Distribution profile and in vivo RNA association of cytoplasmic AU-rich sequence binding proteins in various mammalian cells: effect of the organizational state of cellular architecture

With the aid of sequential detergent fractionation and label transfer techniques, in this study we monitored the distribution of cytoplasmic AU-rich sequence binding proteins (AUBP) in different cell types. We show here that cells of various origin display diverse AUBP profiles when the presence and abundance of AUBPs were compared in two major cytoplasmic compartments, the non-ionic detergent-soluble and -insoluble fractions. We also demonstrate that different RNA probes derived from AU-rich 3'-UTR elements of various cytokine and proto-oncogene mRNAs detect distinct AUBPs within the same cell. When the in vivo association of these proteins with AU-rich RNA was assessed using the combination of in vivo UV-crosslinking and label transfer methods, we found that detachment of monolayer cells by trypsinization or disruption of the microfilament network by Cytochalasin treatment prior to UV light exposure, led to selective changes in both the cytoplasmic distribution and in vivo RNA association of many AUBPs. These data indicate that the organizational state of cytoarchitecture may influence processes involved in AUBP-mediated mRNA metabolism.     

Microfilament-dependent modulation of cytoplasmic protein binding to TNFalpha mRNA AU-rich instability element in human lymphoid cells

Cytoplasmic proteins with binding capability to AU-rich instability determinant sequences (ARE) of tumour necrosis factor alpha (TNFalpha) mRNA 3' untranslated region (3'UTR) were assessed in human lymphoid cells. In vitro label transfer experiments using wild type as well as mutant sequences in which the 70 nucleotide-long AUUUA pentamer-containing portion of the 3'UTR had been deleted conferred binding specificity to five major activities of 22/25-, 38/40-, 50-, 60- and 80-kDa proteins in cytoplasmic extracts of peripheral blood mononuclear cells (PBMCs). Cytochalasin-induced disarrangement of the F-actin-based microfilament system led to a Triton X-100-insoluble to soluble redistribution of these binding activities. No such changes were observed in Jurkat tumour cells. Combination of in vivo UV-crosslinking and in vitro label transfer experiments revealed considerable differences in RNA association between proteins of the same cell type as well as between proteins of identical molecular weight (Mw) derived from either PBMCs or Jurkat cells. Our findings may explain some aspects of differential regulation of interleukin 2 (IL-2) and TNFalpha mRNA stability upon microfilament disruption in human PBMCs observed in an earlier study. These results also suggest that the physical state of cytoplasmic structural environment might contribute to important regulatory processes regarding key elements of eukaryotic mRNA metabolism, such as modulation of stability. Finally, these data highlight the possibility that the often observed disorganization of the cytoskeleton in tumour cells may partly be responsible for the maintenance of the neoplastic state, a phenomenon that potentially involves ARE-AUBP interactions.     

Lymphocyte HEV adhesion variants differ in the expression of multiple gene sequences

Lymphocyte adhesion to high endothelial venule cells in lymphoid organs of mice is mediated by several cell-surface glycoproteins, one of which, gp90MEL-14, is detected by the MEL-14 monoclonal antibody (mAb). The MEL-14 mAb was used to select two variants of the EL4 cell line, EL4MEL-14-hi and EL4-MEL-14-lo, that have disparate cell surface expression of this adhesion receptor. A cDNA library constructed from EL4MEL-14-hi mRNA was enriched for sequences present at higher levels in EL4MEL-14-hi cells than EL4MEL-14-lo cells. Quantitative analysis of candidate differential clones by RNA probe protection methods identified five clones whose steady-state mRNA levels were increased in the EL4MEL-14-hi cells. One of these clones, DIFF6, is derived from an RNA whose expression level is higher in several cell lines producing high amounts of MEL-14-reactive gp90, and absent or present at lower levels in several cell lines expressing low levels of this glycoprotein. However, DIFF6 does not encode gp90MEL-14. The nucleotide sequence of this clone predicts a relatively hydrophilic protein characteristic of a cytoplasmic or nuclear protein. Present experiments indicate that expression of gp90MEL-14, a cell-surface-adhesion receptor molecule, may be coregulated with additional cytoplasmic or nuclear factors.     

Identification of novel argonaute-associated proteins

RNA silencing processes are guided by small RNAs known as siRNAs and microRNAs (miRNAs) . They reside in ribonucleoprotein complexes, which guide the cleavage of complementary mRNAs or affect stability and translation of partial complementary mRNAs . Argonaute (Ago) proteins are at the heart of silencing effector complexes and bind the single-stranded siRNA and miRNA . Our biochemical analysis revealed that Ago2 is present in a pre-miRNA processing complex that is able to transfer the miRNA into a target-mRNA cleaving complex. To gain insight into the function and composition of RNA silencing complexes, we purified Ago1- and Ago2-containing complexes from human cells. Several known Ago1- and/or Ago2-associated proteins including Dicer were identified, but also two novel factors, the putative RNA helicase MOV10, and the RNA recognition motif (RRM)-containing protein TNRC6B/KIAA1093. The new proteins localize, similar to Ago proteins, to mRNA-degrading cytoplasmic P bodies, and they are functionally required to mediate miRNA-guided mRNA cleavage.     

MEL-28, a novel nuclear-envelope and kinetochore protein essential for zygotic nuclear-envelope assembly in C. elegans

The nuclear envelope (NE) of eukaryotic cells separates nucleoplasm from cytoplasm, mediates nucleo-cytoplasmic transport, and contributes to the control of gene expression. The NE consists of three major components: the nuclear membranes, the nuclear pore complexes (NPCs), and the nuclear lamina. The list of identified NE proteins has increased considerably during recent years but is most likely not complete. In most eukaryotes, the NE breaks down and is then reassembled during mitosis. The assembly of NPCs and the association and fusion of nuclear membranes around decondensing chromosomes are tightly coordinated processes. Here, we report the identification and characterization of MEL-28, a large protein essential for the assembly of a functional NE in C. elegans embryos. RNAi depletion or genetic mutation of mel-28 severely impairs nuclear morphology and leads to abnormal distribution of both integral NE proteins and NPCs. The structural defects of the NE were associated with functional defects and lack of nuclear exclusion of soluble proteins. MEL-28 localizes to NPCs during interphase, to kinetochores in early to middle mitosis then is widely distributed on chromatin late in mitosis. We show that MEL-28 is an early-assembling, stable NE component required for all aspects of NE assembly.     

Combined application of in vivo UV-crosslinking and in vitro label transfer in the examination of AU-rich sequence binding protein - RNA interactions.

A combination of in vivo UV light-induced crosslinking of nucleic acids to proteins and in vitro label transfer assay was applied to investigate specific interactions between AU-rich sequences (ARE) in the 3′ UTR of lymphokine mRNAs and cytoplasmic AU-rich sequence element binding proteins (AUBP) in normal human lymphoblasts and MLA 144 gibbon lymphoid tumor cells. We demonstrate that a pool of cytoplasmic AUBP can be effectively crosslinked to RNA in vivo , suggesting a close association of these proteins with ARE sequences in the cytoplasm. We also show that the UV-crosslinked AUBP pool is markedly reduced in malignantly transformed MLA 144 cells compared with normal lymphoblasts, indicating weaker interaction between lymphokine ARE and AUBP in these tumor cells. Similar differences in AUBP-RNA associations were found between the membrane-bound polysomal subfractions of the two cell types where most of the AUBP activity was localized. We suggest that the decreased AUBP-mRNA association in MLA 144 cells might reflect a process concerned with disturbances of mRNA metabolism in the neoplastic phenotype.     

Phenotype and localization of thymocytes expressing the homing receptor-associated antigen MEL-14: arguments for the view that most mature thymocytes are located in the medulla

The monoclonal antibody MEL-14 recognizes a lymphocyte surface structure (the MEL-14 antigen) involved in migration of lymphocytes into lymph nodes. Its use as a maturation marker for T cells within the thymus led to the view that a small population (1 to 2%) of MEL-14high thymocytes located in the inner cortex represented fully mature cells about to exit as thymus emigrants. The medulla, in this view, contained only the phenotypically mature but MEL-14low cells, and was not the source of thymus emigrants. The data we present, derived from flow-cytometric analysis of suspension-stained CBA mouse thymocytes, is not in accordance with this view. A high proportion (approximately 20%) of thymocytes express relatively high levels of MEL-14; these include some immature Ly-2- L3T4- and nonmature Ly-2+ L3T4+ thymocytes. Among the 12 to 14% thymocytes of mature phenotype (PNAlow or H-2Khigh or Ly-2+ L3T4- and Ly-2- L3T4+), more than half express relatively high levels of MEL-14. The mature phenotype and MEL-14moderate-to-high cells (8% of thymocytes) appear too numerous to account for the few percent MEL-14high cells seen in the cortex in frozen sections, and the mature phenotype but MEL-14low cells (2 to 3% of thymocytes) too few to fill the medulla; however, both together account numerically for the medullary population. By section staining, the medulla contains Ly-2- L3T4+ and Ly-2+ L3T4- cells in a characteristic 2:1 ratio; by suspension staining this ratio agrees with that of the total mature phenotype population, but not with that of the MEL-14low subset previously claimed to represent medullary cells. Another paradox is apparent when suspension staining and section staining are compared: suspension staining reveals that many mature phenotype cells coexpress high levels of both MEL-14 and H-2K, yet section staining reveals H-2Khigh cells in the medulla but not in the inner cortex, and reveals scattered MEL-14high cells throughout the cortex but not in the medulla. We suggest that section staining for MEL-14 fails to locate the mature cells that stain for MEL-14 in suspension; the few MEL-14high cells localized in both the inner and the outer cortex on section staining are predominantly immature Ly-2- L3T4- and nonmature Ly-2+ L3T4+ thymocytes; the majority of thymocytes of mature phenotype, whether MEL-14high or MEL-14low on suspension staining, are of medullary location; the medulla is the most likely immediate source of thymic emigrants.     

Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis.

The helicase-like 1a and polymerase-like 2a proteins of brome mosaic virus (BMV) are required for viral RNA replication in vivo, are present in membrane-bound viral RNA polymerase extracts, and share conservation with the many other members of the alphavirus-like superfamily. To better understand BMV RNA replication and BMV-host interactions, we used confocal microscopy and double-label immunofluorescence to determine and compare the sites of 1a, 2a, and nascent viral RNA accumulation in BMV-infected barley protoplasts. 1a and 2a showed nearly complete colocalization throughout infection, accumulating in defined cytoplasmic spots usually adjacent to or surrounding the nucleus. These spots grew throughout infection and by 16 h postinoculation often assumed a vesicle-like appearance. The BMV RNA replication complex incorporated 5-bromouridine 5'-triphosphate into RNA in vitro and in vivo, allowing immunofluorescent detection of nascent RNA. The cytoplasmic sites of BMV-specific RNA synthesis coincided with the sites of 1a and 2a accumulation, and at the resolution of confocal microscopy, all sites of 1a and 2a accumulation were sites of BMV RNA synthesis. Double-label immunofluorescence detection of selected subcellular markers and 1a or 2a showed that BMV replication complexes were tightly associated with markers for the endoplasmic reticulum but not the medial Golgi or later compartments of the cellular secretory pathway. Defining this association of BMV RNA replication complexes with endoplasmic reticulum markers should assist in identifying and characterizing host factors involved in BMV RNA replication.     

Identification of nucleolin as an AU-rich element binding protein involved in bcl-2 mRNA stabilization

bcl-2 mRNA contains an AU-rich element (ARE) that functions in regulating bcl-2 stability. Our earlier studies indicated that taxol- or okadaic acid-induced bcl-2 mRNA destabilization in HL-60 cells is associated with decreased binding of trans-acting factors to the ARE. To identify factors that play a role in the regulation of bcl-2 mRNA stability, bcl-2 ARE-binding proteins were purified from HL-60 cells. Three polypeptides of 100, 70, and 32 kDa were isolated from a bcl-2 ARE affinity matrix. Matrix-assisted laser desorption ionization mass spectroscopy analysis identified these proteins as full-length nucleolin and proteolytic fragments of nucleolin. RNA gel shifts assays indicated that recombinant nucleolin (residues 284-707) binds specifically to bcl-2 ARE RNA. In addition, recombinant nucleolin decreases the rate of decay of mRNA in HL-60 cell extracts in an ARE-dependent manner. Taxol or okadaic acid treatment of HL-60 cells results in proteolysis of nucleolin in a similar time frame as drug-induced bcl-2 mRNA down-regulation. These findings suggest that nucleolin functions as a bcl-2-stabilizing factor and that taxol and okadaic acid treatment induces apoptosis in HL-60 cells through a process that involves down-regulation of nucleolin and destabilization of bcl-2 mRNA.     

Gonadotropin-induced differentiation of granulosa cells is associated with the co-ordinated regulation of cytoskeletal proteins involved in cell-contact formation

The gonadotropin-induced differentiation of granulosa cells in culture was studied, with particular attention being given to the organization and expression of cytoskeletal proteins involved in the formation of cell contacts, as well as to progesterone production. Gonadotropin-treated granulosa cells formed clusters of spherical cells containing few vinculin-containing focal contacts, exhibited a diffuse distribution of actin, and had few adherens junctions but more gap junctions than cells grown without the hormone. In gonadotropin-treated cells, the levels of synthesis of the cytoskeletal proteins, vinculin, alpha-actinin, and actin, were dramatically reduced, but the synthesis of the tubulins and vimentin was unaffected. Decreased levels of synthesis of these cytoskeletal proteins were also observed in an in vitro translation assay using poly(A)+ RNA from gonadotropin-treated cells. The hybridization of cytoplasmic RNA with cloned actin and vimentin cDNAs revealed a marked decrease in actin-RNA levels, but no change in vimentin-RNA levels in these cells. Such alterations in cytoskeletal-protein expression were also observed in cells treated with compounds that cause elevated cellular cAMP levels by acting at a stage beyond gonadotropin receptor stimulation. Furthermore, by keeping the cells in a spherical configuration in suspension culture, or by treating the cells with cytochalasin B, similar changes in the synthesis of these cytoskeletal proteins were observed. During this process, there was a concomitant increased in the production of progesterone (although to a much lesser extent in suspension culture) that occurred in parallel with the appearance of large mitochondria with lamellar-tubular cristae and a well-developed smooth endoplasmic reticulum, these features being characteristic of granulosa-lutein cells in vivo. Our results suggest that changes in cell shape and contact, together with the regulation of cytoskeletal elements that determine cellular morphogenesis, are part of the gonadotropin-controlled differentiation program in granulosa cells and may also occur during the maturation of these cells in vivo.     

RNA chaperone activity of protein components of human Ro RNPs

Ro ribonucleoprotein (RNP) complexes are composed of one molecule of a small noncoding cytoplasmic RNA, termed Y RNA, and the two proteins Ro60 and La. Additional proteins such as hnRNP I, hnRNP K, or nucleolin have recently been shown to be associated with subpopulations of Y RNAs. Ro RNPs appear to be localized in the cytoplasm of all higher eukaryotic cells but their functions have remained elusive. To shed light on possible functions of Ro RNPs, we tested protein components of these complexes for RNA chaperone properties employing two in vitro chaperone assays and additionally an in vivo chaperone assay. In these assays the splicing activity of a group I intron is measured. La showed pronounced RNA chaperone activity in the cis-splicing assay in vitro and also in vivo, whereas no activity was seen in the trans-splicing assay in vitro. Both hnRNP I and hnRNP K exhibited strong chaperone activity in the two in vitro assays, however, proved to be cytotoxic in the in vivo assay. No chaperone activity was observed for Ro60 in vitro and a moderate activity was detected in vivo. In vitro chaperone activities of La and hnRNP I were completely inhibited upon binding of Y RNA. Taken together, these data suggest that the Ro RNP components La, hnRNP K, and hnRNP I possess RNA chaperone activity, while Ro60-Y RNA complexes might function as transporters, bringing other Y RNA binding proteins to their specific targets.     

The cytoplasmic domain of the cellulose-synthesizing complex in vascular plants

The cytoplasmic domain of the rosette terminal complex has been imaged in situ in patches of plasma membrane isolated from tobacco BY-2 protoplasts. By partially extracting the plasma membrane lipids, cellulose microfibrils were observed through the plasma membrane. Rosette terminal complexes were identified on the basis of their association with the ends of these cellulose microfibrils. The cytoplasmic domain of the rosette terminal complex has been shown to be hexagonal in shape and has been measured to be 45-50 nm in diameter and 30-35 nm tall. These findings demonstrate that the terminal complex does indeed have a substantial cytoplasmic component, and that the hexagonal array observed in the lipid bilayer by freeze fracture is actually only a small part of the overall complex. These findings will allow better modeling of the terminal complex and may facilitate predictions of how many proteins are associated with the rosette terminal complex in vivo.     

Memory B cells express a phenotype consistent with migratory competence after secondary but not short-term primary immunization

The cell surface phenotype of dinitrophenol (DNP)-specific memory B cells, defined by their capacity to transfer IgG responses into syngeneic irradiated recipients, was assessed using two markers of relevance to lymphocyte migratory properties: (i) peanut agglutinin, which binds to terminal galactosyl residues expressed at high levels by several nonmigrating lymphocyte subsets and, among lymph node B cells, is highly specific for germinal center cells; and (ii) MEL-14, a monoclonal antibody specific for lymphocyte surface receptors required for migration from the blood into peripheral lymph nodes. At various times after primary or secondary immunization with DNP-keyhole limpet hemocyananin (KLH), lymph node B cells were separated by fluorescence-activated cell sorting on the basis of staining with PNA and/or MEL-14, and the presence of B-memory cells in each fraction was assessed by adoptive transfer with antigen (DNP-KLH) and helper T cells. One week after immunization, most of the memory sorted in the PNAhi population, confirming a previous report by R. F. Coico, B. S. Bhogal, and G. J. Thorbecke (J. Immunol. 131, 2254, 1983) that early memory B cells or their precursors are contained within the germinal center cell population, a population which is known to be MEL-14- and migratory-incompetent. Six weeks after primary stimulation, however, the bulk of memory cells, unlike germinal center cells, were MEL-14hi. After secondary immunization, memory was still predominantly MEL-14+ and PNAlo, although in some experiments adoptive responses were transferred by all sorted fractions. The results are consistent with the hypothesis that antigen-specific B cells initially undergo local (sessile) differentiation and proliferation in germinal centers, where they develop the capacity for adoptive transfer of antigen-specific secondary responses, but that with continued development their long-lived memory-containing progeny express a phenotype permitting their reentry into the recirculating lymphocyte pool.