Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1alpha), and other RDM1 isoforms. We show that RDM1alpha undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response.     

RBEL1 is a novel gene that encodes a nucleocytoplasmic Ras superfamily GTP-binding protein and is overexpressed in breast cancer

Rab family proteins are generally known as regulators of protein transport and trafficking. A number of Rab proteins have been implicated in cancer development and/or progression. Here we report the identification of a novel Rab-like protein, which we have named RBEL1 (Rab-like protein 1) for its higher similarity to the Rab subfamily members. We have characterized two isoforms of RBEL1 including the predominant RBEL1A and the less abundant RBEL1B that results from alternative splicing. Both isoforms harbor conserved N-terminal guanine trinucleotide phosphate (GTP) binding domains and, accordingly, are capable of binding to GTP. Both isoforms contain variable C termini and exhibit differential subcellular localization patterns. Unlike known Rabs that are mostly cytosolic, RBEL1B predominantly resides in the nucleus, whereas RBEL1A is localized primarily to the cytosol. Interestingly, a point mutation affecting RBEL1B GTP binding also alters the ability of mutant protein to accumulate in the nucleus, suggesting GTP binding potential to be important for RBEL1B nuclear localization. Our results also indicate that RBEL1A is overexpressed in about 67% of primary breast tumors. Thus, RBEL1A and RBEL1B are novel Rab-like proteins that localize in the nucleus and cytosol and may play an important role in breast tumorigenesis.     

Divergent N-terminal sequences target an inducible testis deubiquitinating enzyme to distinct subcellular structures

Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-gamma-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action.     

Identification, expression pattern, and subcellular location of human RIP isoforms

XRIP alpha was identified as an adapter protein involved in RAP nuclear import. Several homologs were reported in mammal EST analysis, but the expression pattern and genomic organization of hRIP isoforms were not clarified yet. We isolated nine isoforms of hRIP from a premade human fetal brain library. hRIP alpha is the longest isoform with 219 residues, containing a N-terminal arginine-rich basic region, followed by an acidic region and two C-terminal Zn finger-like structures. hRIP beta deletes one Zn-finger-like structure. Three hRIP alpha isoforms and four hRIP sigma isoforms express truncated proteins due to frame shift. hRIP gamma isoforms lost the C-terminal Zn-finger-like structure. hRIP delta isoforms only contain the N-terminal arginine-rich basic region and the core sequence of the acidic region. The genomic organization of hRIP was identified by bioinformatic analysis. hRIP, containing seven exons, is located at human chromosome 17p13. hRIP was expressed in all 16 detected human tissues with a similar pattern. All EGFP-hRIP fusion proteins were located at the nucleus in the HEK293 cell. The two-polar molecular structure of hRIP might be involved in the basic cell function, and plays a role in the alternative nuclear ingress.     

Investigation into the use of C- and N-terminal GFP fusion proteins for subcellular localization studies using reverse transfection microarrays

Reverse transfection microarrays were described recently as a high throughput method for studying gene function. We have investigated the use of this technology for determining the subcellular localization of proteins. Genes encoding 16 proteins with a variety of functions were placed in Gateway expression constructs with 3' or 5' green fluorescent protein (GFP) tags. These were then packaged in transfection reagent and spotted robotically onto a glass slide to form a reverse transfection array. HEK293T cells were grown over the surface of the array until confluent and GFP fluorescence visualized by confocal microscopy. All C-terminal fusion proteins localized to cellular compartments in accordance with previous studies and/or bioinformatic predictions. However, less than half of the N-terminal fusion proteins localized correctly. Of those that were not in concordance with the C-terminal tagged proteins, half did not exhibit expression and the remainder had differing subcellular localizations to the C-terminal fusion protein. This data indicates that N-terminal tagging with GFP adversely affects the protein localization in reverse transfection assays, whereas tagging with GFP at the C-terminal is generally better in preserving the localization of the native protein. We discuss these results in the context of developing high-throughput subcellular localization assays based on the reverse transfection array technology.     

Sequence requirement for the nucleolar localization of human I-mfa domain-containing protein (HIC p40)

The human I-mfa domain-containing protein (HIC) mRNA produces two protein isoforms, HIC p32 and p40, synthesized from alternative translational initiations. p32 translation is initiated from a standard AUG codon and p40 is an N-terminal extension of p32 generated from an upstream GUG codon. The two isoforms show different subcellular localization: p32 is distributed throughout the cytoplasm whereas p40 can be found both in the cytoplasm and the nucleolus. To investigate the possibility that p40 contains a nucleolus targeting sequence in its N-terminal region, COS cells were transfected with an eukaryotic expression vector coding for green fluorescent protein (GFP) fused to the p40 N terminus. The localization of this fusion protein in the nucleolus indicated that the N-terminal amino acids of p40 probably contain a nucleolar localization signal (NoLS). To find the structural motifs required for nucleolar localization of p40, deletion mutants were expressed in COS cells as fusion polypeptides with GFP. We defined a domain of 19 amino acids near the N terminus that contains an arginine-rich subdomain that conforms to other known NoLS. To demonstrate that this sequence is an authentic NoLS, the sequence was fused to GFP. This fusion protein was observed to migrate into the nucleolus. Taken together, our studies demonstrate that p40 contains a NoLS.     

Intracellular localization of RORalpha is isoform and cell line-dependent

The retinoid-related orphan receptor alpha (RORalpha) belongs to the nuclear receptor superfamily and comprises four isoforms generated by different promotor usage and alternative splicing. To better understand its function, the subcellular distribution of RORalpha was investigated. We could show that subcellular distribution of RORalpha is cell line and isoform-dependent. Isoform specific differences were mediated by the A/B domains which with the exception of RORalpha1 contain a signal that mediates cytoplasmic localization. The lack of this signal in RORalpha1 results in a complete nuclear localization and prevents cell membrane association observed for RORalpha2, 3, and 4. The region responsible for membrane association was identified as the C-terminal alpha-helix 12. Furthermore, the hinge region/ligand binding domain mediates nuclear localization. Our results show that isoform specific activity of RORalpha is not only regulated by different expression and DNA binding affinities but also by different subcellular distribution. Different access to the nucleus reveals an important mechanism regulating the activity of this constitutively active nuclear receptor.     

Characterization of four mammalian numb protein isoforms. Identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain.

Numb is a membrane-associated, phosphotyrosine binding (PTB) domain-containing protein that functions as an intrinsic determinant of cell fate during Drosophila development. We have identified four isoforms of mammalian Numb with predicted molecular masses of 65, 66, 71, and 72 kDa that are generated by alternative splicing of the Numb mRNA. The different isoforms result from the presence of two sequence inserts within the PTB domain and the central region of the protein. The endogenous expression pattern of these isoforms, examined using specific antisera, varied in different tissues and cell lines. In addition, differentiation of P19 cells with retinoic acid leads to the specific loss of expression of the 71- and 72-kDa Numb proteins, suggesting that the expression of certain forms of Numb protein is regulated in a cell type-specific manner. Expression of Numb proteins fused to green fluorescent protein revealed that the form of the PTB domain with the alternatively spliced insert constitutively associated with the plasma membrane in polarized Madin-Darby canine kidney cells. In contrast, the isoform without the insert was cytoplasmic, suggesting that different PTB domain isoforms may regulate the subcellular localization of Numb proteins. The membrane localization may be due, in part, to differential affinity for acidic phospholipids. The distinct expression and localization patterns of the different mammalian Numb isoforms suggest that they have distinct functional properties.     

The rice cold-responsive calcium-dependent protein kinase OsCPK17 is regulated by alternative splicing and post-translational modifications

Plant calcium-dependent protein kinases (CDPKs) are key proteins implicated in calcium-mediated signaling pathways of a wide range of biological events in the organism. The action of each particular CDPK is strictly regulated by many mechanisms in order to ensure an accurate signal translation and the activation of the adequate response processes. In this work, we investigated the regulation of a CDPK involved in rice cold stress response, OsCPK17, to better understand its mode of action. We identified two new alternative splicing (AS) mRNA forms of OsCPK17 encoding truncated versions of the protein, missing the CDPK activation domain. We analyzed the expression patterns of all AS variants in rice tissues and examined their subcellular localization in onion epidermal cells. The results indicate that the AS of OsCPK17 putatively originates truncated forms of the protein with distinct functions, and different subcellular and tissue distributions. Additionally, we addressed the regulation of OsCPK17 by post-translational modifications in several in vitro experiments. Our analysis indicated that OsCPK17 activity depends on its structural rearrangement induced by calcium binding, and that the protein can be autophosphorylated. The identified phosphorylation sites mostly populate the OsCPK17 N-terminal domain. Exceptions are phosphosites T107 and S136 in the kinase domain and S558 in the C-terminal domain. These phosphosites seem conserved in CDPKs and may reflect a common regulatory mechanism for this protein family.     

Conservation of RET proto-oncogene splicing variants and implications for RET isoform function

The RET proto-oncogene encodes a receptor tyrosine kinase required for development of the kidney and neural crest-derived cell types. Alternative splicing of the 3' exons of human RET results in three protein isoforms with distinct C-termini: RET9, RET51, and RET43. These RET isoforms show differential binding to downstream adapter molecules, suggesting they may have distinct signaling functions. We have characterized Ret 3' sequences in mouse and investigated alternative splicing of this region. We found that the organization of Ret 3' sequences is very similar to human RET. The mouse locus also has alternatively spliced C-terminal coding regions, and the sequences corresponding to RET9 and RET51 are highly conserved in both position and sequence with the human locus. Further, we compared the predicted C-terminal amino acids of RET9 and RET51 in seven vertebrate species, and found that they are well conserved. We have identified sequence encoding a putative ret43 isoform in mouse, however the predicted amino acid sequence showed low homology to human RET43. Our data suggest that RET isoforms are evolutionarily highly conserved over a broad range of species, which may indicate that each isoform has a distinct role in normal RET function.     

Subcellular translocation signals regulate Geminin activity during embryonic development

BACKGROUND INFORMATION: Geminin (Gem) is a protein with roles in regulating both the fidelity of DNA replication and cell fate during embryonic development. The distribution of Gem is predominantly nuclear in cells undergoing the cell cycle. Previous studies have demonstrated that Gem performs multiple activities in the nucleus and that regulation of Gem activation requires nuclear import in at least one context. In the present study, we defined structural and mechanistic features underlying subcellular localization of Gem and tested whether regulation of the subcellular localization of Gem has an impact on its activity in cell fate specification during embryonic development. RESULTS: We determined that nuclear localization of Gem is dependent on a bipartite NLS (nuclear localization signal) in the N-terminus of Xenopus Gem protein. This bipartite motif mapped to a Gem N-terminal region previously shown to regulate neural cell fate acquisition. Microinjection into Xenopus embryos demonstrated that import-deficient Gem was incapable of modulating ectodermal cell fate, but that this activity was rescued by fusion to a heterologous NLS. Cross-species comparison of Gem protein sequences revealed that the Xenopus bipartite signal is conserved in many non-mammalian vertebrates, but not in mammalian species assessed. Instead, we found that human Gem employs an alternative N-terminal motif to regulate the protein's nuclear localization. Finally, we found that additional mechanisms contributed to regulating the subcellular localization of Gem. These included a link to Crm1-dependent nuclear export and the observation that Cdt1, a protein in the pre-replication complex, could also mediate nuclear import of Gem. CONCLUSIONS: We have defined new structural and regulatory features of Gem, and showed that the activity of Gem in regulating cell fate, in addition to its cell-cycle-regulatory activity, requires control of its subcellular localization. Our data suggest that rather than being constitutively nuclear, Gem may undergo nucleocytoplasmic shuttling through several mechanisms involving distinct protein motifs. The use of multiple mechanisms for modulating Gem subcellular localization is congruent with observations that Gem levels and activity must be stringently controlled during cell-cycle progression and embryonic development.     

Multiple mechanisms regulate subcellular localization of human CDC6

CDC6 is a protein essential for DNA replication, the expression and abundance of which are cell cycle-regulated in Saccharomyces cerevisiae. We have demonstrated previously that the subcellular localization of the human CDC6 homolog, HsCDC6, is cell cycle-dependent: nuclear during G(1) phase and cytoplasmic during S phase. Here we demonstrate that endogenous HsCDC6 is phosphorylated during the G(1)/S transition. The N-terminal region contains putative cyclin-dependent kinase phosphorylation sites adjoining nuclear localization sequences (NLSs) and a cyclin-docking motif, whereas the C-terminal region contains a nuclear export signal (NES). In addition, we show that the observed regulated subcellular localization depends on phosphorylation status, NLS, and NES. When the four putative substrate sites (serines 45, 54, 74, and 106) for cyclin-dependent kinases are mutated to alanines, the resulting HsCDC6A4 protein is localized predominantly to the nucleus. This localization depends upon two functional NLSs, because expression of HsCDC6 containing mutations in the two putative NLSs results in predominantly cytoplasmic distribution. Furthermore, mutation of the four serines to phosphate-mimicking aspartates (HsCDC6D4) results in strictly cytoplasmic localization. This cytoplasmic localization depends upon the C-terminal NES. Together these results demonstrate that HsCDC6 is phosphorylated at the G(1)/S phase of the cell cycle and that the phosphorylation status determines the subcellular localization.     

Identification and characterization of novel splice variants of the human EPM2A gene mutated in Lafora progressive myoclonus epilepsy

The EPM2A gene, defective in the fatal neurodegenerative disorder Lafora disease (LD), is known to encode two distinct proteins by differential splicing; a phosphatase active cytoplasmic isoform and a phosphatase inactive nuclear isoform. We report here the identification of three novel EPM2A splice variants with potential to code for five distinct proteins in alternate reading frames. These novel isoforms, when ectopically expressed in cell lines, show distinct subcellular localization, interact with and serve as substrates of malin ubiquitin ligase-the second protein defective in LD. Two phosphatase active isoforms interact to form a heterodimeric complex that is inactive as a phosphatase in vitro, suggesting an antagonistic function for laforin isoforms if expressed endogenously in significant amounts in human tissues. Thus alternative splicing could possibly be one of the mechanisms by which EPM2A may regulate the cellular functions of the proteins it codes for.     

Identification of novel nuclear localization signals of Drosophila myeloid leukemia factor

Myeloid leukemia factor 1 (MLF1) was first identified as part of a leukemic fusion protein produced by a chromosomal translocation, and MLF family proteins are present in many animals. In mammalian cells, MLF1 has been described as mainly cytoplasmic, but in Drosophila, one of the dMLF isoforms (dMLFA) localized mainly in the nucleus while the other isoform (dMLFB), that appears to be produced by the alternative splicing, displays both nuclear and cytoplasmic localization. To investigate the difference in subcellular localization between MLF family members, we examined the subcellular localization of deletion mutants of dMLFA isoform. The analyses showed that the C-terminal 40 amino acid region of dMLFA is necessary and sufficient for nuclear localization. Based on amino acid sequences, we hypothesized that two nuclear localization signals (NLSs) are present within the region. Site-directed mutagenesis of critical residues within the two putative NLSs leads to loss of nuclear localization, suggesting that both NLS motifs are necessary for nuclear localization.