Ribosome biogenesis relies on a balance among transcription, processing, and export of the ribosomal particles; it is tightly coupled to cell cycle progression. Defects in any of these steps ultimately leads to nucleolar stress, followed by a G1 cell cycle arrest dependent on the stabilization of the tumor suppressor p53 (Deisenroth and Zhang, 2010 ). Interestingly, proteins involved in tumorigenesis also control ribosome biogenesis at various stages. The protooncogene c-Myc, as well as mTOR, both positively regulate ribosome biogenesis via increased transcription of all three RNA polymerases (Schlosser et al., 2003 ; Mayer et al., 2004 ; Poortinga et al., 2004 ; Arabi et al., 2005 ; Grandori et al., 2005 ; Gomez-Roman et al., 2006 ; Mayer and Grummt, 2006 ; van Riggelen et al., 2010 ). Alternatively, several tumor suppressor proteins have been shown to have a repressive effect on Pol I (Cavanaugh et al., 1995 ; Budde and Grummt, 1999 ; Zhai and Comai, 2000 ; Zhang et al., 2005 ). Nucleophosmin (NPM1), a nucleolar protein required for rRNA processing, is also often found overexpressed, mutated, or deleted in various types of cancer (Tanaka et al., 1992 ; Nozawa et al., 1996 ; Shields et al., 1997 ; Skaar et al., 1998 ; Subong et al., 1999 ; Mendes-da-Silva et al., 2000 ; Tsui et al., 2004 ; Falini et al., 2005 ; Grisendi et al., 2006 ). Together these observations suggest that ribosome biogenesis represents an important pathway that could be targeted to stop the growth of cancer cells.

Recently a panoply of large-scale studies in Saccharomyces cerevisiae have led to the purification of various preribosomal particles and the identification of a considerable number of proteins involved in the maturation and export of both the 60S and 40S ribosomal subunits (Harnpicharnchai et al., 2001 ; Nissan et al., 2002 ; Peng et al., 2003 ; Schafer et al., 2003 ; Krogan et al., 2004 ; Kressler et al., 2008 ; Li et al., 2009 ). Many of the factors identified have putative mammalian homologues, yet most of these proteins remain to be characterized in higher eukaryotes. Although the processing of rRNA requires several proteins, there are few complexes that have been identified in this process in mammals. The mammalian Pes1-Bop1-WDR12 complex was shown to be critical for the processing of the 32S pre-rRNA into the mature 28S rRNA (Strezoska et al., 2000 , 2002 ; Lapik et al., 2004 ; Holzel et al., 2005 , 2010 ; Grimm et al., 2006 ; Rohrmoser et al., 2007 ). Recently the nucleolar small ubiquitin-like modifier (SUMO)-specific proteases SENP3 and SENP5 were shown to be required for efficient rRNA processing via their interaction with the nucleolar chaperone NPM1, indicating a possible role for SUMOylation in the regulation of ribosome synthesis (Yun et al., 2008 ).

We recently identified LAS1L as a nucleolar protein required for the synthesis of the 60S ribosomal subunit and maturation of the 28S rRNA (Castle et al., 2010 ). Depletion of LAS1L results in accumulation of the 32S pre-rRNA intermediate, indicating a pre-rRNA processing defect of the internal transcribed spacer-2 (ITS-2), a region between the 5.8S and 28S rRNAs in the pre-RNA transcript (Castle et al., 2010 ). Interestingly, reduction of LAS1L levels also resulted in nucleolar disruption and increased levels of the tumor suppressor p53 and its transcriptional target p21, resulting in a cell cycle arrest in G1 phase (Castle et al., 2010 ). The stabilization of p53 suggests that LAS1L plays an important role in controlling ribosome biogenesis and nucleolar integrity.

Recently Finkbeiner et al. (2011) determined that PELP1, TEX10, and WDR18 interact together as part of a complex sharing sequence homology with the Rix1 complex (Ipi1p, Rix1p, and Ipi3p) required for ribosome biogenesis in S. cerevisiae (Finkbeiner et al., 2011 ). Indeed, TEX10 contains a conserved N-terminal Ipi1 domain and is ~20% identical to Ipi1p (Finkbeiner et al., 2011 ). Likewise, WDR18 and Ipi3p are ~25% identical, and both proteins contain multiple WD40 repeats (Finkbeiner et al., 2011 ). At its N-terminus, PELP1 displays a Rix1 homology region, and both PELP1 and Rix1 share a C-terminal region rich in acidic residues (Finkbeiner et al., 2011 ). In mammals, PELP1 has been characterized as a transcriptional regulator capable of modulating the functions of diverse nuclear receptors, such as the estrogen (ERα and ERβ), progesterone, and glucocorticoid receptors (Vadlamudi et al., 2001 , 2004 ; Nair et al., 2004 ; Kayahara et al., 2008 ). Our study demonstrates that PELP1 localizes to the nucleus and nucleolus and is important for efficient synthesis of the 28S rRNA (Figures 4 and ​and7).7). Interestingly, PELP1 was found to bind RNA and interact with components of the mRNA splicing machinery (Nair et al., 2006 ), and a recent study has shown that PELP1 can associate with the rDNA promoter (Gonugunta et al., 2011 ). It is possible that, depending on its subcellular compartmentalization, PELP1 regulates either Pol I- or Pol II-specific RNA-processing events.

The Rix1 complex in S. cerevisiae associates with a late pre-60S particle and has been indicated by multiple groups as being required for pre-rRNA processing (Galani et al., 2004 ; Krogan et al., 2004 ; Nissan et al., 2004 ). This complex is thought to be involved in the later steps of pre-rRNA processing that occur after cleavage of the ITS-2 region, and it has been shown to preferentially bind the 7S rRNA (Krogan et al., 2004 ). We demonstrate that the human homologues of the Rix1 complex (PELP1, TEX10, and WDR18) cofractionate with a particle that contains the 32S pre-rRNA (Figure 5), suggesting it could associate with the 60S subunit before cleavage of the ITS-2 occurs. Furthermore, depletion of PELP1, TEX10, and WDR18 showed an increase in the 32S intermediate (Figure 7, B and ​andD),D), suggesting they are also required for the processing of this earlier step. Another possibility is that the Rix1 complex is required in mammals for other ITS-2–processing factors, such as LAS1L and NOL9, to be recruited to the particle. Indeed, we observed that depletion of PELP1, TEX10, or WDR18 leads to a dramatic decrease in LAS1L protein stability (Figure S1A). Whether the Rix1 complex participates directly in the modification of pre-rRNAs or is simply required for the stabilization of other rRNA-processing factors remains to be determined. The possibility also remains that the Rix1 complex functions in a different manner in mammals than in yeast.

In this study, we also identified NOL9 as a novel LAS1L-interacting protein (Figures 1A and ​and2A).2A). NOL9 was recently discovered to be a 5′-polynucleotide kinase that can phosphorylate both single- and double-stranded RNA (Heindl and Martinez, 2010 ). The pre-rRNA–processing defects seen upon depletion of NOL9 and LAS1L are identical. Endonucleolytic cleavage within the ITS-2 in the 32S pre-rRNA generates the 12S and 28S pre-rRNAs, and the lower levels of 12S pre-rRNA and accrual of the 32S intermediate upon depletion implies that both NOL9 and LAS1L are required for efficient ITS-2 scission (Figure 7, B and ​andC).C). Depletion of NOL9 results in a 5′ extended 5.8S rRNA intermediate following ITS-1 cleavage (Heindl and Martinez, 2010 ), suggesting that 5′ phosphorylation of the extended 5.8S is required for subsequent exonucleolytic processing. Indeed, the exonuclease Rat1p (XRN2 in human) is necessary for the 5′-end maturation of the 5.8S and 25S rRNAs following endonucleolytic cleavage (Henry et al., 1994 ; Geerlings et al., 2000 ; Xue et al., 2000 ; Fang et al., 2005 ; El Hage et al., 2008 ) and requires a 5′-monophosphate for efficient hydrolysis of substrates (Stevens, 1980 ). Interestingly, the S. cerevisiae homologues of NOL9 (Grc3p) and LAS1L (Las1p) were both found by tandem affinity purification and mass spectrometry analysis to be interactors of Rai1p, a cofactor for Rat1p (Sydorskyy et al., 2003 ). Although the role of this interaction has not been explored, it indicates that Grc3p and Las1p might act with Rat1p and Rai1p in rRNA processing. The endonuclease performing the ITS-2 cleavage to generate the 12S and extended 28S rRNAs has yet to be identified both in budding yeast and mammals. LAS1L, with the Rix1 complex (PELP1-WDR18-TEX10), could be responsible for recruiting this endonuclease with NOL9 and XRN2 to the 32S rRNA for efficient cleavage, 5′ phosphorylation, and subsequent exonucleolytic processing.

Although Finkbeiner et al. (2011) previously described the mammalian Rix1 complex and LAS1L as interacting, the exact nature of the association between LAS1L and the proteins in the mammalian Rix1 complex has not been fully explored. Our further analysis of these interactions using sucrose gradient fractionation on nuclear extracts and subsequent coimmunoprecipitation experiments has revealed that LAS1L and the Rix1 complex proteins do indeed interact in the pre-60S fractions (Figure 6, B and ​andC).C). However, in the fractions corresponding to free nuclear proteins and smaller protein complexes, LAS1L only associates with NOL9 (Figure 6C). Furthermore, PELP1 interacts only with WDR18 in the fractions corresponding to free proteins (Figure 6B). Based on these coimmunoprecipitation experiments, it is therefore likely that LAS1L and NOL9 form a separate complex that does not include PELP1 and WDR18 when LAS1L and NOL9 are not associated with the pre-60S particle. It is also interesting to note that, in our experiments, TEX10 only associates with PELP1 in fractions that correspond to pre-60S ribosomal particles (Figure 6B). One could then conclude from our experiments that the mammalian Rix1 complex could form only on the pre-60S particle. However, further analysis of the maturation of mammalian pre-60S ribosomal particles will be necessary to investigate the temporal association of these proteins with pre-60S particles in mammalian ribosome biogenesis.

Another protein found to interact with LAS1L in this study is RanBP5 (Figure 2A), a karyopherin β that associates with the nuclear pore complex to facilitate the import of both small and large ribosomal proteins (Jakel and Gorlich, 1998 ; Chou et al., 2010 ). RanBP5 is homologous with the karyopherin-β proteins Pse1p/Kap121p and Yrb4p/Kap123p in S. cerevisiae. Both Kap121p and Kap123p are responsible not only for the nuclear import of ribosomal proteins (Rout et al., 1997 ), but also for the export of preribosomal particles (Moy and Silver, 1999 ; Sydorskyy et al., 2003 ). Furthermore, Kap121p is required for nuclear import of factors involved in preribosomal particle formation (Leslie et al., 2004 ; Lebreton et al., 2006 ). RanBP5 may be involved in importing LAS1L and its interacting proteins to the nucleus, and this could explain the aberrant accumulation of the 32S intermediate observed upon RanBP5 depletion (Figure 7, B and ​andD).D). Furthermore, it is possible that the defects in rRNA processing seen upon depletion of RanBP5 are a downstream effect resulting from failed nuclear import of ribosomal proteins, which leads to nuclear accumulation of immature preribosomal particles (Figure 7, B and ​andD).D). Impending investigations will be aimed toward defining a precise role for RanBP5 in the maturation of the 60S preribosomal particles.

Recent studies in budding yeast have suggested that SUMOylation could play an important role in ribosome biogenesis. Analysis of the 40S and 60S preribosomal particles at different maturation stages has shown that early particles are decorated with SUMO (Panse et al., 2006 ). Moreover, investigation of the SUMO proteome revealed that ribosomal proteins and several processing factors involved in the 40S and 60S synthesis are also modified by SUMOylation (Panse et al., 2006 ). The nucleolar SUMO-deconjugating enzyme SENP3 was recently shown to catalyze the removal of SUMO conjugates on NPM1, a process required for efficient rRNA processing and synthesis of the 60S ribosomal subunit (Haindl et al., 2008 ). NPM1 was also shown to be required for stable accumulation of SENP3 in the nucleolus (Kuo et al., 2008 ; Yun et al., 2008 ). In this study, we found that SENP3 associates with LAS1L, and both SENP3 and NPM1 interact with PELP1 (Figure 2, A and ​andB).B). We further describe LAS1L and PELP1 as SUMOylated substrates of SENP3, wherein loss of SENP3 or NPM1 results in accrual of SUMOylated forms of these proteins (Figure 11), and this is supported by previously published work (Finkbeiner et al., 2011 ). Moreover, we demonstrate that depletion of SENP3 or NPM1 results in a relocalization of LAS1L and PELP1 from the nucleolus with no observed disassembly of the LAS1L complex (Figures 9 and ​and10),10), suggesting that nucleolar localization is not required for the complex to assemble. We further observed that induction of a nucleolar stress through treatment with actinomycin D also results in relocalization of the complex from the nucleolus to the nucleoplasm (Figure 8). Previous work has demonstrated that depletion of NPM1 and SENP3 impairs ribosome biogenesis, which could in turn lead to a nucleolar stress (Haindl et al., 2008 ; Yun et al., 2008). We therefore cannot eliminate the possibility that nucleolar stress in general causes relocalization of LAS1L and PELP1 out of the nucleolus. However, as depletion of SENP3 or NPM1 results in accumulation of SUMOylated LAS1L and PELP1 concomitant with their relocalization from the nucleolus to the nucleoplasm, it is likely that the nucleolar relocalization is due to the accumulation of SUMOylated forms of these proteins. It is also conceivable that SUMOylation of LAS1L and PELP1 is a downstream effect of general nucleolar stress, and it will be interesting to further investigate this possibility. Taken together, our data suggest that SUMOylation partially regulates ribosome biogenesis by modulating the subcellular localization of LAS1L and PELP1. Indeed, studies from Finkbeiner and colleagues demonstrated that expression of a PELP1-SUMO fusion protein results in relocalization of PELP1 from the nucleolus to the nucleoplasm (Finkbeiner et al., 2011 ). Interestingly, we observed that SENP3 mainly interacts with LAS1L and the Rix1 complex in the fractions corresponding to the 60S particle (Figure 6). SENP3 could participate in a quality control checkpoint serving to limit access of SUMOylated ribosome biogenesis factors to the 60S particle. Whether or not the other proteins in the complex can be SUMOylated remains to be determined, and future experiments will be focused on investigating a role for SUMO in regulating the function of the LAS1L complex.