The notion of a “plurifunctional nucleolus” was proposed previously (Pederson, 1998
). However, at present, a comprehensive view of the biological processes that might occur within this subnuclear structure is still missing. Unquestionably, identification of proteins contained within nucleoli represents one of the first indispensable steps to fulfill this tremendous task. For this reason, a proteomic analysis was developed and reported herein to obtain a catalog of proteins contained within nucleoli of human cells. This catalog of 213 proteins seemed complementary to that of the 271 proteins obtained recently by others (Andersen et al., 2002
) and allowed the establishment of a list of 350 different nucleolar proteins. However, establishing a list of nucleolar proteins is not sufficient per se to point out the different biological processes that might occur within nucleoli. This is why, when possible, the biological functions of the 350 proteins found in nucleoli were determined and gathered together according to several functional classes corresponding to well-defined major biological processes.
The first step of our proteomic analysis of human nucleoli was to purify these membraneless nuclear structures from HeLa cells and to verify their purity. While performing the fractionation procedure, integrity and purity of nuclei as well as enrichment of the nucleolar fraction with nucleoli were assessed by analysis of each fraction under light and electron microscopes. This analysis showed clearly that nuclei were the only organelles present in the nuclear fraction and that the nucleolar fraction contained a very high concentration of nucleoli, which conserved their ultrastructural integrity.
The enrichment of proteins specific to nucleolar and/or other fractions was evaluated by separation of the different fractions by 1-DE followed by Coomassie and silver nitrate staining as well as by Western blot. These analyses showed clearly that numerous proteins, such as histones were present almost exclusively in the fractions from nuclear origin, confirming the high quality of the nucleocytoplasmic separation procedure. In addition, the finding that in our experimental conditions, ERK2 was found by Western blot analysis exclusively in the cytoplasmic fraction suggested that extremely low contamination of the nuclear fractions, if any, with cytoplasmic proteins was present. More importantly, these analyses demonstrated that the majority of the proteins visible in the nucleolar fraction were not detected in the other fractions. In particular, the nucleolar fraction was highly enriched in two proteins that have been previously shown to be among the most abundant proteins of nucleoli, B23 and nucleolin (Bugler et al., 1982
; Roussel and Hernandez-Verdun, 1994
Because all of these results indicated very strongly that our nucleolar fraction was highly enriched in nucleoli and in proteins specific for this fraction, the next step of our proteomic analysis was to perform an extensive identification of the proteins contained within this fraction. For this, proteins from this fraction were separated by 1-DE and 2-DE through polyacrylamide gels. The separated proteins were stained with Coomassie Blue. Proteins of interest were in gel digested with trypsin and resulting peptides analyzed by mass spectrometry and for most of them by nano-liquid chromatography-electrospray ionization-Q-q-time of flight mass spectrometry to obtain sequence information. These data permitted the identification of 190 different proteins after separation by 1-DE and 23 supplementary proteins after separation by 2-DE.
The final step of our proteomic analysis was to outline the biological processes in which the 213 proteins could be involved. A bibliographic analysis was performed to obtain functional data for each of the 213 proteins. From this first analysis, it seemed that several functional studies were available for 109 out of the 213 proteins, whereas no functional studies were available for the 104 remaining proteins. This allowed us to determine unambiguously the biological mechanisms in which these 109 proteins are involved. Further analyses were performed to gain insights into the functions of the 104 proteins for which no functional studies were available. A search within protein databases was made to find proteins homologous to each of these 104 proteins. This search allowed the identification of a homologous protein with a well-defined function for 43 proteins of the 104, permitting the assignment of a hypothetical function. No function could be attributed for the remaining 61 proteins representing 28.6% of the 213 proteins.
As expected, numerous proteins found within nucleoli are either ribosomal proteins (15.5%) or proteins involved in ribosome biogenesis (23.5%). Interestingly, this analysis allowed us to propose clearly that 31 proteins of this latter category, which are from human origin, participate to ribosome biogenesis due to their homology with previously characterized proteins, which are for most of them from yeast origin. Moreover, the high proportion in our nucleolar fractions of proteins involved in well-established nucleolar functions strongly indicates that our nucleolar fraction is highly enriched in nucleoli. Several other proteins found within nucleoli in the current analysis are involved in the ultrastructural organization of the nucleus: fibrous proteins (5.1%) and structural proteins of the chromatin (2.8%) (Verheijen et al., 1986
; Gerner et al., 1998
; Belmont et al., 1999
; Jung et al., 2000
; Bergquist et al., 2001
). Five proteins (2.3%) belong to the DNA-dependent protein kinase system, a multifunctional complex shown to be involved in many biological processes such as DNA repair and telomere maintenance (Dynan and Yoo, 1998
; Featherstone and Jackson, 1999
; d'Adda di Fagagna et al., 2001
). Several proteins (6.6%) were arbitrarily regrouped into the class named “others” because they are involved in different biological processes, and each of them would require the construction of a novel specific functional class. For example, importin α-2 subunit, casein kinase II, ubiquitin, poly[ADP-ribose] polymerase-1, or S100 proteins are proteins from this category (Allende and Allende, 1995
; Pickart, 2001
; Tong et al., 2001
; Weis et al., 1995
; Donato, 2001
). One of the features emerging from our classification is the surprising finding that ~12% of the 213 proteins are proteins involved in the regulation of every step of mRNA metabolism, i.e., their synthesis, splicing, editing, nuclear export, and also their translation and degradation. As illustrated in Table , most of these proteins participate in several steps of mRNA metabolism. Indeed, many of these proteins have never been shown to be localized within nucleoli by using other techniques. Therefore, at this stage of the study, we cannot exclude that some of these proteins are found artifactually within the nucleolar fraction. Conversely, we cannot exclude also that these proteins are found in small amount and very transiently within nucleoli rendering them difficult to visualize using other techniques. Many more experiments would be required to address this question.
Table 1 . Nucleolar proteins involved in the regulation of mRNA metabolism
Analysis of the biological functions of the nucleolar proteins identified previously by Andersen et al. (2002)
demonstrated that these proteins could be classified according to the same functional classes elaborated in this study. Therefore, this study, by allowing the classification of a total of ~350 nucleolar proteins according to the biological processes in which they are involved, firmly confirms the plurifunctional nature of nucleoli and outlines biological processes taking place within these nuclear domains (Andersen et al., 2002
; this study). In particular, the present analysis shows clearly that translational regulators, chaperones, and also proteins involved in mRNA processing are found within nucleoli, in addition to other components of the translation machinery such as ribosomes (Leary and Huang, 2001
), tRNAs (Bertrand et al., 1998
; Pederson and Politz, 2000
), signal recognition particle (Politz et al., 2000
; this study), and even mRNAs (Kalland et al., 1991
; Bond and Wold, 1993
; Kadowaki et al., 1994a
). This provides molecular evidence to the recent demonstration that translation can occur within nuclei of human cells (Iborra et al., 2001
) and suggests that nucleoli themselves could play a central role in the control of this process. Indeed, one may suppose that, depending on the physiological or pathological state of the cell, highly specialized translation machines preassemble within nucleoli before being either exported to the cytoplasm or directly used in nuclei to translate certain classes of mRNAs. Alternatively, these preassembled translation machines may participate to the mRNA quality control (Hentze and Kulozik, 1999