Biomedical subjects

Herbert Tschochner

Publications and source records attributed to Herbert Tschochner.

9 recordsLinked to original sources

PubMed2005

Roles of eukaryotic ribosomal proteins in maturation and transport of pre-18S rRNA and ribosome function.

Despite the rising knowledge about ribosome function and structure and how ribosomal subunits assemble in vitro in bacteria, the in vivo role of many ribosomal proteins remains obscure both in pro- and eukaryotes. Our systematic analysis of yeast ribosomal proteins (r-proteins) of the small subunit revealed that most eukaryotic r-proteins fulfill different roles in ribosome biogenesis, making them indispensable for growth. Different r-proteins control distinct steps of nuclear and cytoplasmic pre-18S rRNA processing and, thus, ensure that only properly assembled ribosomes become engaged in translation. Comparative analysis of dynamic and steady-state maturation assays revealed that several r-proteins are required for efficient nuclear export of pre-18S rRNA, suggesting that they form an interaction platform with the export machinery. In contrast, the presence of other r-proteins is mainly required before nuclear export is initiated. Our studies draw a correlation between the in vitro assembly, structural localization, and in vivo function of r-proteins.

Eukaryotic Cells↗

PubMed2004

The composition of the RNA polymerase I transcription machinery switches from initiation to elongation mode.

The amounts of RNA polymerase I (Pol I) and basal rDNA transcription factors were determined in yeast whole cell extracts. A 17-fold excess of Pol I was found compared to the Pol I-specific initiation factors upstream activating factor (UAF) and core factor (CF) which underlines that both initiation factors interact with a minor fraction of Pol I when rDNA transcription is active. Surprisingly, Rrn3p, another Pol I-specific initiation factor, is more abundant in cell lysates than UAF and CF. Our analyses revealed that a large fraction of cellular Rrn3p is not associated with Pol I. However, the amount of initiation-active Rrn3p which forms a stable complex with Pol I corresponds to the levels of UAF and CF which have been shown to bind the promoter. Initiation-active Rrn3p dissociates from the template during or immediately after Pol I has switched from initiation to elongation. Our data support a model in which the elongating Pol I leaves the initiation factors UAF, CF and Rrn3p close by the promoter.

DNA, Ribosomal↗

PubMed2004

Dephosphorylation of RNA polymerase I by Fcp1p is required for efficient rRNA synthesis.

Differently phosphorylated forms of RNA polymerase (Pol) II are required to guide the enzyme through the transcription cycle. Here, we show that a phosphorylation/dephosphorylation cycle is also important for RNA polymerase I-dependent synthesis of rRNA precursors. A key component of the Pol II transcription system is Fcp1p, a phosphatase that dephosphorylates the C-terminal domain of the largest Pol II subunit. Fcp1p stimulates transcription elongation and is required for Pol II recycling after transcription termination. We found that Fcp1p is also part of the RNA Pol I transcription apparatus. Fcp1p is required for efficient rDNA transcription in vivo, and also, recombinant Fcp1p stimulates rRNA synthesis both in promoter-dependent and in nonspecific transcription assays in vitro. We demonstrate that Fcp1 activity is not involved in the formation of the initiation-active form of Pol I (the Pol I-Rrn3p complex) and propose that dephosphorylation of Pol I by Fcp1p facilitates chain elongation during rRNA synthesis.

Phosphoprotein Phosphatases↗

PubMed2003

The pre-ribosomal network.

Recent achievements in yeast functional proteomics have significantly advanced our knowledge about ribosome biogenesis. Here, we present a program developed to integrate data from various proteome analyses with cell biological data on components present in the ribosome producing factories. This program allows users to attribute factors to certain complexes and to specific steps of ribosome biogenesis. Thus, it helps to gain novel insights into the complex network involved in maturation of ribosomal subunits. The database can be accessed at the URL http://www.pre-ribosome.de.

Biomarkers↗

PubMed2003

Pre-ribosomes on the road from the nucleolus to the cytoplasm.

Eukaryotic ribosomes are assembled in the nucleolus before export to the cytoplasm. Ribosome formation is a highly dynamic and coordinated multistep process, which requires synthesis, processing and modification of pre-rRNAs, assembly with ribosomal proteins and transient interaction of numerous non-ribosomal factors with the evolving pre-ribosomal particles. In the past two years, exciting insights into the sequential events occurring during pre-ribosome formation have been obtained, thanks largely to the advances in proteomic analyses. We now have a first biochemical map of the earliest 90S pre-ribosomes and of their daughter pre-40S and pre-60S ribosomal subunits along their path from the nucleolus to the cytoplasm. The future challenge will be to assign functions to the more than 150 non-ribosomal factors that transiently associate with the developing pre-ribosomes.

Active Transport, Cell Nucleus↗

PubMed2002

A Noc complex specifically involved in the formation and nuclear export of ribosomal 40 S subunits.

Formation and nuclear export of 60 S pre-ribosomes requires many factors including the heterodimeric Noc1-Noc2 and Noc2-Noc3 complexes. Here, we report another Noc complex with a specific role in 40 S subunit biogenesis. This complex consists of Noc4p, which exhibits the conserved Noc domain and is homologous to Noc1p, and Nop14p, a nucleolar protein with a role in 40 S subunit formation. Moreover, noc4 thermosensitive mutants are defective in 40 S biogenesis, and rRNA processing is inhibited at early cleavage sites A(0), A(1), and A(2). Using a fluorescence-based visual assay for 40 S subunit export, we observe a strong nucleolar accumulation of the Rps2p-green fluorescent protein reporter in noc4 ts mutants, but 60 S subunit export was normal. Thus, Noc4p and Nop14p form a novel Noc complex with a specific role in nucleolar 40 S subunit formation and subsequent export to the cytoplasm.

Amino Acid Sequence↗

PubMed2002

Localization of the yeast RNA polymerase I-specific subunits.

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.

Cryoelectron Microscopy↗

PubMed2002

TFIIH plays an essential role in RNA polymerase I transcription.

TFIIH is a multisubunit protein complex that plays an essential role in nucleotide excision repair and transcription of protein-coding genes. Here, we report that TFIIH is also required for ribosomal RNA synthesis in vivo and in vitro. In yeast, pre-rRNA synthesis is impaired in TFIIH ts strains. In a mouse, part of cellular TFIIH is localized within the nucleolus and is associated with subpopulations of both RNA polymerase I and the basal factor TIF-IB. Transcription systems lacking TFIIH are inactive and exogenous TFIIH restores transcriptional activity. TFIIH is required for productive but not abortive rDNA transcription, implying a postinitiation role in transcription. The results provide a molecular link between RNA polymerase I transcription and transcription-coupled repair of active ribosomal RNA genes.

Animals↗

PubMed2002

90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors.

We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.

Blotting, Northern↗