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Ralf-Peter Jansen

Publications and source records attributed to Ralf-Peter Jansen.

12 recordsLinked to original sources
PubMed2006

Coordination of endoplasmic reticulum and mRNA localization to the yeast bud.

Localization of messenger RNAs and local protein synthesis contribute to asymmetric protein distribution not only of cytoplasmic but also of membrane or secreted proteins. Since synthesis of the latter protein classes occurs at the rough endoplasmic reticulum (ER), mRNA localization and distribution of ER should be coordinated. However, this coordination is not yet understood. In yeast, mRNA localization to the growing bud depends on the myosin Myo4p, its adaptor She3p, and the specific RNA binding protein She2p. These proteins mediate the localization of 23 mRNAs including ASH1 mRNA and mRNAs encoding membrane proteins. In addition, Myo4p and She3p are required for segregation of cortical ER to the bud. Here we show, with ASH1 mRNA as a model mRNA, that localizing messenger ribonucleoprotein (mRNP) particles comigrate with tubular ER structures to the bud, which requires the RNA binding protein She2p. Coordinated movement of the ASH1 mRNP with ER tubules but not their association with each other depends on Myo4p and She3p. Subcellular fractionation experiments demonstrate a cosegregation of ER and She2p, which is independent of Myo4p, She3p, or polysomes. Our findings suggest a novel model for mRNA localization that involves association of She2p and mRNPs with ER tubules and myosin-dependent cotransport of tubules and localized mRNPs.

Biological Transport↗
PubMed2006

Structural biology of RNA polymerase III: subcomplex C17/25 X-ray structure and 11 subunit enzyme model.

We obtained an 11 subunit model of RNA polymerase (Pol) III by combining a homology model of the nine subunit core enzyme with a new X-ray structure of the subcomplex C17/25. Compared to Pol II, Pol III shows a conserved active center for RNA synthesis but a structurally different upstream face for specific initiation complex assembly during promoter selection. The Pol III upstream face includes a HRDC domain in subunit C17 that is translated by 35 A and rotated by 150 degrees compared to its Pol II counterpart. The HRDC domain is essential in vivo, folds independently in vitro, and, unlike other HRDC domains, shows no indication of nucleic acid binding. Thus, the HRDC domain is a functional module that could account for the role of C17 in Pol III promoter-specific initiation. During elongation, C17/25 may bind Pol III transcripts emerging from the adjacent exit pore, because the subcomplex binds to tRNA in vitro.

Amino Acid Sequence↗
PubMed2005

X-ray structure of RLI, an essential twin cassette ABC ATPase involved in ribosome biogenesis and HIV capsid assembly.

The ABC ATPase RNase-L inhibitor (RLI) emerges as a key enzyme in ribosome biogenesis, formation of translation preinitiation complexes, and assembly of HIV capsids. To help reveal the structural mechanism of RLI, we determined the Mg2+-ADP bound crystal structure of the twin cassette ATPase of P. furiosus RLI at 1.9 A resolution and analyzed functional motifs in yeast in vivo. RLI shows similarities but also differences to known ABC enzyme structures. Twin nucleotide binding domains (NBD1 and NBD2) are arranged to form two composite active sites in their interface cleft, indicating they undergo the ATP-driven clamp-like motion of the NBDs of ABC transporters. An unusual "hinge" domain along the NBD1:NBD2 interface provides a frame for association and possibly ATP-driven conformational changes of the NBDs. Our results establish a first structural basis for ABC domain heterodimers and suggest that RLI may act as mechanochemical enzyme in ribosome and HIV capsid biogenesis.

Adenosine Triphosphatases↗
PubMed2005

A putative nuclear function for mammalian Staufen.

In addition to its role in rRNA processing and ribosome assembly, the nucleolus plays a part in the assembly of non-ribosomal ribonucleoprotein particles (RNPs) that are destined for cytoplasmic RNA delivery. Recent evidence indicates that mammalian Staufen2, a brain-specific RNA-binding protein involved in RNA localization, can--at least transiently--enter the nucleolus. Therefore, the assembly of Staufen2 into transport-competent RNPs might occur in the nucleus before their export into the cytoplasm. This could provide new insights into the mechanisms of subcellular RNA localization.

Active Transport, Cell Nucleus↗
PubMed2005

Intracellular RNA sorting, transport and localization.

RNA localization and translational control are crucial for cellular fine-tuning of gene expression in space and time. A recent meeting in Tucson, Arizona pointed out mechanisms conserved across different species and cell types that contribute to the establishment of cell polarity and cell migration. Furthermore, it is becoming increasingly clear that these post-transcriptional control processes are relevant for various diseases.

Animals↗
PubMed2004

RNA integrity as a quality indicator during the first steps of RNP purifications : a comparison of yeast lysis methods.

BACKGROUND: The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor. RESULTS: We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins. CONCLUSION: We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells.

Cell Fractionation↗
PubMed2004

A novel transport pathway for a yeast plasma membrane protein encoded by a localized mRNA.

Generally, plasma membrane (PM) proteins are cotranslationally inserted into the endoplasmic reticulum (ER) and travel in vesicles via the Golgi apparatus to the PM. In the yeast Saccharomyces cerevisiae, the polytopic membrane protein Ist2p is encoded by an mRNA that is localized to the cortex of daughter cells. It has been suggested that IST2 mRNA localization leads to the accumulation of the protein at the PM of daughter cells. Since small- and medium-sized daughter cells only contain cortical, but not perinuclear ER, this implies the local translation of Ist2p specifically at the cortical ER. Here, we show that localization of constitutively expressed IST2 mRNA is required for delivery of Ist2p to the PM of daughter, but not mother cells and that it does not result in daughter-specific Ist2p accumulation. In contrast to a PM-located hexose transporter (Hxt1p) that follows the standard secretory pathway, the trafficking of Ist2p is independent of myosin-mediated vesicular transport. Furthermore, colocalization experiments in mutants of the secretory pathway demonstrate that trafficking of Ist2p does not require the classical secretory machinery. These data suggest the existence of a novel trafficking pathway connecting specialized domains of the ER with the PM.

Blotting, Western↗
PubMed2004

mRNA localization and the cytoskeleton.

mRNA localization is a widespread post-transcriptional mechanism for targeting protein synthesis to specific cellular sites. It is involved in the generation of cell polarity, asymmetric segregation of cell fate determinants and germ cell specification. Actin and microtubule filaments have key functions during RNA localization, especially during transport of mRNAs and anchoring at target sites. Recent advances in understanding the role of motors and filament systems have mainly resulted from the contribution of live imaging of mRNA movement and from the purification of putative localization ribonucleoproteins. There have also been new findings on the role of centrosomes in RNA localization.

Actin Cytoskeleton↗
PubMed2003

The UCS domain protein She4p binds to myosin motor domains and is essential for class I and class V myosin function.

BACKGROUND: Myosins are motor proteins involved in processes like cell motility, vesicle transport, or cytokinesis. In a variety of organisms, a novel group of proteins forming the UCS (UNC-45/CRO1/SHE4) domain-containing family are essential for proper myosin function. The Saccharomyces cerevisae UCS domain protein She4p is involved in two myosin-requiring events, endocytosis and mRNA localization. RESULTS: In contrast to UCS domain proteins from other organisms that interact with class II myosins, we demonstrate that She4p associates with yeast class I and class V myosins. She4p binds to motor domains of class V myosin Myo4p and class I myosin Myo5p, and this binding depends on She4p's UCS domain. In vivo, She4p is essential for the function and localization of Myo3p, Myo4p, and Myo5p (but not of Myo2p) and for colocalization of class I myosins with cortical actin patches. In vitro, She4p stimulates binding of Myo5p to filamentous actin. Wild-type She4p, but not a mutant lacking the UCS domain, accumulates in a cap-like structure at the bud tip. This localization requires Myo2p and actin, suggesting a Myo2-dependent mechanism by which She4p is targeted to the bud cap. Localization of She4p is essential for proper positioning and myosin-actin association of cortical Myo5p. CONCLUSIONS: Our results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins.

Actins↗
PubMed2002

Ribonucleoprotein-dependent localization of the yeast class V myosin Myo4p.

Class V myosins are motor proteins with functions in vesicle transport, organelle segregation, and RNA localization. Although they have been extensively studied, only little is known about the regulation of their spatial distribution. Here we demonstrate that a GFP fusion protein of the budding yeast class V myosin Myo4p accumulates at the bud cortex and is a component of highly dynamic cortical particles. Bud-specific enrichment depends on Myo4p's association with its cargo, a ribonucleoprotein complex containing the RNA-binding protein She2p. Cortical accumulation of Myo4p at the bud tip can be explained by a transient retention mechanism that requires SHE2 and, apparently, localized mRNAs bound to She2p. A mutant She2 protein that is unable to recognize its cognate target mRNA, ASH1, fails to localize Myo4p. Mutant She2p accumulates inside the nucleus, indicating that She2p shuttles between the nucleus and cytoplasm and is exported in an RNA-dependent manner. Consistently, inhibition of nuclear mRNA export results in nuclear accumulation of She2p and cytoplasmic Myo4p mislocalization. Loss of She2p can be complemented by direct targeting of a heterologous lacZ mRNA to a complex of Myo4p and its associated adaptor She3p, suggesting that She2p's function in Myo4p targeting is to link an mRNA to the motor complex.

Cell Nucleus↗
PubMed2002

Characterization of Candida albicans ASH1 in Saccharomyces cerevisiae.

Several fungi can undergo a dimorphic switch from yeast-like to hyphal or pseudohyphal forms in response to environmental cues. The Saccharomyces cerevisae GATA-type transcription factor Ash1p is required for diploid pseudohyphal differentiation during nitrogen starvation. In haploid cells, Ash1p is essential for the cell-type specific repression of the HO gene. We cloned CaAsh1, a putative homologue of Ash1p from the pathogen Candida albicans. Although the overall similarity of CaAsh1p and Ash1p is modest, both proteins show significant homology in their carboxy termini. Upon expression in budding yeast, CaASH1 mRNA is asymmetrically sorted during cell division, which is similar to the fate of ASH1 mRNA. Cell-specific CaASH1 mRNA localization results in an asymmetric CaAsh1 protein distribution and an accumulation of the protein in daughter cell nuclei. CaAsh1 is unable to repress HO expression in S. cerevisiae but can replace ScAsh1 function during pseudohyphal growth. This suggests a partial functional similarity of the two Ash1 proteins.

Amino Acid Sequence↗