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Torben Heick Jensen

Publications and source records attributed to Torben Heick Jensen.

15 recordsLinked to original sources
PubMed2007

Identification and characterization of GFAPkappa, a novel glial fibrillary acidic protein isoform.

Glial fibrillary acidic protein (GFAP) is the principal component of the intermediary filaments in mature astrocytes of the central nervous system (CNS). The protein consists of three domains: the head, the coiled-coil, and the tail. Here, we describe the isolation of an evolutionary conserved novel GFAP isoform, GFAPkappa, produced by alternative splicing and polyadenylation of the 3'-region of the human GFAP pre-mRNA. As a consequence, the resulting human GFAPkappa protein harbors a nonconserved C-terminal tail sequence distinct from the tails of GFAPalpha, the predominant GFAP isoform, and GFAPepsilon, an isoform which also results from alternative splicing. The head and coiled-coil rod domains are identical between the three GFAP isoforms. Interestingly, GFAPkappa is incapable of forming homomeric filaments, and increasing GFAPkappa expression levels causes a collapse of intermediate filaments formed by GFAPalpha. In searching for a biological relevance of GFAPkappa, we noticed that mRNA expression levels of GFAPalpha, GFAPepsilon, and GFAPkappa are gradually increased during development of the embryonic pig brain. However, whereas the GFAPalpha/GFAPepsilon ratio is constant, the GFAPkappa/GFAPepsilon ratio decreases during brain development. Furthermore, in glioblastoma tumors, an increased GFAPkappa/GFAPepsilon ratio is detected. Our results suggest that the relative expression level of the GFAPkappa isoform could modulate the properties of GFAP intermediate filaments and perhaps thereby influencing the motility of GFAP positive astrocytes and progenitor cells within the CNS.

Alternative Splicing↗
PubMed2006

A novel plasmid-based microarray screen identifies suppressors of rrp6Delta in Saccharomyces cerevisiae.

Genetic screens in Saccharomyces cerevisiae provide novel information about interacting genes and pathways. We screened for high-copy-number suppressors of a strain with the gene encoding the nuclear exosome component Rrp6p deleted, with either a traditional plate screen for suppressors of rrp6Delta temperature sensitivity or a novel microarray enhancer/suppressor screening (MES) strategy. MES combines DNA microarray technology with high-copy-number plasmid expression in liquid media. The plate screen and MES identified overlapping, but also different, suppressor genes. Only MES identified the novel mRNP protein Nab6p and the tRNA transporter Los1p, which could not have been identified in a traditional plate screen; both genes are toxic when overexpressed in rrp6Delta strains at 37 degrees C. Nab6p binds poly(A)+ RNA, and the functions of Nab6p and Los1p suggest that mRNA metabolism and/or protein synthesis are growth rate limiting in rrp6Delta strains. Microarray analyses of gene expression in rrp6Delta strains and a number of suppressor strains support this hypothesis.

Down-Regulation↗
PubMed2006

Structure of the nuclear exosome component Rrp6p reveals an interplay between the active site and the HRDC domain.

The multisubunit eukaryotic exosome is an essential RNA processing and degradation machine. In its nuclear form, the exosome associates with the auxiliary factor Rrp6p, which participates in both RNA processing and degradation reactions. The crystal structure of Saccharomyces cerevisiae Rrp6p displays a conserved RNase D core with a flanking HRDC (helicase and RNase D C-terminal) domain in an unusual conformation shown to be important for the processing function of the enzyme. Complexes with AMP and UMP, the products of the RNA degradation process, reveal how the protein specifically recognizes ribonucleotides and their bases. Finally, in vivo mutational studies show the importance of the domain contacts for the processing function of Rrp6p and highlight fundamental differences between the protein and its prokaryotic RNase D counterparts.

Binding Sites↗
PubMed2006

The role of the Brr5/Ysh1 C-terminal domain and its homolog Syc1 in mRNA 3'-end processing in Saccharomyces cerevisiae.

The cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae is thought to provide the catalytic activities of the mRNA 3'-end processing machinery, which include endonucleolytic cleavage at the poly(A) site, followed by synthesis of an adenosine polymer onto the new 3'-end by the CPF subunit Pap1. Because of similarity to other nucleases in the metallo-beta-lactamase family, the Brr5/Ysh1 subunit has been proposed to be the endonuclease. The C-terminal domain of Brr5 lies outside of beta-lactamase homology, and its function has not been elucidated. We show here that this region of Brr5 is necessary for cell viability and mRNA 3'-end processing. It is highly homologous to another CPF subunit, Syc1. Syc1 is not essential, but its removal improves the growth of other processing mutants at restrictive temperatures and restores in vitro processing activity to cleavage/ polyadenylation-defective brr5-1 extract. Our findings suggest that Syc1, by mimicking the essential Brr5 C-terminus, serves as a negative regulator of mRNA 3'-end formation.

Amino Acid Sequence↗
PubMed2006

A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis.

Nuclear-cytoplasmic partitioning and traffic between cytoplasmic and nuclear compartments are fundamental processes in eukaryotic cells. Nuclear pore complexes mediate transport of proteins, RNAs and ribonucleoprotein particles in and out of the nucleus. Here we present positional cloning of a plant nucleoporin gene, Nup133, essential for a symbiotic signal transduction pathway shared by Rhizobium bacteria and mycorrhizal fungi. Mutation of Nup133 results in a temperature sensitive nodulation deficient phenotype and absence of mycorrhizal colonization. Root nodules developing with reduced frequency at permissive temperatures are ineffective and electron microscopy show that Rhizobium bacteria are not released from infection threads. Measurement of ion fluxes using a calcium-sensitive dye show that Nup133 is required for the Ca2+ spiking normally detectable within minutes after application of purified rhizobial Nod-factor signal molecules to root hairs. Localization of NUP133 in the nuclear envelope of root cells and root hair cells shown with enhanced yellow fluorescent protein fusion proteins suggests a novel role for NUP133 nucleoporins in a rapid nuclear-cytoplasmic communication after host-plant recognition of symbiotic microbes. Our results identify a component of an intriguing signal process requiring interaction at the cell plasma membrane and at intracellular nuclear and plastid organelle-membranes to induce a second messenger.

Alleles↗
PubMed2005

Dramatically improved RNA in situ hybridization signals using LNA-modified probes.

In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)(+) RNA accumulation within the nucleus/ nucleolus of wild-type cells. LNA-based probes should be readily applicable to a diverse array of cells and tissue samples.

DNA-Directed DNA Polymerase↗
PubMed2005

Reviving the exosome.

In the nucleus of eukaryotic cells, RNA decay in the 3'-5' direction is carried out by a complex of exonucleases called the exosome. Surprisingly, the purified exosome shows only weak activity in vitro. Two papers in this issue of Cell, by LaCava et al. (2005) and Wyers et al. (2005), and a third report by Vanacova et al. (2005) help to solve this riddle by characterizing a new RNA decay-activating complex containing a poly(A) polymerase. In addition, they identify new unconventional RNA polymerase II transcription units.

Cell Nucleus↗
PubMed2005

A link between transcription and mRNP quality in Saccharomyces cerevisiae.

The transcription machinery plays a direct role in the assembly of messenger ribonucleoprotein particles (mRNPs), contributing to the loading of proteins onto nascent transcripts. Such mRNP biogenesis is linked to the THO complex that operates at the boundary between transcription and nuclear export. Early mRNP assembly events are subject to surveillance by the nuclear exosome that retains, and degrades, aberrant mRNAs. A yeast strain that carries deletions of Hpr1p and Rrp6p of the THO complex and the nuclear exosome, respectively, grows slowly, possibly due to lack of Rrp6p-dependent mRNA quality control. We selected a number of spontaneous revertant strains from the slow growth phenotype. Interestingly, transcriptional activity was reduced in all revertants. These data corroborate earlier findings that transcriptional down regulation improves growth of cells containing a crippled mRNP formation/surveillance system and illustrates the impact transcriptional activity can have on early mRNP assembly.

Models, Biological↗
PubMed2005

Formation of export-competent mRNP: escaping nuclear destruction.

In eukaryotic cells, primary transcripts are processed and bound by proteins before export to the cytoplasm. Nuclear production of export-competent messenger ribonucleoprotein particles (mRNPs) is a complicated process, and mRNP biogenic events that function sub-optimally are rapidly attacked by surveillance leading to degradation of the mRNA. Export of nuclear mRNAs is therefore constantly challenged by the opposing force of mRNA retention and decay. This balance ensures that only 'perfect' transcripts persist, and that non-functional and potentially deleterious transcripts are removed early in their biogenesis. Thus, eukaryotic systems of mRNP quality control can be viewed as simple Darwinian principles operating at the molecular level.

Active Transport, Cell Nucleus↗
PubMed2004

Modulation of transcription affects mRNP quality.

Cotranscriptional loading of proteins onto nascent transcripts contributes to the formation of messenger ribonucleoprotein particles (mRNPs) competent for nuclear export. The transcription machinery is believed to play a pivotal role in mRNP assembly, which is at least partially linked to the function of the THO/TREX complex and the mRNA termination/polyadenylation apparatus. Here we demonstrate a prominent role for the rate of transcription in the production of export-competent mRNPs. We show that a transcription-defective allele of the Rad3p helicase, a component of the TFIIH transcription initiation factor, suppresses several phenotypes associated with defective mRNA processing and export. Strikingly, the effects of compromised Rad3p activity can be phenocopied by a transcription elongation drug as well as by other mutations affecting transcription. Our results suggest that efficient mRNP assembly is under a kinetic control that is influenced by the rate of transcription.

Adenosine Triphosphatases↗
PubMed2003

Early formation of mRNP: license for export or quality control?

Eukaryotic mRNA is processed by enzymes and packaged with proteins within nuclei to generate functional messenger ribonucleoprotein (mRNP) particles. Processing and packaging factors can interact with mRNA cotranscriptionally to form an early mRNP. Erroneous mRNP formation leads to nuclear retention and degradation of the mRNA. It therefore appears that one function of cotranscriptional mRNP assembly is to discard aberrant mRNPs early in their biogenesis. Cotranscriptional mRNP assembly may also enable the transcription machinery to respond to improper mRNP formation.

Cell Nucleus↗
PubMed2003

Co-transcriptional monitoring of mRNP formation.

In eukaryotes, newly manufactured mRNA is subject to strict quality control. If certain standards are not met, offending messages are retained in the nucleus and degraded by the exosome complex. New data suggest that exosome-mediated processes are intimately coupled to the transcription machinery, which makes the mRNA in the first place.

Animals↗
PubMed2003

Localization of nuclear retained mRNAs in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated in retaining RNAs in these foci; on deletion of the exosome component Rrp6p, the RNA is released. To determine the exact nuclear location of retained as well as released mRNAs, we have used mRNA export mutant strains to analyze the spatial relationship between newly synthesized heat shock mRNA, the chromosomal site of transcription, and known S. cerevisiae nuclear structures such as the nucleolus and the nucleolar body. Our results show that retained SSA4 RNA localizes to an area in close proximity to the SSA4 locus. On deletion of Rrp6p and release from the genomic locus, heat shock mRNAs produced in the rat7-1 strain colocalize predominantly with nucleolar antigens. Bulk poly(A)(+) RNA, on the other hand, is localized primarily to the nuclear rim. Interestingly, the RNA binding nucleocytoplasmic shuttle protein Npl3p shows strong colocalization with bulk poly(A)(+) RNA, regardless of its nuclear location. Taken together, our data show that retention occurs close to the gene and indicate distinct nuclear fates of different mRNAs.

Cell Nucleus↗
PubMed2002

Interactions between mRNA export commitment, 3'-end quality control, and nuclear degradation.

Several aspects of eukaryotic mRNA processing are linked to transcription. In Saccharomyces cerevisiae, overexpression of the mRNA export factor Sub2p suppresses the growth defect of hpr1 null cells, yet the protein Hpr1p and the associated THO protein complex are implicated in transcriptional elongation. Indeed, we find that a pool of heat shock HSP104 transcripts are 3'-end truncated in THO complex mutant as well as sub2 mutant backgrounds. Surprisingly, however, this defect can be suppressed by deletion of the 3'-5' exonuclease Rrp6p. This indicates that incomplete RNAs result from nuclear degradation rather than from a failure to efficiently elongate transcription. RNAs that are not degraded are retained at the transcription site in a Rrp6p-dependent manner. Interestingly, the addition of a RRP6 deletion to sub2 or to THO complex mutants shows a strong synthetic growth phenotype, suggesting that the failure to retain and/or degrade defective mRNAs is deleterious. mRNAs produced in the 3'-end processing mutants rna14-3 and rna15-2, as well as an RNA harboring a 3' end generated by a self-cleaving hammerhead ribozyme, are also retained in Rrp6p-dependent transcription site foci. Taken together, our results show that several classes of defective RNPs are subject to a quality control step that impedes release from transcription site foci and suggest that suboptimal messenger ribonucleoprotein assembly leads to RNA degradation by Rrp6p.

Active Transport, Cell Nucleus↗