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.

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.

Brefeldin A resistance factor 1 (Bfr1p) is a nonessential RNA-binding protein and multicopy suppressor of brefeldin A sensitivity in Saccharomyces cerevisiae Deletion of BFR1 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. In the present study, we show that mutations in RNA-binding residues of Bfr1p impede its RNA-dependent colocalization with ER, yet do not mimic the known cellular defects seen upon BFR1 deletion. However, a Bfr1 RNA-binding mutant is impaired in binding to ERG4 mRNA, which encodes an enzyme required for the final step of ergosterol biosynthesis. Consistently, bfr1Δ strains show a strong reduction in Erg4p protein levels, most likely because of degradation of misfolded Erg4p. Polysome profiling of bfr1Δ or bfr1 mutant strains reveals a strong shift of ERG4 mRNA to polysomes, consistent with a function of Bfr1p in elongation or increased ribosome loading. Collectively, our data reveal that Bfr1 has at least two separable functions: one in RNA binding and cotranslational protein translocation into the ER and one in ploidy control or chromosome segregation.

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. Interaction of Scp160p with these mRNAs requires the conserved K homology domains 13 and 14. Transfer RNA pairing index analysis of Scp160p target mRNAs indicates a high degree of consecutive use of iso-decoding codons. As shown for one target mRNA encoding the glycoprotein Pry3p, Scp160p depletion results in translational downregulation but increased association with polysomes, suggesting that it is required for efficient translation elongation. Depletion of Scp160p also decreased the relative abundance of ribosome-associated tRNAs whose codons show low potential for autocorrelation on mRNAs. Conversely, tRNAs with highly autocorrelated codons in mRNAs are less impaired. Our data indicate that Scp160p might increase the efficiency of tRNA recharge, or prevent diffusion of discharged tRNAs, both of which were also proposed to be the likely basis for the translational fitness effect of tRNA pairing.

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. We used ASH1-mRNA transport in yeast to provide a first assessment of where and how localizing mRNAs are specifically recognized and incorporated into mRNPs. By using in vitro-interaction and reconstitution assays, we found that none of the implicated mRNA-binding proteins showed highly specific cargo binding. Instead, we identified the cytoplasmic myosin adapter She3p as additional RNA-binding protein. We further found that only the complex of the RNA-binding proteins She2p and She3p achieves synergistic cargo binding, with an at least 60-fold higher affinity for localizing mRNAs when compared to control RNA. Mutational studies identified a C-terminal RNA-binding fragment of She3p to be important for synergistic RNA binding with She2p. The observed cargo specificity of the ternary complex is considerably higher than previously reported for localizing mRNAs. It suggests that RNA binding for mRNP localization generally exhibits higher selectivity than inferred from previous in vitro data. This conclusion is fully consistent with a large body of in vivo evidence from different organisms. Since the ternary yeast complex only assembles in the cytoplasm, specific mRNA recognition might be limited to the very last steps of mRNP assembly. Remarkably, the mRNA itself triggers the assembly of mature, motor-containing complexes. Our reconstitution of a major portion of the mRNA-transport complex offers new and unexpected insights into the molecular assembly of specific, localization-competent mRNPs and provides an important step forward in our mechanistic understanding of mRNA localization in general.

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. Recently, an active role of neuronal glycogen and even its contribution to neuronal survival could be demonstrated. We used the neuronal cell lines NSC-34 and N18TG2 and could demonstrate that they express the key-enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase and contain glycogen which is mobilized on glucose deprivation and elevated potassium concentrations, but not by hormones stimulating cAMP formation. Conditions of metabolic stress, namely hypoxia, oxidative stress and pH lowering, induce glycogen degradation. Our studies revealed that glycogen can contribute to the energy supply of neuronal cell lines in situations of metabolic stress. These findings shed new light on the so far neglected role of neuronal glycogen. The key-enzyme in glycogen degradation is glycogen phosphorylase. Neurons express only the brain isoform of the enzyme that is supposed to be activated primarily by the allosteric activator AMP and less by covalent phosphorylation via the cAMP cascade. Our results indicate that neuronal glycogen is not degraded upon hormone action but by factors lowering the energy charge of the cells directly.

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.

Type V myosin (MyoV)-dependent transport of cargo is an essential process in eukaryotes. Studies on yeast and vertebrate MyoV showed that their globular tails mediate binding to the cargo complexes. In Saccharomyces cerevisiae, the MyoV motor Myo4p interacts with She3p to localize asymmetric synthesis of HO 1 (ASH1) mRNA into the bud of dividing cells. A recent study showed that localization of GFP-MS2-tethered ASH1 particles does not require the Myo4p globular tail, challenging the supposed role of this domain. We assessed ASH1 mRNA and Myo4p distribution more directly and found that their localization is impaired in cells expressing globular tail-lacking Myo4p. In vitro studies further show that the globular tail together with a more N-terminal linker region is required for efficient She3p binding. We also determined the x-ray structure of the Myo4p globular tail and identify a conserved surface patch important for She3p binding. The structure shows pronounced similarities to membrane-tethering complexes and indicates that Myo4p may not undergo auto-inhibition of its motor domain.

Cytoplasmic localization and localized translation of messenger RNAs contribute to asymmetrical protein distribution. Recognition of localized mRNAs by RNA-binding proteins can occur in the cytoplasm or, alternatively, co- or post-transcriptionally in the nucleus. In budding yeast, mRNAs destined for localization are bound by the She2 protein before their nuclear export. Here, we show that a specific transcript, known as ASH1 mRNA, and She2 localize specifically to the nucleolus when their nuclear export is blocked. Nucleolar She2 localization is enhanced in a She2 mutant that cannot bind to RNA. A fusion protein of the amino terminus of She3 and She2 (She3N-She2) fails to enter the nucleus, but does not impair ASH1 mRNA localization. Instead, these cells fail to distribute Ash1 protein asymmetrically, which is caused by a defective translational control of ASH1 mRNA. Our results indicate that the nucleolar transit of RNA-binding proteins such as She2 is necessary for the correct assembly of translationally silenced localizing messenger ribonucleoproteins.

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.