Human Molecular Genetics Advance Access originally published online on March 28, 2006
Human Molecular Genetics 2006 15(9):1525-1538; doi:10.1093/hmg/ddl074
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The nuclear MicroSpherule protein 58 is a novel RNA-binding protein that interacts with fragile X mental retardation protein in polyribosomal mRNPs from neurons
1Unité de Recherche en Génétique Humaine et Moléculaire, Centre de recherche Hôpital Saint-François d'Assise, le CHUQ, Québec, Canada G1L 3L5, 2Département de biologie médicale, Faculté de médecine, Université Laval, Québec, Canada, 3UMR 6543, Faculté de Médecine, Université de Nice, Nice 06107, France and 4Département de Psychiatrie, Faculté de médecine, Centre de recherche Université Laval Robert-Giffard, Québec, Canada G1J 2G3
* To whom correspondence should be addressed at: URGHM, Centre de recherche Hôpital St François d'Assise 10 rue de l'Espinay, Québec, PQ, Canada G1L 3L5. Tel: +1 4185254402; Fax: +1 4185254195; Email: edward.khandjian{at}crsfa.ulaval.ca
Received February 14, 2006; Accepted March 19, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The fragile X syndrome, the leading cause of inherited mental retardation, is due to the inactivation of the fragile mental retardation 1 gene (FMR1) and the subsequent absence of its gene product FMRP. This RNA-binding protein is thought to control mRNA translation and its absence in fragile X cells leads to alteration in protein synthesis. In neurons, FMRP is thought to repress specific mRNAs during their transport as silent ribonucleoparticles (mRNPs) from the cell body to the distant synapses which are the sites of local synthesis of neuro-specific proteins. The mechanism by which FMRP sorts out its different mRNAs targets might be tuned by the intervention of different proteins. Using a yeast two-hybrid system, we identified MicroSpherule Protein 58 (MSP58) as a novel FMRP-cellular partner. In cell cultures, we found that MSP58 is predominantly present in the nucleus where it interacts with the nuclear isoform of FMRP. However, in neurons but not in glial cells, MSP58 is also present in the cytoplasmic compartment, as well as in neurites, where it co-localizes with FMRP. Biochemical evidence is given that MSP58 is associated with polyribosomal poly(A)+ mRNPs. We also show that MSP58, similar to FMRP, is present on polyribosomes prepared from synaptoneurosomes and that it behaves as an RNA-binding protein with a high affinity to the G-quartet structure. We propose that this novel cellular partner for FMRP escorts FMRP-containing mRNP from the nucleus and nucleolus to the somato-dendritic compartment where it might participate in neuronal translation regulation.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The fragile X syndrome (FXS) is the leading cause of inherited mental retardation, due to the silencing of the fragile X mental retardation 1 gene (FMR1) and the subsequent absence of FMRP, its gene product. FXS affects 1/4000 males and 1/7000 females worldwide and is characterized by moderate to severe mental retardation. The phenotype is complex and is also accompanied by physical abnormalities such as facial dysmorphism, postpubertal macro-orchidism in males and mild connective tissue dysplasia (1
–4). FMRP is an RNA-binding protein widely expressed in mammalian tissues (5) and particularly abundant in neurons (6) and is an element of messenger ribonucleoprotein (mRNP) complexes associated with brain polyribosomes (7–9). Its presence within the translational apparatus suggests that it is involved in the translational control of certain mRNAs. In addition, FMRP is present in mRNA cargoes that are transported along neurites and dendrites (10,11). Its interaction with the RNA-induced silencing complex in the cytoplasm suggests that it can also play a role in the control of the stability of mRNA and/or it acts as translational inhibitor via the interaction with the AGO proteins (12).
Two KH domains and an RGG box modulate the ability of FMRP to bind RNA. The RGG box has high affinity for the RNA G-quartet structure (13,14) present in several of FMRP's mRNA targets (15), whereas the KH2 domain seems to recognize synthetic RNAs aptamers presenting a loop–loop pseudoknot-specific motif or ‘kissing complex’ (16). In Fmr1 null mice, a series of mRNA display change, both in localization and abundance, pointing out the critical role that FMRP plays in the targeting of specific mRNAs (17,18). It is proposed that the absence of FMRP would lead to alterations in the local synaptic synthesis of proteins that are essential for synaptic development and maturation. One of the consequences of the absence of FMRP is the presence of abnormal looking immature and surnumerary neuronal dendritic spines in the brains of fragile X patients (19,20), which ultimately lead to mental retardation in FXS patients.
FMRP is able to interact directly through its N-terminal domain with a series of protein partners. Among these, FXR1P and FXR2P are two closely related paralogues that show high levels of homology with FMRP (21,22) and can form heterodimers with FMRP. The FMRP-interacting protein 82-FIP is an RNA-binding protein that is present in polyribosomal mRNPs (23), whereas NUFIP1, also an RNA-binding protein, is a nucleocytoplasmic shuttling protein that is associated with active synaptoneurosomes (24). Two paralogous proteins, CYFIP1 and CYFIP2, that interact with FMRP also provide a link to the Rho GTPase pathway that controls actin cytoskeleton dynamics (25,26). More recently, IMP1, a zip-code protein involved in mRNA transport, has also been found to directly interact with FMRP (27). RanBPM, a protein involved in the microtubule-organizing centre has been reported to bind to the C-terminus of FMRP (28). In addition, other RNA-binding proteins such as nucleolin, YB-1/p50, Pur-
and Staufen have been detected in complexes containing FMRP, but it is not known whether they interact directly or indirectly with FMRP (29–31). Only a few non-RNA-binding proteins have been shown to interact with FMRP, including myosin Va (31) and Lgl in Drosophila (32).
In this study, we describe a novel FMRP-interacting protein named MicroSpherule Protein 58 (MSP58). We provide evidence that MSP58 associates with polyribosomal poly(A+) mRNP and is also found as a polyribosome component in synaptoneurosomes. Moreover, we show that MSP58 is a novel nuclear RNA-binding protein able to bind G-quartet RNA.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
MSP58 is a novel FMRP-interacting proteins
To identify new FMRP-interacting proteins specific to neurons, we screened a human fetal brain cDNA library using a yeast two-hybrid system, with the major FMRP Iso7 form as a bait. From
2.8x106 clones screened, 78 positive clones displayed adenine and histidine prototrophy as well as ß-galactosidase activity. PCR and restriction analysis of selected colonies showed that among these, three clones were redundant and carried a 1.7 kb insert. This insert corresponded to the full-length cDNA of a human nuclear/nucleolar protein of 58 kDa called MSP58 (33). The predicted open-reading frame encodes a protein of 462 residues. The cDNA obtained from the yeast two-hybrid screening also harboured the 5' and 3'-untranslated regions which were identical to the previously established sequence (33). The specificity of the interaction of MSP58 with FMRP was confirmed in yeast by co-transforming the AH109 strain with plasmids encoding MSP58 and FMRP (Table 1). Given the high degree of similarity between all members of the FXR family, we also tested the ability of MSP58 to interact with FXR1P and FXR2P, the two other members of the FXR family. MSP58 was found to interact with FMRP, FXR1P and FXR2P (Table 1), whereas no interaction was detected with control plasmids bearing unrelated proteins cDNA. The strong interaction between MSP58 and the FXR proteins induced us to further investigate FMRP/MSP58 interaction.
|
MSP58 is a highly evolutionarily conserved nuclear protein
MSP58 was initially shown to interact with the nucleolar protein p120, and its overexpression led to enlargement of nucleoli (33
). Also, MSP58 was reported to play a role in modulation of Daxx-dependent transcriptional repression (34). Recently, a study revealed that MSP58 behaves as an oncogene and that its transformation activity can be inhibited by physical interaction with the PTEN tumour suppressor (35). Moreover, it was reported that MSP58 associates to and stabilizes the transcriptional activity of the bHLH trancription factor STRA13 (36). Finally, MSP58 was shown to interact with Mi-2ß in the nucleolus and to up-regulate ribosomal gene transcription (37). However, despite of all these data, the cellular role of MSP58 remains unclear because divergent properties/functions have been attributed to the protein.
We searched for sequences displaying similarities with human MSP58 cDNA and found homologues in species as distant as human (hMSP58, also called MCRS1), mouse (mMSP58), Xenopus laevis (xMSP58 or xMCRS1), zebrafish (zMSP58 or zMCRS1), quail (qMSP58 or qTOJ3) and finally Drosophila melanogaster (dMSP58 or CG1135-PA) (Fig. 1A). Alignment of all sequences revealed that all homologues share a high level of similarity. Indeed, human and mouse MSP58 proteins are 98.7% identical, differing by only six residues over 462, whereas human shares 81% identity with its frog counterpart. As the presence of highly conserved residues stretches should delineate domains essential for MSP58 function(s), we performed computational analysis of these sequences. The N-terminal part contains a highly conserved domain bearing a putative bipartite nuclear-localization signal (amino acids 32–46), as well as a putative nucleolar-localization signal (amino acids 44–56). This stretch of residues 44–56 KRRSSR-IKRKKFDDELVSS is 100% conserved over species, showing slight divergences only in Drosophila, suggesting functional relevance of this domain. Further down the sequence, a putative monopartite nuclear-localization signal (amino acids 113–123) is detected, whereas a coil–coil domain is predicted at position 301–350. Despite the high level of conservation of the central part of MSP58, i.e. amino acids 125–294, no similarity to known functional domains were found. The C-terminus of all MSP58 homologues contains a highly conserved forkhead-associated (FHA) domain (Fig. 1B). FHA domains are known to interact with phosphorylated residues and indeed, MSP58 FHA domain was shown to specifically bind phosphorylated residues of STRA13 and PTEN (35,36).
|
MSP58 N-terminal domains interact with the phosphorylation domain of FMRP
To confirm the results obtained in yeast and to verify whether the FMRP/MSP58 interaction was direct and specific, we performed a series of pulldown assays using the FMRP full-length protein, as well as FXR1P (Iso7) and FXR2P. These proteins were produced and labelled with [35S]methionine by in vitro transcription–translation using the rabbit reticulocyte lysate, whereas human MSP58 was produced as a fusion protein with glutathione S-transferase (GST) in bacteria. The GST–MSP58 fusion protein immobilized on glutathione–Sepharose was incubated with either in vitro-translated FMRP, FXR1P, FXR2P or luciferase as a negative control. The three members of the FXR family were shown to bind to the immobilized GST–MSP58, whereas luciferase did not (Fig. 2A). No binding to GST was observed, confirming the specificity of the interaction. To verify that the interaction between FMRP and MSP58 was direct and not RNA-dependent, GST pulldowns between in vitro-translated FMRP and GST–MSP58 bound to beads were performed in the presence of RNase A, RNase T1 and DNase I. This treatment did not alter FMRP binding to MSP58 (data not shown), indicating that the interaction occurs at the protein–protein level.
|
To refine the area of interaction between MSP58 and FMRP, MSP58 fragments were fused to GST and tested for their ability to retain 35S-radiolabelled FMRP. As shown in Figure 2B, MSP58 N-terminus encompassing amino acids 1–124 was sufficient to bind FMRP, and a fortiori the stretch of amino acids 1–294 as well as the full-length MSP58, indicating that FMRP-binding domain lies in the N-terminus. In contrast, to determine the region of FMRP that binds MSP58, several FMRP variants and deletion constructs were tested for their ability to bind GST–MSP58. As MSP58 has been previously reported to behave as a nuclear protein, we also tested its ability to bind the nuclear FMRP isoform (Iso6) lacking exon 14 (amino acids 426–490) that encompasses the NES. As shown in Figure 2C, FMRP full-length (Iso7) and Iso6 and all FMRP versions truncated before (amino acids 1–526) and after the RGG box (1–554) were able to bind recombinant GST–MSP58. Because FMRP version truncated at amino acids 426 was not able to bind MSP58, although FMRP Iso6 lacking amino acids 426–490 still interacts with MSP58, we concluded that the binding domain to MSP58 lies between amino acids 490 and 526 (Fig. 2E). Interestingly, it encompasses the phosphorylation domain of FMRP which has been mapped at amino acids 444–526 (38
–40). This domain is also present in FXR1P and FXR2P and displays a highly conserved block of residues among the three proteins (Fig. 2D).
We then questioned whether point mutations in the serine 500 residue of FMRP, known to be phosphorylated (38), would affect its binding to MSP58. Substitution of the S500 by the unphosphorylable residue alanine or by the acidic residue aspartate, which mimics a charged phosphorylated serine, did not, at least in vitro, affect the binding of MSP58.
Subcellular localization of MSP58
According to the few reports available in the literature, MSP58 is supposed to be either exclusively nucleolar or nucleolar and nucleoplasmic (33,34,36,37) or even cytoplasmic (35). It is worth mentioning that most of these studies utilized cells transiently expressing an HA-tagged MSP58 that was revealed with anti-HA antibodies, whereas the cellular distribution of the bona fide endogenous MSP58 has not been fully documented. We first raised an anti-MSP58 antibody in rabbit against the central part of human protein (amino acids 125–294). One antiserum was obtained, and after immuno-affinity purification, its specificity towards MSP58 was determined by both immunoblot analyses and immunofluorescence staining using HeLa and Cos-1 cells transfected with an expression vector for hMSP58. In addition, we observed that different staining patterns were obtained depending on the procedures used to fix the cells prior to the immunofluorescence stainings, and we assumed that the discrepancies observed in its subcellular localization reported by others were due to the fixation procedures. A classical 10 min 4% PFA fixation followed by a 0.5% Triton permeabilization treatment revealed an exclusively nucleolar staining (Fig. 3A), whereas a 10 min fixation with 4% PFA in the presence of 0.5% Triton showed both nuclear and nucleolar localization (Fig. 3B). In contrast, a combined fixation and permeabilization procedure using formaldehyde in a mixture of acetone and methanol (2/19/19 by vol) allowed to visualize both nucleoplasmic and nucleolar distribution of MSP58 and also a faint cytoplasmic staining. In control analyses, no staining was detected when the antibodies were pre-incubated with recombinant purified GST–MSP58 amino acids 125–294 that was used for immunization (Fig. 3D). On the basis of these results, we concluded that MSP58 lies predominantly in the nucleus, whereas a small fraction is detected in the cytoplasm.
|
MSP58 recruits FMRP nuclear Iso6 into the nucleolus
To better visualize in vivo the subcellular localization of MSP58, we fused its cDNA to the red fluorescent protein (RFP) and transfected Cos-1 cells with this construct. Exogenous RFP–MSP58 strongly accumulates in the nucleolus but is also present in the nucleoplasm (Fig. 4A). We also noticed that upon overexpression of RFP–MSP58 nucleoli became enlarged, in agreement with Ren et al. (33
). This nucleolar accumulation was also reproducible in HeLa and Fmr1–/– STEK cells (data not shown). The specificity of the distribution was assessed by immunostaining of the RFP–MSP58 fusion protein with anti-MSP58 and indeed a co-labelling of RFP–MSP58 and the endogenous MSP58 was observed (not shown). In this series of analyses, we were not able to detect a cytoplasmic staining due to the relative extremely strong nucleolar accumulation of exogenous RFP–MSP58. Alternatively, we speculate that high levels of RFP–MSP58 present in the nucleolus might have deleterious effects on the metabolism of the cell and prevents normal nuclear export of the protein.
|
To study in vivo the interaction between MSP58 and FMRP, we used in transfection experiments the two constructs encoding, respectively, RFP–MSP58 and the nuclear Iso6 FMRP fused to GFP. In single transfected Cos-1 cells, GFP–FMRP Iso6 localizes in the nucleoplasm and is excluded from the nucleolus (Fig. 4B). Interestingly, co-expression of RFP–MSP58 and GFP–FMRP Iso6 drastically altered the nucleoplasmic distribution of the latter. Indeed, in co-transfected cells, GFP–FMRP Iso6 was clearly recruited into the nucleolus (Fig. 4C). To ascertain that this phenomenon was not artefactual owing to the presence of the fusion proteins RFP and GFP, which are known to form homo and heteromultimers in vivo, we transfected Cos-1 cells with untagged versions of pTL1-FMRP Iso6 together with pTL1-MSP58 and revealed both proteins by immunofluorescence staining using anti-MSP58 antibody and mAb1C3 to FMRP, and the same phenomenon was observed (data not shown). To assess whether the ability of MSP58 to attract FMRP Iso6 in the nucleolus was not cell-type-specific, RFP–MSP58 and GFP–FMRP Iso6 were also co-expressed in different cell lines, i.e. HeLa and Fmr1–/– STEK cells, and similar observations were made suggesting that MSP58 has the ability to drag and retain FMRP Iso6 in the nucleolus.
In a second set of experiments, we co-transfected RFP–MSP58 with the cytoplasmic GFP–FMRP Iso7 and observed that GFP–FMRP Iso7 remained in the cytoplasm, whereas RFP–MSP58 accumulated in the nucleoplasm and the nucleolus (Fig. 4D). These results strongly suggest that MSP58 recruits the nuclear FMRP Iso6 while leaving the FMRP full-length in the cytoplasm. Indeed, MSP58 failed also to translocate the cytoplasmic FXR1P and FXR2P as seen in transfection assays with expression vectors coding for these homologues (data not shown).
Expression of MSP58 in rat tissues
Because MSP58 expression was previously studied in murine tissues by RT–PCR or northern blot analyses (36), we determined the distribution of the protein by immunoblotting analyses using our new specific anti-MSP58 antibody. In agreement with previous studies at the mRNA level, MSP58 is detected in all tissues tested, albeit at different levels (Fig. 5). The highest levels of MSP58 were detected in spleen and testis, although strong signals were also present in brain. In muscular tissues such as cardiac and skeletal striated muscle, only trace amounts of MSP58 could be detected.
|
MSP58 co-localizes with FMRP in the cytoplasm of neurons
Because high levels of MSP58 are detected in brain extract, we determined its distribution in adult rat brain sections by immunohistochemical approaches using the MSP58 antibody. We observed strong staining in all nuclei throughout the brain with a specific nuclear localization in all glial cells (Fig. 6). Surprisingly, strong MSP58 staining was also detected in all cortical pyramidal neurons present in the cortex, and in neurons of the CA3 region of the hippocampus as well as in Purkinje cells in the cerebellum, where it co-localized with FMRP. We failed to detect MSP58 in the nucleolus, most probably due to the difficulties of penetration of the antibody in a fixed tissue. Essentially, the same distributions of MSP58 and FMRP were seen in paraffin-embedded sections of rat brain (data not shown). These observations illustrate that MSP58 is abundant in the cytoplasm of neurons, contrary to other cells, and co-localizes with FMRP and are in favour of an interaction between MSP58 and FMRP.
|
To further document the cytoplasmic localization of MSP58 in neurons, electron microscopy analyses were performed on immunogold-labelled rat brain sections using the purified MSP58 antibody. In granular cells of the cerebellum, MSP58 was restricted to the nuclei, whereas in Purkinje cells additional cytoplasmic localizations could be observed (Fig. 7A). At higher magnification, MSP58 could be detected associated with the ER, both in Purkinje cells (Fig. 7B) and in CA3 hippocampal neurons (Fig. 7C).
|
To verify whether MSP58 was also present in neurites, as is the case for FMRP, we used primary cultures of rat hippocampal neurons and double stained them with anti-MSP58 (revealed in red) and anti-FMRP (in green) and analysed both distribution by confocal microscopy. In neurons in primary culture, the majority of MSP58 is detected in the nucleus and nucleolus whereas FMRP was not. In contrast, while FMRP was predominantly detected in the cytoplasm, a substantial amount of MSP58 co-localized with FMRP in the cytoplasm (Fig. 8A). When a high gain that resulted in the saturation of the fluorescent signal in the cell body was used, a granular-like punctuate MSP58 staining was also clearly observed in the dendritic arborizations. At higher magnification, co-localization of FMRP and MSP58 was evident in the majority of granule-like structures in neurites (Fig. 8B).
|
MSP58 is present in polyribosomal mRNP complexes
It is well established that FMRP is present in mRNP complexes associated with heavy sedimenting polyribosomes prepared from total brain (8
,9,41). Because MSP58 is strongly expressed in brain and is also encountered in the cytoplasm of neurons close to the ER (Figs 6 and 7), we asked whether it could be present within the translation machinery. We prepared brain polyribosomes as previously described and analysed by velocity sedimentation through sucrose density gradients (8). In the presence of Mg2+, MSP58 was detected in fractions corresponding to heavy sedimenting polyribosomes and its distribution along the gradient mirrors that of FMRP (Fig. 9A). In the presence of EDTA that dissociates ribosomes into their subunits concomitant with the release of free mRNP complexes, MSP58 and FMRP were detected sedimenting in the same fractions, suggesting that MSP58 is present in mRNP complexes that also carry FMRP (Fig. 9B).
|
In neurons, polyribosomal aggregates are also present in dendritic spines and synapses and these structures contain FMRP (7
,42). We therefore asked whether MSP58 would also escort mRNPs to synaptic polyribosomes. To test this hypothesis, we prepared a polyribosomal fraction from purified synaptoneurosomes and analysed this fraction by velocity sedimentation through sucrose density gradients. Two main results were obtained: first, FMRP is indeed associated with synaptosomal polyribosomes, in contradiction with the results obtained by Zalfa et al. (43), and secondly that MSP58 also is present in these structures (Fig. 9C). FMRP as well as MSP58 could be released from synaptosomal polyribosomes after treatment with the chelating EDTA agent (data not shown).
MSP58 is a novel RNA-binding protein associated with poly(A+) mRNP
We questioned the ability of MSP58 to be present in poly(A)+ mRNPs by performing oligo(dT) selection on purified 150–500S rat brain polyribosomes treated with EDTA to dissociate ribosomal subunits and to release their associated mRNPs. We observed that MSP58 is retained onto the oligo(dT) column and is eluted together with FMRP at 0.5 M NaCl. These results suggest that, similar to FMRP, MSP58 is an RNA-binding protein associated with poly(A+) mRNPs pointing out its ability to bind RNA in vivo as well as with other interacting proteins present in RNP complexes (Fig. 10A). Because most of the proteins interacting with FMRP present on polyribosomes are known to be RNA-binding proteins (8), we searched for the ability of MSP58 to bind RNA and performed RNA-homopolymers binding assays with purified recombinant GST–MSP58. Indeed, GST–MSP58 could selectively bind to polyG and polyU, but not to polyA or polyC (Fig. 10B), a pattern of binding to RNA homopolymers similar to that observed for FMRP (44). Interestingly, the binding activity to polyG and polyU was retained in a truncated version of MSP58, suggesting that the RNA-binding domain lies within the N-terminal domain of MSP58 encompassing amino acids 1–124. It should be noted that this domain appears also to bind FMRP (Fig. 2E). Despite our efforts, no consensus RNA-binding sequence could be detected in this domain, suggesting that it should contain a yet uncharacterized sequence.
|
Because RNA homopolymers correspond to synthetic RNA segments, the affinity of MSP58 to these structures might not reflect its ability to bind biologically relevant mRNA structures encountered in vivo. Therefore, we questioned whether it could have affinity to a well-known mRNA structure called G-quartet. This structure is a recognition motif that allows binding with high affinity to FMRP and is present in several FMRP-mRNA targets, and particularly in the coding region of FMR1 mRNA (13
,14). Electrophoretic mobility shift assays were performed with recombinant GST–MSP58 in the presence of the N19 fragment corresponding to FMR1 mRNA G-quartet minimal binding site (14). The results showed that MSP58 was able to bind N19, whereas a fragment corresponding to amino acids 125–294 was not (Fig. 10C). To ascertain that MSP58 binding to the G-quartet structure was not due to a general affinity for RNA, we performed nitrocellulose filter-binding assays (13,14) which allowed to titrate by competition assays the specificity of the binding of MSP58 to the G-quartet N19 sequence. Indeed, similar to FMRP, the binding of MSP58 to the N19 RNA was efficiently competed by the same sequence, whereas an unrelated fragment of FMR1 mRNA called N8 was unable to displace the binding (Fig. 10D). The binding of MSP58 to the N19 sequence was clearly dependent on the presence of the cation K+, known to stabilize the G-quartet structure as previously demonstrated (13,14), whereas reduced binding was observed in the presence of Na+ (Fig. 10E). All these results taken together suggest that MSP58 is a novel RNA-binding protein with high affinity to a G-quartet motif. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
In this study, we have identified a novel partner for FMRP. MSP58 is evolutionarily conserved from fly to man. Its N-terminal part contains putative nucleolar and nuclear localization signals which seem to control its targeting to the nuclear compartment and is necessary to bind FMRP, whereas FMRP appears to bind MSP58 through its C-terminal domain (amino acids 444–526) that contains a phosphorylation domain. Despite the fact that mutations on the putative phosphorylated serine 500 did not seem to affect the binding to MSP58 in vitro, we cannot exclude that in vivo phosphorylation might modulate the interaction between MSP58 and FMRP. Additional serines (Ser 494, 497, 504) that are highly evolutionary conserved in the phosphorylation domain of the FXR proteins have not been yet tested for their putative phosphorylation.
Previous studies have suggested that MSP58's functions are related to transcriptional regulation in the nucleus and nucleolus. This assumption is based on the observation that it has been exclusively detected in the nuclear compartment where it was reported to interact with transcription factors (34,36) and to up-regulate ribosomal gene expression (37). Our study reveals an additional important function of MSP58 as an RNA-binding protein presumably involved in translation regulation. We have shown that MSP58 is present in the cytoplasm of neurons in association with poly(A+) mRNP present in heavy sedimenting brain polyribosomes. The case of such a nuclear protein that is also present in association with the translation machinery is not isolated, because several proteins that have been characterized initially as nuclear proteins, such as hnRNP A1, PTB, La, hnRNP, YB-1/p50 and nucleolin, turned out to be present also in the cytoplasm and even implicated in translation control [for discussion and cross-references see (45)]. Of particular interest is YB-1/p50, which has been originally defined as a transcription factor, but which is the major protein of cytoplasmic mRNPs (46). Moreover, several FMRP-interacting proteins such as FXR1P, FXR2P, 82FIP and NUFIP are also nucleocytoplasmic shuttling proteins associated with polyribosomes (23,24). However, to our knowledge, MSP58 is the only FMRP partner which is also present in the nucleolus.
In co-transfection experiments, we observed a clear co-localization between MSP58 and FMRP nuclear Iso6 in the nucleolus. FMRP Iso6 being normally exclusively nucleoplasmic and excluded from the nucleolus (47), we conclude that overexpression of MSP58 results in the translocation of FMRP Iso6 into the nucleolar compartment. This suggests that in overexpression assays a fraction of FMRP can enter the nucleolus, as previously shown by immunoelectron microscopy studies performed on Cos-1 cells (48,49). Even though FMRP lacks the nucleolar targeting sequence present in FXR1P and FXR2P (50), the exceptional detection of FMRP in the nucleolus, if relevant, might result from an alternate mechanism, perhaps by interaction with FXR1P or FXR2P or even with other nuclear proteins. The binding of MSP58 to FMRP could directly participate in the translocation of FMRP-containing mRNP in the nucleolus.
Recent data indicate that the initial steps of RNA recognition and the packaging of mRNP complexes by RNA-binding proteins to be transported in the cytoplasm occur in the nucleus (51–53). We propose that FMRP binds its target mRNA in the nucleoplasm to be assembled in an mRNP with MSP58 and other RNA-binding proteins and RNA. The binding of MSP58 might change the conformation of FMRP and therefore affect the binding of its RNA targets on the RGG box, owing to the close proximity of the phosphorylation MSP58-binding domain and the RGG box. Moreover, the ability of MSP58 to bind with high affinity RNA G-quartet structures suggests that it might, in vivo, compete with FMRP for the binding of mRNA harbouring this structure. The protein composition of the FMRP-containing mRNP complexes might indeed modulate the type of mRNA targeted to the nascent mRNP. In addition to its central role in ribosome biogenesis, the nucleolus has been proposed to be the site of the assembly of pre-mRNP complexes (54,55). According to this point of view, the nucleolus would function as a nuclear checkpoint to verify the functional integrity and relevancy of these particles. Following this attractive model, it can be envisioned that the FMRP-containing mRNPs transit in the nucleolus where their potential functionality are verified before they are exported in the cytoplasm. Together with the other known FMRP-interacting proteins, such as FXR1P, FXR2P and 82-FIP, the interaction between MSP58 and FMRP in mRNP at the polyribosomal level might modulate FMRP function in the control of translation.
In neurons, a population of FMRP/MSP58-containing mRNP will be sorted from the neuronal cell body at a hypothetic RNP-triage centre (56,57) to be translocated at very distant locations in the form of mRNP granules. These mobile structures contain a reservoir of mRNAs that are maintained in a repressed state during migration (58) until they reach the synapse where FMRP is thought to play a key role in the control of their local translation. The presence of MSP58 in polyribosomes at the synapse as well as its ability to bind G-quartet containing mRNA raises the possibility that it would play a role, in concert with FMRP and/or other factors, in the control of local de novo protein synthesis, an essential phenomenon for synaptic development and maturation.
In summary, we have shown that MSP58 is a nucleocytoplasmic shuttling protein able to bind RNA and that it is associated with the cytoplasmic translation apparatus both in the cytoplasm and at the synapse. We therefore propose that in neurons MSP58 may be part of structures that are transported from the nucleus to translation sites, in a similar way as FMRP. However, the present study does not allow us to conclude whether MSP58 is an active essential partner to translocate mRNPs or whether it is dispensable to the cellular machinery. Also, it will be necessary to study whether MSP58 neuronal distribution is altered in Fmr1 KO neurons. Throughout this paper, we have presented FMRP as a central key molecule and hypothesized that its affinity to RNA could in theory be modulated by protein interactors. An other diametrically opposite view would be that FMRP modulates the activity of MSP58. As the world of RNA-binding proteins is expanding, understanding the role of MSP58 and of other RNA-binding FMRP interactors, will be essential to unravel the functions altered by the absence of FMR1 expression in the FXS.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Yeast two-hybrid screen
Full-length cDNA of human FMRP Iso7 from the pTL1-FMR1 Iso7 was subcloned in-frame with the DNA-binding domain of the transcription factor Gal4 (Gal4 DNA-BD) into the EcoRI/Pst1 sites of the yeast expression vector pGBKT7 (Yeast Two-Hybrid System, Clontech). Human fetal brain cDNA library screening was performed by mating the AH109 Mat
expressing the bait with the Y187 strain pre-transformed with the human fetal brain library encoded by the pACT2 vector (Matchmaker pre-transformed library, Clontech). Strains resulting from the mating carried the ADE3, HIS3 and LacZ reporters under the control of Gal4 responsive elements. From 2.8x106 clones screened, 78 positive colonies showed adenine and histidine prototrophy and ß-galactosidase activity. Among these, PCR and restriction analysis showed that three colonies were redundant and carried a 1.7 kb insert. This insert corresponded to the full-length cDNA of human MicroSpherule protein of 58 kDa (MSP58 or MCRS1, GenBank accession no. Q96EZ8).
Purification of recombinant fusion proteins and production of anti-MSP58 antibodies
For expression of recombinant GST–MSP58, the coding sequence of MSP58 or the fragments corresponding to amino acids 1–124, 1–294 and 125–294 were amplified by PCR using the primers (EcoRI F: 5'-CGG GAA TTC GAC AAA GAT TCT CAG GGG CT-3', XhoI R: 5'-GGC GAG CTC TCA CTG TGG TGT GAT CTT GG-3', 125EcoRI F: 5'-GCC GAA TTC CCA CTT CAG GTG ACC AAG G-3', 294Bam R: 5'-CGG GGA TCC CTT GAG CTT ACT GTC ATC AAT C-3' and 124Bam R: 5'-CGG GGA TCC CTG TTT ACT CTT CTT CAC ACG C-3') and subcloned into the EcoRI/XhoI or EcoRI/BamHI sites of pGex-4T-1 (Amersham). Fusion proteins were expressed in BL21(DE3) Escherichia coli strain (Stratagene) grown in liquid LB until OD
1 and induced overnight with 1 mM IPTG. Bacteria were then collected and the expressed fusion proteins purified in non-denaturing conditions on glutathione–Sepharose beads, according to manufacturers's protocole (Amersham Pharmacia Biotech). Fusion protein was eluted from the beads with 10 mM reduced glutathione in 50 mM Tris–HCl (pH 8.0). Protein yields were estimated by Coomassie staining using as standard different concentrations of bovine serum albumin (BSA) ranging from 0.2 to 1 µg/µl. The GST–MSP58 amino acids 125–294 were used to produce antibodies in rabbit using standard protocol, and anti-MSP58 IgG were affinity purified with the same fusion protein used for immunization.
GST-pulldown assays
FMRP and its truncated variants, FXR1P and FXR2P, were produced by in vitro transcription–translation in rabbit reticulocyte system in the presence of [35S]methionine (Amersham) according to manufacturer's instruction (Promega, Madison, WI, USA). Five microlitres of in vitro-translated proteins were mixed with 1 µg of GST–MSP58 and its variant recombinant proteins bound to beads and incubated 2 h, at room temperature, under constant rotation in 500 µl of pulldown buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP-40, Protease Inhibitor Cocktail, Roche). Beads were collected by spinning (3000 g, at room temperature for 2 min) and washed four times with the pulldown buffer. Final wash was removed and beads were resuspended in 50 µl of SDS sample buffer. One-third of the sample was loaded on a 7.5% SDS–PAGE. Gel was then dried and exposed for 3 days to a Biomax film (Kodak).
RNA studies
Homopolymer binding assays
Binding assays were performed according to established procedures. Briefly, 0.5 µg of recombinant GST-tagged MSP58 were incubated with immobilized poly(G), poly(U), poly(A) or poly(C) polyacrylhydrazido-agarose beads (Sigma) in 0.5 ml of binding buffer containing 20 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.5% NP-40 and 2.5 mM MgCl2 supplemented with Protease Inhibitor Cocktail (Roche) for 1 h at room temperature. After incubation, the beads were washed four times with binding buffer and bound proteins eluted by addition of SDS sample buffer followed by heat denaturation. Proteins were separated by SDS–PAGE and blotted either with anti-GST antibody (Amersham) used at 1:25 000 or with anti-MSP58 serum. Control analyses were also performed with 35S-labelled MSP58 produced in the rabbit reticulocyte lysate system (data not shown). In vitro-translated proteins were detected after exposure of the gel to an X-ray film.
RNA-binding assays
The different RNA fragments N19 and N8 derived from FMR1 RNA were synthesized by in vitro transcription with T7 RNA polymerase from pTL1 (47) derivative plasmids linearized with PstI. The RNAs were purified using the NucAway Spin columns (Ambion). RNAs were then ethanol precipitated and resuspended in the appropriate buffer. For binding experiments, N19 was labelled co-transcriptionally by incorporation of [-32P]ATP. Labelled RNAs were purified on a 1% low-melting agarose gel (Ambion). Labelled RNAs (80 000 c.p.m., 5 fmol) were renatured for 10 min at 40°C in 40 µl of binding buffer [50 mM Tris–HCl pH 7.4 at 4°C, 1 mM MgCl2, 1 mM EDTA, 150 mM KCl, 1 mM dithiothreitol (DTT)] with 8 U of RNasin (Invitrogen), 10 µg of E. coli total tRNA and 0.01% BSA. The RNA was then added to increasing amount of proteins. RNA–protein complexes were formed for 10 min at 0°C. After incubation, binding solutions were passed through MF-membrane filters (0.45 HA, Millipore) and washed with 2 ml binding buffer. Filters were air-dried and the amount of radioactivity was measured by Cerenkov counting. Data were plotted as percentage of total RNA bound versus the protein concentration. Competition experiments to determine the relative binding strength of MSP58 to G-quartets were carried using 32P-labelled N19 RNA incubated with MSP58 (0.1 pmol) in the presence of increasing concentrations of unlabelled competitors. FMRP was used as an internal positive control. Binding of FMRP and MSP58 to the G-quartet were performed in the presence of 150 mM of either K+ or Na+.
Cell culture, primary neuron culture and transient transfection assays
HeLa S3, Cos-1 and STEK Fmr1 KO/TSV40 cells were propagated and maintained in DMEM supplemented with 10% FBS and antibiotics (100 U/ml penicillin, 50 mg/ml streptomycin). Transfection assays with different vectors were performed in the presence of Effectene according to manufacturer's recommendations (Qiagen). Primary neuron culture was prepared from rat hippocampi as described (59).
Protein studies
Organs were removed from animals, processed according to standard protocols and protein extracts prepared for SDS–PAGE as described (60). Immunoblot analyses were performed using mAb1C3 to FMRP, rabbit polyclonal antisera directed against MSP58 and anti-L7 ribosomal protein. Detection of bound antibodies was performed with HRP-coupled secondary antibodies followed by ECL reaction.
Immunofluorescence studies
Immunocytofluorescence
Cos-1 cells grown on glass cover slips were washed three times with ice-cold phosphate-buffered saline (PBS) then fixed with the fixation and permeabilization protocols described in Results. To detect endogenous MSP58, the anti-MSP58 was used at 1:200 (overnight at 4°C), followed by incubation (90 min at room temperature) with Alexa fluor 488 goat anti-rabbit IgG (Molecular Probes). Control experiments were performed in the presence of antibodies that were pre-incubated with the recombinant purified GST–MSP58 amino acids 125–294 used for immunization. For direct visualization of MSP58 in eukaryotic cells, MSP58 was fused to the RFP by subcloning its cDNA from the pGex-4T-1/MSP58 full-length in the EcoRI–SalI sites of the polylinker of a modified version of peRFP graciously provided by Paul De Koninck.
Immunohistofluorescence
Adult rats, deeply anaesthetized with ketamine xylol (40 mg/kg, i.p.) were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain was removed, transferred to cryoprotective solution (HistoPrep, Fisher Scientific), frozen and serially cut into longitudinal sections (5 µm) with a Leica CM1900 cryostate. Sections were treated as described (23) and mounted on slides and processed for double immunofluorescence using rabbit anti-MSP58 (at 1:200 dilution) and anti-FMRP mAb1C3 antibody. Double immunofluorescence staining were performed by separate and sequential incubations of each primary antibody diluted in PBS at 4°C overnight, followed by the respective secondary antibody coupled to Alexa594 or Alexa 488 (Molecular Probes) and incubated at room temperature for 3 h. Samples were analysed with a Nikon EclipseE800 microscope equipped with a Hammamatsu CCD camera. Images were then treated with the Adobe PhotoShop software program.
Electron microscopy
Pre-embedding immunogold labelling was performed as described (61). Briefly, free floating fixed rat brain sections were blocked for 30 min in 0.8% BSA, 0.1% cold fish skin gelatin (Amersham, Piscataway, NJ, USA) and 5% normal goat serum in Tris-buffered saline (TBS), followed by incubation with MSP58 antiserum for 48 h at 4°C. Sections were washed and incubated with 0.5 nm gold-conjugated secondary antibody (AuroProbe, Amersham, 1:80 in TBS) for 12 h at 4°C and the gold particles were enhanced with silver solution (IntenSE, Amersham). Sections were treated with 1% OsO4, dehydrated in graded ethanol, then in propylene oxide and embedded in Durcupan ACM (Fluka). Specimens were sectioned and examined using a Philips Tecnai 12 electron microscope.
Polyribosome preparation and analysis
Total brain polyribosomes were prepared from young Sprague-Dawley rats (7 days old) and purified as described (8). For EDTA–mRNPs studies, the fractions of sucrose gradients corresponding to 150–500S were pooled, diluted with 1 volume of buffer (20 mM Tris–HCl, pH 7.4, 100 mM KCl, 1.25 mM MgCl2) and the particles pelleted by centrifugation in a Sorvall TH-641 rotor for 2 h at 34 000 r.p.m. at 4°C. The polyribosomal pellets were resuspended in 20 mM Tris–HCl, pH 7.4, 100 mM KCl, 1% NP-40 containing 25 mM EDTA and analysed after centrifugation in a linear 5–35% (w/w) sucrose density gradient for 3.5 h at 34 000 r.p.m. at 4°C.
For preparation of synaptosomes, 16 cortices from young animals (7 days old) were homogenized by hand using a glass homogenizer (30 strokes) in a buffer containing 20 mM Tris–HCl (pH 7.4), 100 mM NaCl, 1.25 mM MgCl2, 0.32 M sucrose, 1 mM DTT, 50 µg/ml cycloheximide, 5 U/ml RNasine (Amersham Pharmacia) and protease inhibitors (Mini Complete, Roche Biochemicals). Purification of synaptosomes followed a two-step procedure. After centrifugation at 640g for 10 min, the supernatant was layered on Percoll gradient and the synaptosomal fraction obtained as described (62). The second step of purification was performed by floatation in an Optiprep gradient (63). The purified synaptosomal fraction was then resuspended in buffer containing 1% NP-40 and the released polyribosomes were concentrated after ultracentrifugation through a sucrose pad (45% w/w) in a Sorval TH-641 rotor at 34 000 r.p.m. for 3 h. The polyribosomal pellet was resuspended in buffer containing 1% NP-40 and analysed by velocity sedimentation in a 15–45% (w/w) isokinetic sucrose gradient and centrifuged in a 4 ml tube with a Sorvall TST 60.4 rotor at 30 000 r.p.m. for 2 h at 4°C. All gradients were fractionated by upward displacement using an ISCO UA-5 flow-through spectrophotometer set at 254 nm and connected to a gradient collector.
Each collected fraction was precipitated overnight at –20°C after addition of 2 volumes of ethanol. The precipitated material was collected by centrifugation at 12 000 r.p.m. for 20 min and solubilized in SDS sample buffer before immunoblot analyses. FMRP was detected with mAb1C3, MSP58 with its corresponding antiserum and ribosomal L7 protein with rabbit anti-L7 serum. As synaptosomes account for a very minor fraction of the brain, we used a total of 16 cortices per extraction, and performed the sedimentation analyses in small 4 ml tubes instead of the standard 11 ml used to analyse total polyribosomes extracted from three brains. Finally, the sensivity of the UV detector used to follow the polyribosomal profile was tuned to scale 0.5 (optical density at 254 nm) instead of 2.0 (compare the profile of total brain versus synaptosomes in Fig. 8).
|
ACKNOWLEDGEMENTS |
|---|
We thank Roumiana Gulemetova and Richard Kinkead for helpful discussions concerning brain fixation and preparation, Paul De Koninck for the neuron primary cultures and for the modified peGFP and peRFP plasmids and Philippe Lemieux and J. Grosgeorge for technical assistance. L.D. was a recipient of a postdoctoral fellowship from the FRAXA Research Foundation (USA) and was supported by traveling grants from the Conquer fragile X Foundation and the Boerhinger Ingelheim Fund. M.G. holds a Jerome Lejeune (France) fellowship. This work was supported by grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada (to E.W.K.) and from La Fondation de la Recherche Médicale, GIS Maladies Rares and FRAXA Foundation (to B.B.).
Conflict of Interest statement. None declared.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
- Hagerman, R.J. and Cronister, A. (1996) Fragile X Syndrome: Diagnosis, treatement and Research, University Press, Baltimore.
- Bardoni, B. and Mandel, J.L. (2002) Advances in understanding of fragile X pathogenesis and FMRP function, and in identification of X linked mental retardation genes. Curr. Opin. Genet. Dev., 12, 284–293.[CrossRef][ISI][Medline]
- O'Donnell, W.T. and Warren, S.T. (2002) A decade of molecular studies of fragile X syndrome. Annu. Rev. Neurosci., 25, 315–338.[Medline]
- Khandjian, E.W., Bechara, E., Davidovic, L. and Bardoni, B. (2005) Fragile X mental retardation protein: many partners and multiple targets for a promiscuous function? Curr. Genomics, 6, 515–522.[CrossRef]
- Davidovic, L., Tremblay, S., Gravel, M., De Koninck, P. and Khandjian, E.W. (2006) The fragile X syndrome: one protein missing and 1001 disoriented mRNAs. Med. Sci., 22, 41–46.
- Devys, D., Lutz, Y., Rouyer, N., Bellocq, J.P. and Mandel, J.L. (1993) The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation. Nat. Genet., 4, 335–340.[CrossRef][ISI][Medline]
-
Feng, Y., Gutekunst, C.A., Eberhart, D.E., Yi, H., Warren, S.T. and Hersch, S.M. (1997) Fragile X mental retardation protein: nucleocytoplasmic shuttling and association with somatodendritic ribosomes. J. Neurosci., 17, 1539–1547.
[Abstract/Free Full Text] -
Khandjian, E.W., Huot, M.E., Tremblay, S., Davidovic, L., Mazroui, R. and Bardoni, B. (2004) Biochemical evidence for the association of fragile X mental retardation protein with brain polyribosomal ribonucleoparticles. Proc. Natl Acad. Sci. USA, 101, 13357–13362.
[Abstract/Free Full Text] -
Stefani, G., Fraser, C.E., Darnell, J.C. and Darnell, R.B. (2004) Fragile X mental retardation protein is associated with translating polyribosomes in neuronal cells. J. Neurosci., 24, 7272–7276.
[Abstract/Free Full Text] -
De Diego Otero, Y., Severijnen, L.A., van Cappellen, G., Schrier, M., Oostra, B. and Willemsen, R. (2002) Transport of fragile X mental retardation protein via granules in neurites of PC12 cells. Mol. Cell. Biol., 22, 332–8341.
[Abstract/Free Full Text] -
Antar, L.N., Afroz, R., Dictemberg, J.B., Carroll, R.C. and Bassell, G.J. (2004) Metabotropic glutamate receptor activation regulates fragile X mental retardation protein and FMR1 mRNA localization differentially in dendrites and at synapses. J. Neurosci., 24, 2648–2655.
[Abstract/Free Full Text] - Jin, P., Alisch, R.S. and Warren, S.T. (2004) RNA and microRNAs in fragile X mental retardation. Nat. Cell Biol., 6, 1048–1053.[CrossRef][ISI][Medline]
- Darnell, J.C., Jensen, K.B., Jin, P., Brown, V., Warren, S.T. and Darnell, R.B. (2001) Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function. Cell, 107, 489–499.[CrossRef][ISI][Medline]
- Schaeffer, C., Bardoni, B., Mandel, J.L., Ehresmann, B., Ehresmann, C. and Moine, H. (2001) The fragile X mental retardation protein binds specifically to its mRNA via a purine quartet motif. EMBO J., 20, 4803–4813.[CrossRef][ISI][Medline]
- Darnell, J.C., Warren, S.T. and Darnell, R.B. (2004) The fragile X mental retardation protein, FMRP, recognizes G-quartets. Ment. Retard. Dev. Disabil. Res. Rev., 10, 49–52.[CrossRef][ISI][Medline]
-
Darnell, J.C., Fraser, C.E., Mostovetsky, O., Stefani, G., Jones, T.A., Eddy, S.R. and Darnell, R.B. (2005) Kissing complex RNAs mediate interaction between the fragile-X mental retardation protein KH2 domain and brain polyribosomes. Genes Dev., 19, 903–918.
[Abstract/Free Full Text] - Brown, V., Jin, P., Ceman, S., Darnell, J.C., O'Donnell, W.T., Tenenbaum, S.A., Jin, X., Feng, Y., Wilkinson, K.D., Keene, J.D., Darnell, R.B. and Warren, S.T. (2001) Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell, 107, 477–487.[CrossRef][ISI][Medline]
- Miyashiro, K.Y., Beckel-Mitchener, A., Purk, T.P., Becker, K.G., Barret, T., Liu, L., Carbonetto, S., Weiler, I.J., Greenough, W.T. and Eberwine, J. (2003) RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice. Neuron, 37, 417–431.[CrossRef][ISI][Medline]
-
Irwin, S.A., Galvez, R. and Greenough, W.T. (2000) Dendritic spine structural anomalies in fragile-X mental retardation syndrome. Cereb. Cortex, 10, 1038–1044.
[Abstract/Free Full Text] - Greenough, W.T., Klintsova,