Multifunctional RNA nanoparticles.

Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA-DNA hybrids aimed to conditionally activate multiple split functionalities inside cells.

Structural basis for inhibition of translation by the tumor suppressor Pdcd4.

The tumor suppressor function of Programmed Cell Death 4 (Pdcd4) is achieved through interactions between Pdcd4 and components of the translation initiation complex, namely, the RNA helicase eIF4A and the scaffolding protein eIF4G. These interactions are mediated through two MA3 domains on the Pdcd4 molecule and result in inhibition of protein synthesis. We have solved the high-resolution crystal structure of the C-terminal MA3 (cMA3) domain of Pdcd4 in several crystal forms and demonstrated its similarity to the MA3 domain of eIF4G. As predicted by the structure, the cMA3 domain competes with eIF4Gc for binding to eIF4A and surprisingly is sufficient to inhibit translation initiation. Mutations that abolish eIF4A binding negate both functions of the cMA3. Interestingly mutations in the Akt phosphorylation site influenced neither cMA3 binding to eIF4A nor its ability to inhibit translation initiation. Finally, our structural analysis reveals MA3 domains to be a novel subfamily of VHS domains.

Role of 3'UTRs in the translation of mRNAs regulated by oncogenic eIF4E--a computational inference.

Eukaryotic cap-dependent mRNA translation is mediated by the initiation factor eIF4E, which binds mRNAs and stimulates efficient translation initiation. eIF4E is often overexpressed in human cancers. To elucidate the molecular signature of eIF4E target mRNAs, we analyzed sequence and structural properties of two independently derived polyribosome recruited mRNA datasets. These datasets originate from studies of mRNAs that are actively being translated in response to cells over-expressing eIF4E or cells with an activated oncogenic AKT: eIF4E signaling pathway, respectively. Comparison of eIF4E target mRNAs to mRNAs insensitive to eIF4E-regulation has revealed surprising features in mRNA secondary structure, length and microRNA-binding properties. Fold-changes (the relative change in recruitment of an mRNA to actively translating polyribosomal complexes in response to eIF4E overexpression or AKT upregulation) are positively correlated with mRNA G+C content and negatively correlated with total and 3'UTR length of the mRNAs. A machine learning approach for predicting the fold change was created. Interesting tendencies of secondary structure stability are found near the start codon and at the beginning of the 3'UTR region. Highly upregulated mRNAs show negative selection (site avoidance) for binding sites of several microRNAs. These results are consistent with the emerging model of regulation of mRNA translation through a dynamic balance between translation initiation at the 5'UTR and microRNA binding at the 3'UTR.

Alteration of the protein kinase binding domain enhances function of the Saccharomyces cerevisiae molecular chaperone Cdc37.

Cdc37 is a molecular chaperone that has a general function in the biogenesis of protein kinases. We identified mutations within the putative "protein kinase binding domain" of Cdc37 that alleviate the conditional growth defect of a strain containing a temperature-sensitive allele, tpk2(Ts), of the cyclic AMP-dependent protein kinase (PKA). These dominant mutations alleviate the temperature-sensitive growth defect by elevating PKA activity, as judged by their effects on PKA-regulated processes, localization and phosphorylation of the PKA effector Msn2, as well as in vitro PKA activity. Although the tpk2(Ts) growth defect is also alleviated by Cdc37 overproduction, the CDC37 dominant mutants contain wild-type Cdc37 protein levels. In addition, Saccharomyces cerevisiae Ste11 protein kinase has an elevated physical interaction with the altered Cdc37 protein. These results implicate specific amino-terminal residues in the interaction between Cdc37 and client protein kinases and provide further genetic and biochemical support for a model in which Cdc37 functions as a molecular chaperone for protein kinases.

PP2A phosphatase activity is required for stress and Tor kinase regulation of yeast stress response factor Msn2p.

In response to stress and nutrient starvation, the Saccharomyces cerevisiae transcription factor Msn2p accumulates in the nucleus and activates expression of a broad array of genes. Here, we analyze the role of the Tor (target of rapamycin) signaling pathway in mediating these responses. Inactivation of the Tor pathway component Tap42p using tap42(Ts) alleles causes a sustained nuclear localization similar to that after the addition of the Tor kinase inhibitor rapamycin. Effects of Tap42p inactivation and rapamycin addition could be suppressed by deletion of TIP41, which encodes a Tap42p-interacting protein. These results support the notion that rapamycin affects Msn2p by inactivating Tap42p function. Tap42p interacts with the catalytic subunit of PP2A (protein phosphatase 2A) and PP2A-like phosphatases. Deletion of either the catalytic or regulatory subunit that forms the PP2A phosphatase complex prevents nuclear accumulation of Msn2p in the tap42(Ts) strain and in wild-type strains treated with rapamycin. These results suggest that Tap42p is an inhibitor of PP2A phosphatase, which in turn inhibits nuclear export of Msn2p. Interestingly, PP2A function is also required for nuclear accumulation of Msn2p in response to stresses, such as heat and osmotic shock, as well as nitrogen (but not glucose) starvation. Thus, PP2A and the Tor kinase pathway transduce stress and nitrogen starvation signals to Msn2p. Finally, Msn2p localization is unaffected by conditional loss of 14-3-3 protein function, ruling out the possibility that 14-3-3 proteins act as a scaffold to sequester Msn2p in the cytoplasm.

Multiple roles of Tap42 in mediating rapamycin-induced transcriptional changes in yeast.

Tor proteins, targets of the antiinflammatory drug rapamycin, mediate a conserved signaling pathway required for cell growth and proliferation in eukaryotes. By global transcriptional analysis of Saccharomyces cerevisiae, we have examined the role of the essential protein Tap42 in transcriptional regulation by Tor. We find that Tap42 inactivation, like rapamycin addition, prolongs activation of stress response genes. In contrast, Tap42 inactivation, as does inactivation of the protein phosphatases Sit4 and Pph21/22, blocks rapamycin induction of nitrogen discrimination pathway genes. Tap42 inactivation neither affects ribosomal protein gene expression nor blocks rapamycin-induced repression of these genes. These results indicate that Tap42 can both inhibit and activate protein phosphatases and provide insight into the complex events underlying TOR regulation of transcription.