal subunit RPL37A that was retained on Ni-NTA agarose beads in the absence of SENP3/5 (, top), suggesting that it was likewise subject to enhanced modification. Other nucleolar proteins (fibrillarin, DDX3, DDX17, and NVL2; unpublished data) did not show evidence of SUMOylation in this assay. Although this is an incomplete survey of potential conjugation targets within the 60S ribosomal biogenesis pathway, it demonstrates that multiple pathway components are SUMOylation targets, as in yeast. More importantly, enhanced RPL37A and GNL2 SUMOylation after depletion of SENP3 and SENP5 implicates these proteases in the control of ribosome biogenesis through this modification.
Figure 5. Enhanced SUMO modification of RPL37A and GNL2 after depletion of SENP3 and SENP5. U2OS stably expressing full-length His-tagged versions of SUMO-1 and SUMO-2 (His-SUMO1F and His-SUMO2F) were transfected with siRNAs for codepletion of SENP3 and SENP5 or (more ...)
While this manuscript was under revision, Haindl et al. (2008)
reported that SENP3 and B23/nucleophosmin physically interact and that loss of SENP3 disrupts rRNA processing. They argued that SENP3 acts upstream of B23/nucleophosmin, causing inhibition of ribosome biogenesis through increased B23/nucleophosmin SUMOylation. In our hands, depletion of SENP3 and SENP5 by RNAi, either singly (not depicted) or in combination (), did not cause a substantial SUMOylation of B23/nucleophosmin. We do not know why our results differ from those of Haindl et al. (2008)
, but suspect that the relatively low levels of His-tagged SUMO proteins expressed in our stable cell lines may mimic physiological conditions more closely than conditions of transient expression. In any case, our results do not suggest that B23/nucleophosmin is itself the primary target of SENP3 or SENP5. As an alternative model, we note that B23/nucleophosmin interacts with ribosomes (Grisendi et al., 2006
), so that it might act as an adaptor for targeting of SUMO proteases to ribosomal proteins. Consistent with this idea, 60S ribosomal components Rpl26 and RplP0 are coprecipitated from XEEs with xSENP3 in a xB23/nucleophosmin-dependent manner ().
The requirement for B23/nucleophosmin in ribosome synthesis has been proposed to include both action as an endonuclease (Savkur and Olson, 1998
) and as a chaperone (Szebeni and Olson, 1999
). Our data show that regulation of SUMOylation through SENP3 and SENP5 is another major aspect of B23/nucleophosmin function in this process. The capacity of xB23/nucleophosmin to link xSENP3 with ribosomes suggests that it may recruit SUMO proteases to maturing ribosomal particles. Interestingly, B23/nucleophosmin has been implicated in a variety of apparently unrelated cellular processes (Grisendi et al., 2006
), many of which are also dependent on the SUMO conjugation (Hay, 2005
). We therefore speculate that altered SUMOylation may be a simple, unifying mechanism that underlies much of the apparent plurality of B23/nucleophosmin functions.