Changes in the subnuclear distribution of two RNA metabolism-related proteins can be detected in nuclear scaffold or matrix prepared by different techniques

The nuclear scaffold or matrix is a mainly proteinaceous structure thought to act as a nucleoskeleton determining the higher order organization of eukaryotic chromatin. These structures are prepared from isolated nuclei by a series of extraction steps involving the use of ionic detergents or high salt, and restriction enzymes or non-specific nucleases to remove chromatin and other loosely bound components. Since these treatments are harsh and unphysiological, the question remains open as to whether or not these structures, isolated in vitro, correspond to a nucleoskeleton existing in vivo. Recently, it has been demonstrated that the majority of nuclear matrix proteins are involved in RNA metabolism. In this study we have employed a morphological approach involving the use of confocal laser scanning microscopy and indirect immunofluorescence techniques to analyze whether two widely employed methods to prepare the nuclear scaffold or matrix can maintain the spatial distribution of two polypeptides involved in RNA metabolism, i.e., a 105-kDa component of spliceosomes and a ribonucleoprotein antigen. We demonstrate that the localization of these polypeptides changes, in some cases dramatically, in the final nucleoskeletal structures when compared with intact cells. Only when isolated nuclei were stabilized in vitro with the cross-linking agent sodium tetrathionate (NaTT) prior to extraction with 2 M NaCl and DNase I digestion, were the immunofluorescent patterns displayed by the nuclear matrix indistinguishable from those detected in intact cells. These results emphasize the usefulness of NaTT in studying putative nucleoskeletal structures, but also show that the methods currently employed to prepare the nuclear scaffold or matrix may create in vitro artifacts.     

Deposition of storage proteins

Plants store amino acids for longer periods in the form of specific storage proteins. These are deposited in seeds, in root and shoot tubers, in the wood and bark parenchyma of trees and in other vegetative organs. Storage proteins are protected against uncontrolled premature degradation by several mechanisms. The major one is to deposit the storage proteins into specialized membrane-bounded storage organelles, called protein bodies (PB). In the endosperm cells of maize and rice prolamins are sequestered into PBs which are derived from the endoplasmic reticulum (ER). Globulins, the typical storage proteins of dicotyledonous plants, and prolamins of some cereals are transported from the ER through the Golgi apparatus and then into protein storage vacuoles (PSV) which later become transformed into PBs. Sorting and targeting of storage proteins begins during their biosynthesis on membrane-bound polysomes where an N-terminal signal peptide mediates their segregation into the lumen of the ER. After cleavage of the signal peptide, the polypeptides are glycosylated and folded with the aid of chaperones. While still in the ER, disulfide bridges are formed which stabilize the structure and several polypeptides are joined to form an oligomer which has the proper conformation to be either deposited in ER-derived PB or to be further transferred to the PSV. At the trans-Golgi cisternae transport vesicles are sequestered which carry the storage proteins to the PSV. Several storage proteins are also processed after arriving in the PSVs in order to generate a conformation that is capable of final deposition. Some storage protein precursors have short N- or C-terminal targeting sequences which are detached after arrival in the PSV. Others have been shown to have internal sequence regions which could act as targeting information. In some cases positive targeting information is known to mediate sorting into the PSV whereas in other cases aggregation and membrane association seem to be major sorting mechanisms.     

Molecular basis for the differential subcellular localization of the 38- and 39-kilodalton structural proteins of Borna disease virus

Borna disease virus (BDV) is a nonsegmented negative-strand (NNS) RNA virus that is unusual because it replicates in the nucleus. The most abundant viral protein in infected cells is a 38/39-kDa doublet that is presumed to represent the nucleocapsid. Infectious particles also contain high levels of this protein, accounting for at least 50% of the viral proteins. The two forms of the protein differ by an additional 13 amino acids that are present at the amino terminus of the 39-kDa form and missing from the 38-kDa form. To examine whether this difference in amino acid content affects the localization of this protein in cells, the 39- and 38-kDa proteins were expressed in transfected cells. The 39-kDa form was concentrated in the nucleus, whereas the 38-kDa form was found in both the nucleus and cytoplasm. Inspection of the extra 13 amino acids present in the 39-kDa form revealed a sequence (Pro-Lys-Arg-Arg) that is very similar to the nuclear localization signals (in both sequence homology and amino-terminal location) of the VP1 proteins of simian virus 40 and polyomavirus. Primer extension analysis of total RNA from infected cells suggests that there are two mRNA species encoding the two forms of the nucleocapsid protein. In infected cells, the 39-kDa form is expressed at about twofold-higher levels than the 38-kDa form at both the RNA and protein levels. The novel nuclear localization of the 39-kDa nucleocapsid-like protein suggests that this form of the protein is targeted to the nucleus, the site for viral RNA replication, and that it may associate with genomic RNA.     

Comparative analysis of the virion structural proteins of 3 nuclear polyhedrosis viruses

The composition of structural proteins of virions of nuclear polyhedrosis viruses (NPV) of Barathra brassicae L., Lymantria dispor L. and Aporia crataegi L. was studied by polyacrylamide gel-SDS electrophoresis (PAGE-SDS). The object of the study included fractions of virion bundles in the envelope. All the baculoviruses under study were shown to have in virion structural proteins 23-24 polypeptides with molecular weights ranging from 12500 to 95000 daltons. Alongside with polypeptides of similar molecular weight, each baculovirus was shown to have a specific set of structural proteins allowing them to be readily identified. Specific distribution of major polypeptides of the virions was found. All the protein fractions were readily reproducible in repeated analyses with the exception of two minor polypeptides of NPV of Barathra brassicae L. The study showed that the composition of structural proteins of virions may be an important criterion for identification of baculoviruses under identical electrophoresis conditions.     

Identification of three novel unique proteins in seed oil bodies of sesame

Plant seeds store triacylglycerols in discrete organelles called oil bodies. An oil body preserves a matrix of triacylglycerols surrounded by a monolayer of phospholipids embedded with abundant structural proteins termed oleosins and probably some uninvestigated minor proteins of higher molecular mass. Three polypeptides of 27, 37, and 39 kDa (temporarily denominated as Sop1, Sop2, and Sop3) were regularly co-purified with seed oil bodies of sesame. Comparison of amino acid composition indicated that they were substantially less hydrophobic than the known oleosins, and thus should not be aggregated multimers of oleosins. The results of immuno-recognition to sesame proteins extracted from subcellular fractions of mature seeds, various tissues, and oil bodies purified from different stages of seed formation revealed that these three polypeptides were unique proteins gathered in oil bodies, accompanying oleosins and triacylglycerols, during the active assembly of the organelles in maturing seeds. Both in vivo and in intro, immunofluorescence labeling using secondary antibodies conjugated with FITC (fluorescein isothiocyanate) confirmed the localization of these three polypeptides in oil bodies.     

The germinal vesicle nucleus of Xenopus laevis oocytes as a selective storage receptacle for proteins

The amorphous nucleoplasm of the germinal vesicle nucleus of Xenopus laevis oocytes has been selectively extracted under conditions which leave the nuclear formed elements morphologically intact. The nucleoplasm contains about 97% of the total nuclear proteins and on SDS-polyacrylamide gels some 68 polypeptides can be distinguished. On the basis of solubility differences, the nucleoplasmic proteins can be classified into two categories. The first consists of soluble or easily solubilized proteins which comprise about 34 polypeptides making up 87% of the nucleoplasm. A few of these proteins show electrophoretic mobilities similar to those of soluble proteins of the cytoplasm, but most are unique to the nucleus. The residual 13% of the nucleoplasmic proteins are tightly bound to a nucleoplasmic gel and can be extracted only by solubilizing the gel. The solubility characteristics of the proteinaceous gel suggest a complex held together by salt, nonpolar, hydrogen, and possibly disulfide bonding. Some 34 polypeptides can be distinguished in this gel fraction, including prominent and highly enriched polypeptides of about 115,000 and 46,000 daltons. The relatively soluble fraction of the nucleoplasm does not contain informofers and contains little or no nucleic acid. Evidence is presented that if histones are present in the germinal vesicle, they can comprise no more than about 8% of the total protein. The possibility is discussed that the unique polypeptides of the nucleoplasm may be sequestered there by selective adsorption to or in the nuclear gel.     

Intranuclear distribution of HMGI/Y proteins. An immunocytochemical study

The intranuclear distribution of HMGI/Y proteins was analyzed by immunofluorescent staining in several cell lines using a polyclonal antibody that stained a fibrogranular network. In actively growing 3T3 fibroblasts, HMGI/Y proteins were mainly localized to heterochromatin masses, whereas in quiescent cells they were more diffusely distributed. Double labeling experiments showed a co-localization of HMGI/Y with DNA topoisomerase IIalpha. These results are in agreement with previously published biochemical data and indicate a possible involvement of HMGI/Y proteins in several nuclear functions, including chromatin organization and gene expression.     

Common nuclear matrix proteins in rat tissues

Nuclear matrix proteins have been defined as insoluble residual proteins resulting from treatment of isolated nuclei with nucleases, detergents and high ionic strength buffers. They are considered as in part representing the proteins constituting the three-dimensional framework of the interphase nucleus. Though cell-specific nuclear matrix proteins have been differentiated from ubiquitously occurring (common) nuclear matrix proteins, the number and types of common nuclear matrix proteins have not yet been unequivocally established. In the present study nuclear matrix proteins were prepared from isolated nuclei of rat kidney, liver, lung, spleen and testes. The matrix proteins were separated by two-dimensional (2-D) electrophoresis and silver stained. Then the spot patterns were compared by computer-assisted image analysis. Composite images were derived for nuclear matrix proteins of individual tissues. Finding between 396-483 spots per tissue, a total of 964 individual spots were registered. Of these, 102 were common nuclear matrix proteins, as appearing in each of the tissue-characteristic images. The apparent molecular mass and pI data may serve for further identification of these nuclear proteins.     

Nuclear export of proteins and RNAs

Our understanding of protein export from the nucleus to the cytoplasm has been advanced recently by the discovery of active, signal-mediated export pathways. Nuclear export signals have been identified in several proteins, the majority of which are RNA-binding proteins. Nuclear export of RNA molecules is likely to be driven by protein-based nuclear export signals.     

Regions of the haptoglobin 5' flanking gene sequence show different binding affinities to nuclear matrix proteins during the acute phase response

The effect of the acute phase response on the affinity of binding between nuclear matrix proteins and the rat haptoglobin (Hp) gene region was examined. Nuclear matrices isolated from acute phase livers were enriched with the 5' Hp gene flanking region (-705/+159), but not with the spliced, protein-coding sequence. Reassociation experiments with isolated nuclear protein matrix spheres and end-labeled fragments I (-146/+156), II (-146/-541), and III (-541/-705) revealed that the matrix proteins displayed an increased binding potential during the acute phase response for all of the examined regions, this being most pronounced for fragment II. BAL 31 digestion of fragment II showed that the sequence element that was responsible for the increased association with nuclear matrix proteins during the acute phase response was a tract of 38 adenine bases. The DNA region established stable associations with nuclear lamin B (67 kDa, pI 5.7) in the controls, and with lamins A (69 kDa, pI 7.0), B, isoforms of lamin C (62 kDa, pI 6.55-6.95), and a 55-kDa (pI 5.9) polypeptide during the acute phase response. Sequence ABC (-165/-56), which overlaps fragments I and II and represents the Hp cis-acting element, did not bind to the non-histone nuclear matrix proteins.     

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.     

Involvement of the Xenopus laevis Ro60 autoantigen in the alternative interaction of La and CNBP proteins with the 5'UTR of L4 ribosomal protein mRNA

In vertebrates the synthesis of ribosomal proteins is co-ordinately regulated at the translational level. The 5'-untranslated region (5'UTR) of this class of mRNAs contains conserved regions that are necessary and sufficient for translational regulation. Recently, we found that two proteins, the Xenopus laevis La autoantigen and the cellular nucleic acid binding protein (CNBP), are able to bind in vitro a pyrimidine tract at the 5' end and a downstream region, respectively. These regions are considered the common cis-acting elements of translational regulation. It was previously observed that the binding of both these putative trans-acting factors to their RNA sequences is assisted by a protease-sensitive factor(s) that dissociates from the complex after its formation. Here we provide evidence that the requirement for an ancillary factor assisting La binding to the pyrimidine tract of ribosomal protein mRNAs is typical of this RNA, and secondly that it may involve an RNA recognition motif of the La protein not clearly characterized previously. We also show that the Ro60 autoantigen is involved in the common factor activity necessary for the binding of La and CNBP proteins to their respective sequences. In addition, our findings suggest that an RNA also participates in this process. We show that CNBP can multimerise and that it binds to the 5'UTR as a dimer. Both La and CNBP compete for the interaction with the factor, and their binding to the 5'UTR is mutually exclusive. Our results from the binding analysis of mutations in the 5'UTR, which are known to disrupt the translational control in vivo, suggest a model in which the protein interactions and the 5'UTR RNA structure may co-operate in regulating the translational fate of ribosomal protein mRNAs.     

Localization of human cytomegalovirus structural proteins to the nuclear matrix of infected human fibroblasts

The intranuclear assembly of herpesvirus subviral particles remains an incompletely understood process. Previous studies have described the nuclear localization of capsid and tegument proteins as well as intranuclear tegumentation of capsid-like particles. The temporally and spatially regulated replication of viral DNA suggests that assembly may also be regulated by compartmentalization of structural proteins. We have investigated the intranuclear location of several structural and nonstructural proteins of human cytomegalovirus (HCMV). Tegument components including pp65 (ppUL83) and ppUL69 and capsid components including the major capsid protein (pUL86) and the small capsid protein (pUL48/49) were retained within the nuclear matrix (NM), whereas the immediate-early regulatory proteins IE-1 and IE-2 were present in the soluble nuclear fraction. The association of pp65 with the NM resisted washes with 1 M guanidine hydrochloride, and direct binding to the NM could be demonstrated by far-Western blotting. Furthermore, pp65 exhibited accumulation along the nuclear periphery and in far-Western analysis bound to proteins which comigrated with proteins of the size of nuclear lamins. A direct interaction between pp65 and lamins was demonstrated by coprecipitation of lamins in immune complexes containing pp65. Together, our findings provide evidence that major virion structural proteins localized to a nuclear compartment, the NM, during permissive infection of human fibroblasts.