Biomedical subjects

Miroslav Dundr

Publications and source records attributed to Miroslav Dundr.

17 recordsLinked to original sources

PubMed2006

Nucleocytoplasmic shuttling of the retinoblastoma tumor suppressor protein via Cdk phosphorylation-dependent nuclear export.

The retinoblastoma (RB) tumor suppressor protein is a negative regulator of cell proliferation that is functionally inactivated in the majority of human tumors. Elevated Cdk activity via RB pathway mutations is observed in virtually every human cancer. Thus, Cdk inhibitors have tremendous promise as anticancer agents although detailed mechanistic knowledge of their effects on RB function is needed to harness their full potential. Here, we illustrate a novel function for Cdks in regulating the subcellular localization of RB. We present evidence of significant cytoplasmic mislocalization of ordinarily nuclear RB in cells harboring Cdk4 mutations. Our findings uncover a novel mechanism to circumvent RB-mediated growth suppression by altered nucleocytoplasmic trafficking via the Exportin1 pathway. Cytoplasmically mislocalized RB could be efficiently confined to the nucleus by inhibiting the Exportin1 pathway, reducing Cdk activity, or mutating the Cdk-dependent phosphorylation sites in RB that result in loss of RB-Exportin1 association. Thus RB-mediated tumor suppression can be subverted by phosphorylation-dependent enhancement of nuclear export. These results support the notion that tumor cells can modulate the protein transport machinery thereby making the protein transport process a viable therapeutic target.

Active Transport, Cell Nucleus↗

PubMed2006

Human T-cell leukemia virus type I p30 nuclear/nucleolar retention is mediated through interactions with RNA and a constituent of the 60 S ribosomal subunit.

Human T-cell leukemia virus type I is the etiological agent of adult T-cell leukemia/lymphoma, an aggressive and fatal lymphoproliferative malignancy. The virus has evolved strategies to escape immune clearance by remaining latent in most infected cells in vivo. We demonstrated previously that virally encoded p30 protein is a potent post-transcriptional inhibitor of virus replication (Nicot, C., Dundr, M., Johnson, J. M., Fullen, J. R., Alonzo, N., Fukumoto, R., Princler, G. L., Derse, D., Misteli, T., and Franchini, G. (2004) Nat. Med. 10, 197-201). p30 is unable to shuttle out of the nucleus in heterokaryon assays, suggesting the existence of specific retention signals. Because suppression of virus replication relies on nuclear retention of the tax/rex mRNA by p30, determining the retention features of p30 will offer hints to break latency in infected cells and insights into new therapeutic approaches. In this study, we used live cell imaging technologies to study the kinetics of p30 and to delineate its retention signals and their function in virus replication. Notably, this is the first study to identify p30 nucleolar retention domains. Using mutants of p30 that localized in different cellular compartments, we show that post-transcriptional control of virus replication by p30 occurs in the nucleoplasm. We further demonstrate that p30 nuclear/nucleolar retention is dependent upon de novo RNA transcripts and interactions with components of the ribosomal machinery.

Amino Acid Sequence↗

PubMed2006

Conserved zinc fingers mediate multiple functions of ZFP100, a U7snRNP associated protein.

Formation of the 3' end of replication-dependent histone mRNAs is most robust during S phase and is mediated by both the stem-loop binding protein (SLBP) and the U7 snRNP. We previously identified a 100-kDa zinc finger protein (ZFP100) as a component of U7 snRNP that interacts with the SLBP/pre-mRNA complex. Here, we show that myc- or GFP-tagged ZFP100 overexpressed after transfection is concentrated in Cajal bodies (CBs), and unlike components of the spliceosomal snRNPs, photobleaching experiments demonstrate that ZFP100 is stably associated with CBs. Of the 18 zinc fingers contained within ZFP100, the region encompassing fingers 2-6 is sufficient to maintain CB localization. Zn fingers 5-10 are required for maximal binding of ZFP100 to a 20-amino-acid region of Lsm11, a U7 snRNP core protein. Expression of ZFP100 stimulates histone mRNA processing in vivo, assayed by activation of a reporter gene that encodes a GFP mRNA ending in a histone 3' end. Importantly, the domain that is required for CB localization and Lsm11 binding is also sufficient to stimulate histone pre-mRNA processing in vivo. Comparisons with other mammalian ZFP100 orthologs show that the central Zn fingers sufficient for in vivo activity are most highly conserved, whereas the number and sequence of the Zn fingers in the N- and C-terminal domains vary.

Amino Acid Sequence↗

PubMed2006

The road much traveled: trafficking in the cell nucleus.

Trafficking of RNA molecules and proteins within the cell nucleus is central to genome function. Recent work has revealed the nature of RNA and protein motion within the nucleus and across the nuclear membrane. These studies have given insight into how molecules find their destinations within the nucleus and have uncovered some of the structural properties of the nuclear microenvironment. Control of RNA and protein trafficking is now emerging as a physiological regulatory mechanism in gene expression and nuclear function.

Active Transport, Cell Nucleus↗

PubMed2006

FBPs are calibrated molecular tools to adjust gene expression.

The three far-upstream element (FUSE) binding protein (FBP) family members have been ascribed different functions in gene regulation. They were therefore examined with various biochemical, molecular biological, and cell biological tests to evaluate whether their sequence differences reflect functional customization or neutral changes at unselected residues. Each FBP displayed a characteristic profile of intrinsic transcription activation and repression, binding with protein partners, and subcellular trafficking. Although some differences, such as weakened FBP3 nuclear localization, were predictable from primary sequence differences, the unexpected failure of FBP3 to bind the FBP-interacting repressor (FIR) was traced to seemingly conservative substitutions within a small patch of an N-terminal alpha-helix. The transactivation strength and the FIR-binding strength of the FBPs were in the opposite order. Despite their distinguishing features and differential activities, the FBPs traffic to shared subnuclear sites and regulate many common target genes, including c-myc. Though a variety of functions have been attributed to the FBPs, based upon their panel of shared and unique features, we propose that they constitute a molecular regulatory kit that tunes the expression of shared targets through a common mechanism.

Amino Acid Sequence↗

PubMed2006

Potential roles for ubiquitin and the proteasome during ribosome biogenesis.

We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.

Cell Line↗

PubMed2005

The moving parts of the nucleolus.

The cell nucleolus is the subnuclear body in which ribosomal subunits are assembled, and it is also the location of several processes not related to ribosome biogenesis. Recent studies have revealed that nucleolar components move about in a variety of ways. One class of movement is associated with ribosome assembly, which is a vectorial process originating at the sites of transcription in the border region between the fibrillar center and the dense fibrillar component. The nascent preribosomal particles move outwardly to become the granular components where further maturation takes place. These particles continue their travel through the nucleoplasm for eventual export to the cytoplasm to become functional ribosomes. In a second kind of motion, many nucleolar components rapidly exchange with the nucleoplasm. Thirdly, nucleolar components engage in very complex movements when the nucleolus disassembles at the beginning of mitosis and then reassembles at the end of mitosis. Finally, many other cellular and viral macromolecules, which are not related to ribosome assembly, also pass through or are retained by the nucleolus. These are involved in nontraditional roles of the nucleolus, including regulation of tumor suppressor and oncogene activities, signal recognition particle assembly, modification of small RNAs, control of aging, and modulating telomerase function.

Animals↗

PubMed2004

Condensed mitotic chromatin is accessible to transcription factors and chromatin structural proteins.

During mitosis, chromosomes are highly condensed and transcription is silenced globally. One explanation for transcriptional repression is the reduced accessibility of transcription factors. To directly test this hypothesis and to investigate the dynamics of mitotic chromatin, we evaluate the exchange kinetics of several RNA polymerase I transcription factors and nucleosome components on mitotic chromatin in living cells. We demonstrate that these factors rapidly exchange on and off ribosomal DNA clusters and that the kinetics of exchange varies at different phases of mitosis. In addition, the nucleosome component H1c-GFP also shows phase-specific exchange rates with mitotic chromatin. Furthermore, core histone components exchange at detectable levels that are elevated during anaphase and telophase, temporally correlating with H3-K9 acetylation and recruitment of RNA polymerase II before the onset of bulk RNA synthesis at mitotic exit. Our findings indicate that mitotic chromosomes in general and ribosomal genes in particular, although highly condensed, are accessible to transcription factors and chromatin proteins. The phase-specific exchanges of nucleosome components during late mitotic phases are consistent with an emerging model of replication independent core histone replacement.

Acetylation↗

PubMed2004

In vivo kinetics of Cajal body components.

Cajal bodies (CBs) are subnuclear domains implicated in small nuclear ribonucleoprotein (snRNP) biogenesis. In most cell types, CBs coincide with nuclear gems, which contain the survival of motor neurons (SMN) complex, an essential snRNP assembly factor. Here, we analyze the exchange kinetics of multiple components of CBs and gems in living cells using photobleaching microscopy. We demonstrate differences in dissociation kinetics of CB constituents and relate them to their functions. Coilin and SMN complex members exhibit relatively long CB residence times, whereas components of snRNPs, small nucleolar RNPs, and factors shared with the nucleolus have significantly shorter residence times. Comparison of the dissociation kinetics of these shared proteins from either the nucleolus or the CB suggests the existence of compartment-specific retention mechanisms. The dynamic properties of several CB components do not depend on their interaction with coilin because their dissociation kinetics are unaltered in residual nuclear bodies of coilin knockout cells. Photobleaching and fluorescence resonance energy transfer experiments demonstrate that coilin and SMN can interact within CBs, but their interaction is not the major determinant of their residence times. These results suggest that CBs and gems are kinetically independent structures.

Animals↗

PubMed2004

HTLV-1-encoded p30II is a post-transcriptional negative regulator of viral replication.

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) persists despite a vigorous virus-specific host immune response, and causes adult T-cell leukemia and lymphoma in approximately 2% of infected individuals. Here we report that HTLV-1 has evolved a genetic function to restrict its own replication by a novel post-transcriptional mechanism. The HTLV-1-encoded p30(II) is a nuclear-resident protein that binds to, and retains in the nucleus, the doubly spliced mRNA encoding the Tax and Rex proteins. Because Tex and Rex are positive regulators of viral gene expression, their inhibition by p30(II) reduces virion production. p30(II) inhibits virus expression by reducing Tax and Rex protein expression.

Cell Line↗

PubMed2004

Repression of human T-cell leukemia virus type 1 and type 2 replication by a viral mRNA-encoded posttranscriptional regulator.

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are complex retroviruses that persist in the host, eventually causing leukemia and neurological disease in a small percentage of infected individuals. In addition to structural and enzymatic proteins, HTLV encodes regulatory (Tax and Rex) and accessory (open reading frame I and II) proteins. The viral Tax and Rex proteins positively regulate virus production. Tax activates viral and cellular transcription to promote T-cell growth and, ultimately, malignant transformation. Rex acts posttranscriptionally to facilitate cytoplasmic expression of viral mRNAs that encode the structural and enzymatic gene products, thus positively controlling virion expression. Here, we report that both HTLV-1 and HTLV-2 have evolved accessory genes to encode proteins that act as negative regulators of both Tax and Rex. HTLV-1 p30(II) and the related HTLV-2 p28(II) inhibit virion production by binding to and retaining tax/rex mRNA in the nucleus. Reduction of viral replication in a cell carrying the provirus may allow escape from immune recognition in an infected individual. These data are consistent with the critical role of these proteins in viral persistence and pathogenesis in animal models of HTLV-1 and HTLV-2 infection.

Cell Line↗

PubMed2004

Nonphosphorylated human La antigen interacts with nucleolin at nucleolar sites involved in rRNA biogenesis.

La is a RNA-binding protein implicated in multiple pathways related to the production of tRNAs, ribosomal proteins, and other components of the translational machinery (D. J. Kenan and J. D. Keene, Nat. Struct. Mol. Biol. 11:303-305, 2004). While most La is phosphorylated and resides in the nucleoplasm, a fraction is in the nucleolus, the site of ribosome production, although the determinants of this localization are incompletely known. In addition to its conserved N-terminal domain, human La harbors a C-terminal domain that contains an atypical RNA recognition motif and a short basic motif (SBM) adjacent to phosphoserine-366. We report that nonphosphorylated La (npLa) is concentrated in nucleolar sites that correspond to the dense fibrillar component that harbors nascent pol I transcripts as well as fibrillarin and nucleolin, which function in early phases of rRNA maturation. Affinity purification and native immunoprecipitation of La and fluorescence resonance energy transfer in the nucleolus reveal close association with nucleolin. Moreover, La lacking the SBM does not localize to nucleoli. Lastly, La exhibits SBM-dependent, phosphorylation-sensitive interaction with nucleolin in a yeast two-hybrid assay. The data suggest that interaction with nucleolin is, at least in part, responsible for nucleolar accumulation of La and that npLa may be involved in ribosome biogenesis.

Amino Acid Motifs↗

PubMed2002

A kinetic framework for a mammalian RNA polymerase in vivo.

We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.

Animals↗

PubMed2002

Rrn3 phosphorylation is a regulatory checkpoint for ribosome biogenesis.

Cycloheximide inhibits ribosomal DNA (rDNA) transcription in vivo. The mouse homologue of yeast Rrn3, a polymerase-associated transcription initiation factor, can complement extracts from cycloheximide-treated mammalian cells. Cycloheximide inhibits the phosphorylation of Rrn3 and causes its dissociation from RNA polymerase I. Rrn3 interacts with the rpa43 subunit of RNA polymerase I, and treatment with cycloheximide inhibits the formation of a Rrn3.rpa43 complex in vivo. Rrn3 produced in Sf9 cells but not in bacteria interacts with rpa43 in vitro, and such interaction is dependent upon the phosphorylation state of Rrn3. Significantly, neither dephosphorylated Rrn3 nor Rrn3 produced in Escherichia coli can restore transcription by extracts from cycloheximide-treated cells. These results suggest that the phosphorylation state of Rrn3 regulates rDNA transcription by determining the steady-state concentration of the Rrn3.RNA polymerase I complex within the nucleolus.

3T3 Cells↗

PubMed2002

Nucleolomics: an inventory of the nucleolus.

In the January 8 issue of Current Biology, two papers from the Lamond and Mann laboratories describe the largest proteomics analysis to date of a cellular compartment, the nucleolus. As a byproduct of this tour de force, a novel nuclear compartment, the paraspeckles, was identified.

Animals↗

PubMed2002

Aberrant nuclear trafficking of La protein leads to disordered processing of associated precursor tRNAs.

Eukaryotic precursor tRNAs undergo extensive processing prior to nuclear export. The first of multiple factors to interact with pre-tRNAs and other nascent transcripts is the La protein. Using suppressor and wild-type tRNAs, we demonstrate that the normal distribution of cellular end-processed and spliced tRNA species is disordered by La proteins that lack a conserved nuclear retention element. Fission yeast or human La mutants that lack this element enter nuclei and stabilize nascent pre-tRNA but are aberrantly exported and fail to support normal tRNA processing. Instead, anomalous 5' and 3' end-containing, spliced tRNAs accumulate, complexed with the mutant La protein. Thus, appropriate nuclear trafficking by La affects the normal order of pre-tRNA processing.

Active Transport, Cell Nucleus↗

PubMedDate not supplied

Quantitation of GFP-fusion proteins in single living cells.

The green fluorescent protein (GFP) has revolutionized cell biology. The ability to observe genetically encoded fluorescently tagged fusion proteins in intact cells has made virtually any biological process amenable to investigation in living cells. However, most in vivo imaging studies are qualitative and little information about the number of fluorescently labeled molecules observed in a cell or a cellular structure is available. This deficiency severely limits the interpretation of imaging experiments and it impedes the application of in vivo imaging methods for biophysical purposes. Here we describe a simple method for the quantitative determination of the number of GFP-tagged molecules in cellular structures in single living cells. The method is based on the use of rotavirus-like particles containing a known number of GFP molecules as an internal calibration standard during in vivo imaging. We have applied this method to estimate in single living cells the number of fluorescent transcription factor molecules on RNA polymerase I and polymerase II genes. In addition, we have estimated the number of molecules for several proteins in subnuclear compartments and in exocytic vesicles. VLP-GFP calibration is a simple, convenient, rapid, and noninvasive method for routine quantification of GFP-labeled molecules in single, living cells.

Animals↗