Association of NASP with HSP90 in mouse spermatogenic cells: stimulation of ATPase activity and transport of linker histones into nuclei

NASP (nuclear autoantigenic sperm protein) is a linker histone-binding protein found in all dividing cells that is regulated by the cell cycle (Richardson, R. T., Batova, I. N., Widgren, E. E., Zheng, L. X., Whitfield, M., Marzluff, W. F., and O'Rand, M. G. (2000) J. Biol. Chem. 275, 30378-30386), and in the nucleus linker histones not bound to DNA are bound to NASP (Alekseev, O. M., Bencic, D. C., Richardson R. T., Widgren E. E., and O'Rand, M. G. (2003) J. Biol. Chem. 278, 8846-8852). In mouse spermatogenic cells tNASP binds the testis-specific linker histone H1t. Utilizing a cross-linker, 3,3'-dithiobissulfosuccinimidyl propionate, and mass spectrometry, we have identified HSP90 as a testis/embryo form of NASP (tNASP)-binding partner. In vitro assays demonstrate that the association of tNASP with HSP90 stimulated the ATPase activity of HSP90 and increased the binding of H1t to tNASP. HSP90 and tNASP are present in both nuclear and cytoplasmic fractions of mouse spermatogenic cells; however, HSP90 bound to NASP only in the cytoplasm. In vitro nuclear import assays on permeabilized HeLa cells demonstrate that tNASP, in the absence of any other cytoplasmic factors, transports linker histones into the nucleus in an energy and nuclear localization signal-dependent manner. Consequently we hypothesize that in the cytoplasm linker histones are bound to a complex containing NASP and HSP90 whose ATPase activity is stimulated by binding NASP. NASP-H1 is subsequently released from the complex and translocates to the nucleus where the H1 is released for binding to the DNA.     

Characterization of the histone H1-binding protein, NASP, as a cell cycle-regulated somatic protein

Nuclear autoantigenic sperm protein (NASP), initially described as a highly autoimmunogenic testis and sperm-specific protein, is a histone-binding protein that is a homologue of the N1/N2 gene expressed in oocytes of Xenopus laevis. Here, we report a somatic form of NASP (sNASP) present in all mitotic cells examined, including mouse embryonic cells and several mouse and human tissue culture cell lines. Affinity chromatography and histone isolation demonstrate that NASP from myeloma cells is complexed only with H1, linker histones. Somatic NASP is a shorter version of testicular NASP (tNASP) with two deletions in the coding region arising from alternative splicing and differs from tNASP in its 5' untranslated regions. We examined the relationship between NASP mRNA expression and the cell cycle and report that in cultures of synchronized mouse 3T3 cells and HeLa cells sNASP mRNA levels increase during S-phase and decline in G(2), concomitant with histone mRNA levels. NASP protein levels remain stable in these cells but become undetectable in confluent cultures of nondividing CV-1 cells and in nonmitotic cells in various body tissues. Expression of sNASP mRNA is regulated during the cell cycle and, consistent with a role as a histone transport protein, NASP mRNA expression parallels histone mRNA expression.     

Overexpression of the Linker histone-binding protein tNASP affects progression through the cell cycle

NASP is an H1 histone-binding protein that is cell cycle-regulated and occurs in two major forms: tNASP, found in gametes, embryonic cells, and transformed cells; and sNASP, found in all rapidly dividing somatic cells (Richardson, R. T., Batova, I. N., Widgren, E. E., Zheng, L. X., Whitfield, M., Marzluff, W. F., and O'Rand, M. G. (2000) J. Biol. Chem. 275, 30378-30386). When full-length tNASP fused to green fluorescent protein (GFP) is transiently transfected into HeLa cells, it is efficiently transported into the nucleus within 2 h after translation in the cytoplasm, whereas the NASP nuclear localization signal (NLS) deletion mutant (NASP-DeltaNLS-GFP) is retained in the cytoplasm. In HeLa cells synchronized by a double thymidine block and transiently transfected to overexpress full-length tNASP or NASP-DeltaNLS, progression through the G(1)/S border is delayed. Cells transiently transfected to overexpress the histone-binding site (HBS) deletion mutant (NASP-DeltaHBS) or sNASP were not delayed in progression through the G(1)/S border. By using a DNA supercoiling assay, in vitro binding data demonstrate that H1 histone-tNASP complexes can transfer H1 histones to DNA, whereas NASP-DeltaHBS cannot. Measurement of NASP mobility in the nucleus by fluorescence recovery after photobleaching indicates that NASP mobility is virtually identical to that reported for H1 histones. These data suggest that NASP-H1 complexes exist in the nucleus and that tNASP can influence cell cycle progression through the G(1)/S border through mediation of DNA-H1 histone binding.     

Analysis of gene expression profiles in HeLa cells in response to overexpression or siRNA-mediated depletion of NASP

Background  

NASP (Nuclear Autoantigenic Sperm Protein) is a linker histone chaperone required for normal cell division. Changes in NASP expression significantly affect cell growth and development; loss of gene function results in embryonic lethality. However, the mechanism by which NASP exerts its effects in the cell cycle is not understood. To understand the pathways and networks that may involve NASP function, we evaluated gene expression in HeLa cells in which NASP was either overexpressed or depleted by siRNA.     

Features generated for computational splice-site prediction correspond to functional elements

Background  

Accurate selection of splice sites during the splicing of precursors to messenger RNA requires both relatively well-characterized signals at the splice sites and auxiliary signals in the adjacent exons and introns. We previously described a feature generation algorithm (FGA) that is capable of achieving high classification accuracy on human 3' splice sites. In this paper, we extend the splice-site prediction to 5' splice sites and explore the generated features for biologically meaningful splicing signals.     

The H3 chaperone function of NASP is conserved in Arabidopsis

Histones are abundant cellular proteins but, if not incorporated into chromatin, they are usually bound by histone chaperones. Here, we identify Arabidopsis NASP as a chaperone for histones H3.1 and H3.3. NASP interacts in vitro with monomeric H3.1 and H3.3 as well as with histone H3.1–H4 and H3.3–H4 dimers. However, NASP does not bind to monomeric H4. NASP shifts the equilibrium between histone dimers and tetramers towards tetramers but does not interact with tetramers in vitro. Arabidopsis NASP promotes [H3–H4]₂ tetrasome formation, possibly by providing preassembled histone tetramers. However, NASP does not promote disassembly of in vitro preassembled tetrasomes. In contrast to its mammalian homolog, Arabidopsis NASP is a predominantly nuclear protein. In vivo, NASP binds mainly monomeric H3.1 and H3.3. Pulldown experiments indicated that NASP may also interact with the histone chaperone MSI1 and a HSC70 heat shock protein.     

A computational survey of candidate exonic splicing enhancer motifs in the model plant <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

Algorithmic approaches to splice site prediction have relied mainly on the consensus patterns found at the boundaries between protein coding and non-coding regions. However exonic splicing enhancers have been shown to enhance the utilization of nearby splice sites.     

Resolving deconvolution ambiguity in gene alternative splicing

Background  

For many gene structures it is impossible to resolve intensity data uniquely to establish abundances of splice variants. This was empirically noted by Wang et al. in which it was called a "degeneracy problem". The ambiguity results from an ill-posed problem where additional information is needed in order to obtain an unique answer in splice variant deconvolution.     

Conserved intron positions in ancient protein modules

Background  

The timing of the origin of introns is of crucial importance for an understanding of early genome architecture. The Exon theory of genes proposed a role for introns in the formation of multi-exon proteins by exon shuffling and predicts the presence of conserved splice sites in ancient genes. In this study, large-scale analysis of potential conserved splice sites was performed using an intron-exon database (ExInt) derived from GenBank.     

Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro

Background  

Incorporation of exon 11 of the insulin receptor gene is both developmentally and hormonally-regulated. Previously, we have shown the presence of enhancer and silencer elements that modulate the incorporation of the small 36-nucleotide exon. In this study, we investigated the role of inherent splice site strength in the alternative splicing decision and whether recognition of the splice sites is the major determinant of exon incorporation.     

A comprehensive survey of human polymorphisms at conserved splice dinucleotides and its evolutionary relationship with alternative splicing

Background  

Alternative splicing (AS) is a key molecular process that endows biological functions with diversity and complexity. Generally, functional redundancy leads to the generation of new functions through relaxation of selective pressure in evolution, as exemplified by duplicated genes. It is also known that alternatively spliced exons (ASEs) are subject to relaxed selective pressure. Within consensus sequences at the splice junctions, the most conserved sites are dinucleotides at both ends of introns (splice dinucleotides). However, a small number of single nucleotide polymorphisms (SNPs) occur at splice dinucleotides. An intriguing question relating to the evolution of AS diversity is whether mutations at splice dinucleotides are maintained as polymorphisms and produce diversity in splice patterns within the human population. We therefore surveyed validated SNPs in the database dbSNP located at splice dinucleotides of all human genes that are defined by the H-Invitational Database.     

Cancer characterization and feature set extraction by discriminative margin clustering

Background  

A central challenge in the molecular diagnosis and treatment of cancer is to define a set of molecular features that, taken together, distinguish a given cancer, or type of cancer, from all normal cells and tissues.     

A method of precise mRNA/DNA homology-based gene structure prediction

Background  

Accurate and automatic gene finding and structural prediction is a common problem in bioinformatics, and applications need to be capable of handling non-canonical splice sites, micro-exons and partial gene structure predictions that span across several genomic clones.     

Tenascin-C enhances pancreatic cancer cell growth and motility and affects cell adhesion through activation of the integrin pathway

Background

Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells.

Methods

Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition.

Results

Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt.

Conclusion

TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and might therefore play a role in PDAC spreading and metastasis in vivo.     

Selection against tandem splice sites affecting structured protein regions

Background  

Alternative selection of splice sites in tandem donors and acceptors is a major mode of alternative splicing. Here, we analyzed whether in-frame tandem sites leading to subtle mRNA insertions/deletions of 3, 6, or 9 nucleotides are under natural selection.     

Increased large conductance calcium-activated potassium (BK) channel expression accompanied by STREX variant downregulation in the developing mouse CNS

Background  

Large conductance calcium- and voltage activated potassium (BK) channels are important determinants of neuronal excitability through effects on action potential duration, frequency and synaptic efficacy. The pore- forming subunits are encoded by a single gene, KCNMA1, which undergoes extensive alternative pre mRNA splicing. Different splice variants can confer distinct properties on BK channels. For example, insertion of the 58 amino acid stress-regulated exon (STREX) insert, that is conserved throughout vertebrate evolution, encodes channels with distinct calcium sensitivity and regulation by diverse signalling pathways compared to the insertless (ZERO) variant. Thus, expression of distinct splice variants may allow cells to differentially shape their electrical properties during development. However, whether differential splicing of BK channel variants occurs during development of the mammalian CNS has not been examined.