The high-density lipoprotein binding protein HDLBP is an unusual RNA-binding protein with multiple roles in cancer and disease.

The high-density lipoprotein binding protein (HDLBP) is the human member of an evolutionarily conserved family of RNA-binding proteins, the vigilin protein family. These proteins are characterized by 14 or 15 RNA-interacting KH (heterologous nuclear ribonucleoprotein K homology) domains. While mainly present at the cytoplasmic face of the endoplasmic reticulum, HDLBP and its homologs are also found in the cytosol and nucleus.

Proteomic Mapping by APEX2-Catalyzed Proximity Labeling in Saccharomyces cerevisiae Semipermeabilized Cells.

Enzyme-catalyzed proximity labeling (PL) has proven to be a valuable resource for proteomic mapping of subcellular compartments and protein networks in living cells. We have used engineered ascorbate peroxidase (APEX2) to develop a PL approach for budding yeast. It is based on semipermeabilized cells to overcome poor cellular permeability of the APEX2 substrate biotin-phenol and difficulties in its delivery into the cell.

RNA Proximity Labeling: A New Detection Tool for RNA-Protein Interactions.

Multiple cellular functions are controlled by the interaction of RNAs and proteins. Together with the RNAs they control, RNA interacting proteins form RNA protein complexes, which are considered to serve as the true regulatory units for post-transcriptional gene expression. To understand how RNAs are modified, transported, and regulated therefore requires specific knowledge of their interaction partners.

The Motor Neuron-Like Cell Line NSC-34 and Its Parent Cell Line N18TG2 Have Glycogen that is Degraded Under Cellular Stress.

Brain glycogen has a long and versatile history: Primarily regarded as an evolutionary remnant, it was then thought of as an unspecific emergency fuel store. A dynamic role for glycogen in normal brain function has been proposed later but exclusively attributed to astrocytes, its main storage site. Neuronal glycogen had long been neglected, but came into focus when sensitive technical methods allowed quantification of glycogen at low concentration range and the detection of glycogen metabolizing enzymes in cells and cell lysates.

RNA Interactome Identification via RNA-BioID in Mouse Embryonic Fibroblasts.

Cytoplasmic localization of mRNAs is common to all organisms and serves the spatial expression of genes. Cis-acting RNA signals (mostly found in the mRNA's 3'-UTR), called zipcodes recruit trans-acting RNA-binding proteins that facilitate the localization of the mRNA. UV-cross-linking or affinity purification has been applied to identify such proteins but suffer from the need for stable RNA-protein binding or direct contact of protein and RNA.

Local translation of yeast mRNA at the endoplasmic reticulum requires the brefeldin A resistance protein Bfr1.

Brefeldin A resistance factor 1 (Bfr1p) is a nonessential RNA-binding protein and multicopy suppressor of brefeldin A sensitivity in Deletion of leads to multiple defects, including altered cell shape and size, change in ploidy, induction of P-bodies and chromosomal missegregation. Bfr1p has been shown to associate with polysomes, binds to several hundred mRNAs, and can target some of them to P-bodies. Although this implies a role of Bfr1p in translational control of mRNAs, its molecular function remains elusive.

APEX2-mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells.

Enzyme-catalyzed proximity labeling (PL) with the engineered ascorbate peroxidase APEX2 is a novel approach to map organelle compartmentalization and protein networks in living cells. Current procedures developed for mammalian cells do not allow delivery of the cosubstrate, biotin-phenol, into living yeast cells. Here, we present a new method based on semipermeabilized yeast cells.

β-Actin mRNA interactome mapping by proximity biotinylation.

The molecular function and fate of mRNAs are controlled by RNA-binding proteins (RBPs). Identification of the interacting proteome of a specific mRNA in vivo remains very challenging, however. Based on the widely used technique of RNA tagging with MS2 aptamers for RNA visualization, we developed a RNA proximity biotinylation (RNA-BioID) technique by tethering biotin ligase (BirA*) via MS2 coat protein at the 3' UTR of endogenous MS2-tagged β-actin mRNA in mouse embryonic fibroblasts.

Membrane-Associated RNA-Binding Proteins Orchestrate Organelle-Coupled Translation.

Proteins are positioned and act at defined subcellular locations. This is particularly important in eukaryotic cells that deliver proteins to membrane-bound organelles such as the endoplasmic reticulum (ER), mitochondria, or endosomes. It is axiomatic that organelle targeting depends mainly on polypeptide signals.

The RNA-Binding Protein Scp160p Facilitates Aggregation of Many Endogenous Q/N-Rich Proteins.

The RNA-binding protein Scp160p is the yeast homolog of the conserved vigilin protein family. These proteins influence a variety of nuclear and cytoplasmic functions. One of Scp160p's reported roles is to increase translation elongation efficiency in a manner related to codon usage.

Signal sequence-independent targeting of MID2 mRNA to the endoplasmic reticulum by the yeast RNA-binding protein Khd1p.

Localization of mRNAs depends on specific RNA-binding proteins (RBPs) and critically contributes not only to cell polarization but also to basal cell function. The yeast RBP Khd1p binds to several hundred mRNAs, the majority of which encodes secreted or membrane proteins. We demonstrate that a subfraction of Khd1p associates with artificial liposomes and endoplasmic reticulum (ER), and that Khd1p endomembrane association is partially dependent on its binding to RNA.

A jack of all trades: the RNA-binding protein vigilin.

The vigilin family of proteins is evolutionarily conserved from yeast to humans and characterized by the proteins' 14 or 15 hnRNP K homology (KH) domains, typically associated with RNA-binding. Vigilin is the largest RNA-binding protein (RBP) in the KH domain-containing family and one of the largest RBP known to date. Since its identification 30 years ago, vigilin has been shown to bind over 700 mRNAs and has been associated with cancer progression and cardiovascular disease.

Molecular architecture and dynamics of ASH1 mRNA recognition by its mRNA-transport complex.

mRNA localization is an essential mechanism of gene regulation and is required for processes such as stem-cell division, embryogenesis and neuronal plasticity. It is not known which features in the cis-acting mRNA localization elements (LEs) are specifically recognized by motor-containing transport complexes. To the best of our knowledge, no high-resolution structure is available for any LE in complex with its cognate protein complex.

Here, there, everywhere. mRNA localization in budding yeast.

mRNA localization and localized translation is a common mechanism that contributes to cell polarity and cellular asymmetry. In metazoan, mRNA transport participates in embryonic axis determination and neuronal plasticity. Since the mRNA localization process and its molecular machinery are rather complex in higher eukaryotes, the unicellular yeast Saccharomyces cerevisiae has become an attractive model to study mRNA localization.

mRNA transport meets membrane traffic.

Active transport and local translation of mRNAs ensure the appropriate spatial organization of proteins within cells. Recent work has shown that this process is intricately connected to membrane trafficking. Here, we focus on new findings obtained in fungal model systems.

Yeast phospholipid biosynthesis is linked to mRNA localization.

Regulation of the localization of mRNAs and local translation are universal features in eukaryotes and contribute to cellular asymmetry and differentiation. In Saccharomyces cerevisiae, localization of mRNAs that encode membrane proteins requires the She protein machinery, including the RNA-binding protein She2p, as well as movement of the cortical endoplasmic reticulum (cER) to the yeast bud. In a screen for ER-specific proteins necessary for the directional transport of WSC2 and EAR1 mRNAs, we have identified enzymes that are involved in phospholipid metabolism.

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons.

Background: Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes.

Results: Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes.

Scp160p is required for translational efficiency of codon-optimized mRNAs in yeast.

The budding yeast multi-K homology domain RNA-binding protein Scp160p binds to >1000 messenger RNAs (mRNAs) and polyribosomes, and its mammalian homolog vigilin binds transfer RNAs (tRNAs) and translation elongation factor EF1alpha. Despite its implication in translation, studies on Scp160p's molecular function are lacking to date. We applied translational profiling approaches and demonstrate that the association of a specific subset of mRNAs with ribosomes or heavy polysomes depends on Scp160p.

Role of Loc1p in assembly and reorganization of nuclear ASH1 messenger ribonucleoprotein particles in yeast.

Directional transport of mRNA is a universal feature in eukaryotes, requiring the assembly of motor-dependent RNA-transport particles. The cytoplasmic transport of mRNAs is preceded by the nuclear assembly of pre-messenger ribonucleoprotein particles (mRNPs). In budding yeast, the asymmetric synthesis of HO 1 (ASH1) pre-mRNP originates already cotranscriptionally and passes through the nucleolus before its nuclear export.

Association of the yeast RNA-binding protein She2p with the tubular endoplasmic reticulum depends on membrane curvature.

Localization of mRNAs contributes to the generation and maintenance of cellular asymmetry in a wide range of organisms. In Saccharomyces cerevisiae, the so-called locasome complex with its core components Myo4p, She2p, and She3p localizes more than 30 mRNAs to the yeast bud tip. A significant fraction of these mRNAs encodes membrane or secreted proteins.

Take the (RN)A-train: localization of mRNA to the endoplasmic reticulum.

Protein translocation into the endoplasmic reticulum (ER) generally requires targeting of mRNAs encoding secreted or membrane proteins to the ER membrane. The prevalent view is that these mRNAs are delivered co-translationally, using the signal recognition particle (SRP) pathway. Here, SRP delivers signal sequence-containing proteins together with associated ribosomes and mRNA to the SRP receptor present on the ER surface.

Localization of a subset of yeast mRNAs depends on inheritance of endoplasmic reticulum.

Localization of messenger RNA (mRNAs) contributes to generation and maintenance of cellular asymmetry, embryonic development and neuronal function. The She1-3 protein machinery in Saccharomyces cerevisiae localizes >30 mRNAs to the bud tip, including 13 mRNAs encoding membrane or secreted proteins. Ribonucleoprotein (RNP) particles can co-localize with tubular endoplasmic reticulum (ER) structures that form the initial elements for segregation of cortical ER (cER), suggesting a coordination of mRNA localization and cER distribution.

Assembly of mRNA-protein complexes for directional mRNA transport in eukaryotes--an overview.

At all steps from transcription to translation, RNA-binding proteins play important roles in determining mRNA function. Initially it was believed that for the vast majority of transcripts the role of RNA-binding proteins is limited to general functions such as splicing and translation. However, work from recent years showed that members of this class of proteins also recognize several mRNAs via cis-acting elements for their incorporation into large motor-containing particles.

A cytoplasmic complex mediates specific mRNA recognition and localization in yeast.

In eukaryotes, hundreds of mRNAs are localized by specialized transport complexes. For localization, transcripts are recognized by RNA-binding proteins and incorporated into motor-containing messenger ribonucleoprotein particles (mRNPs). To date, the molecular assembly of such mRNPs is not well understood and most details on cargo specificity remain unresolved.