Genetic Interactions between the Yeast RNA Helicase Homolog Prp16 and Spliceosomal Snrnas Identify Candidate Ligands for the Prp16 RNA-Dependent Atpase

Pre-mRNA splicing occurs in a large and dynamic ribonucleoprotein complex, the spliceosome. Several protein factors involved in splicing are homologous to a family of RNA-dependent ATPases, the so-called DEAD/DEAH proteins. A subset of these factors exhibit RNA helicase activity in vitro. The DEAD/DEAH proteins involved in splicing are thought to mediate RNA conformational rearrangements during spliceosome assembly. However, the RNA ligands for these factors are currently unknown. Here, we present genetic evidence in Saccharomyces cerevisiae for a functional interaction between the DEAH protein Prp16, and the U6 and U2 spliceosomal snRNAs. Using a library of mutagenized U6 snRNA genes, we have identified 14 strong suppressors of the cold-sensitive (cs) allele, prp16-302. Remarkably, each suppressor contains a single nucleotide deletion of 1 of the 6 residues that lie immediately upstream of a sequence in U6 that interacts with the 5' splice site. Analysis of site-directed mutations revealed that nucleotide substitutions in the adjacent U2-U6 helix I structure also suppress prp16-302, albeit more weakly. The U6 suppressors tested also partially reverse the phenotype of two other cs alleles, prp16-1 and prp16-301, but not the four temperature-sensitive alleles tested. Finally, overexpression of each cs allele exacerbates its recessive growth phenotype and confers a dominant negative cs phenotype. We propose that the snRNA suppressors function by destabilizing an interaction between the U2-U6 complex and a hypothetical factor (X), which is trapped by cs mutants of PRP16. The phenotypes of overexpressed prp16 alleles are consistent with the model that this trapped interaction inhibits the dissociation of Prp16 from the spliceosome. We discuss the intriguing possibility that factor X is Prp16 itself.     

Is the atherosclerotic phenotype of preeclamptic placentas due to altered lipoprotein concentrations and placental lipoprotein receptors? Role of a small-for-gestational-age phenotype

Atherosis of spiral arteries in uteroplacental beds from preeclamptic women resemble those of atherosclerosis, characterized by increased plasma lipids and lipoproteins. We hypothesized that: 1) lipoprotein receptors/transporters in the placenta would be upregulated in preeclampsia, associated with increased maternal and fetal lipoprotein concentrations; and 2) expression of these would be reduced in preeclamptic placentae from women delivering small-for-gestational-age (SGA) infants. Placental biopsies and maternal and umbilical serum samples were taken from 27 normotensive and 24 preeclamptic women. Maternal/umbilical cord serum LDL, HDL, total cholesterol, and triglycerides were measured. Placental mRNA expression of lipoprotein receptors/transporters were quantified using quantitative RT-PCR. Protein localization/expression of LDL receptor-related protein 1 (LRP-1) in the preeclamptic placentae with/without SGA was measured by immunohistochemistry. Placental mRNA expression of all genes except paraoxonase-1 (PON-1), microsomal triglyceride transfer protein (MTTP), and protein disulfide isomerase family A member 2 (PDIA2) were observed. No differences for any lipoprotein receptors/transporters were found between groups; however, in the preeclamptic group placental LRP-1 expression was lower in SGA delivering mothers (n = 7; P = 0.036). LRP-1 protein was localized around fetal vessels and Hofbauer cells. This is the first detailed study of maternal/fetal lipoprotein concentrations and placental lipoprotein receptor mRNA expression in normotensive and preeclamptic pregnancies. These findings do not support a role of altered lipid metabolism in preeclampsia, but may be involved in fetal growth.     

Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin

Boundary elements hinder the spread of heterochromatin, yet these sites do not fully account for the preservation of adjacent euchromatin. Histone variant H2A.Z (Htz1 in yeast) replaces conventional H2A in many nucleosomes. Microarray analysis revealed that HTZ1-activated genes cluster near telomeres. The reduced expression of most of these genes in htz1Delta cells was reversed by the deletion of SIR2 (sir2Delta) suggesting that H2A.Z antagonizes telomeric silencing. Other Htz1-activated genes flank the silent HMR mating-type locus. Their requirement for Htz1 can be bypassed by sir2Delta or by a deletion encompassing the silencing nucleation sites in HMR. In htz1Delta cells, Sir2 and Sir3 spread into flanking euchromatic regions, producing changes in histone H4 acetylation and H3 4-methylation indicative of ectopic heterochromatin formation. Htz1 is enriched in these euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin. Thus, euchromatin and heterochromatin each contains components that antagonize switching to the opposite chromatin state.     

PchC thioesterase optimizes nonribosomal biosynthesis of the peptide siderophore pyochelin in Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the antibiotic dihydroaeruginoate (Dha) and the siderophore pyochelin are produced from salicylate and cysteine by a thiotemplate mechanism involving the peptide synthetases PchE and PchF. A thioesterase encoded by the pchC gene was found to be necessary for maximal production of both Dha and pyochelin, but it was not required for Dha release from PchE and could not replace the thioesterase function specified by the C-terminal domain of PchF. In vitro, 2-aminobutyrate, a cysteine analog, was adenylated by purified PchE and PchF proteins. In vivo, this analog strongly interfered with Dha and pyochelin formation in a pchC deletion mutant but affected production of these metabolites only slightly in the wild type. Exogenously supplied cysteine overcame the negative effect of a pchC mutation to a large extent, whereas addition of salicylate did not. These data are in agreement with a role for PchC as an editing enzyme that removes wrongly charged molecules from the peptidyl carrier protein domains of PchE and PchF.     

Functional analysis of protein kinase networks in living cells: beyond "knock-outs" and "knock-downs"

The identification of over 500 protein kinases encoded by the human genome sequence offers one measure of the importance of protein kinase networks in cell biology. High throughput technologies for inactivating genes are producing an awe-inspiring amount of data on the cellular and organismal effects of reducing the levels of individual protein kinases. Despite these technical advances, our understanding of kinase networks remains imprecise. Major challenges include correctly assigning kinases to particular networks, understanding how they are regulated, and identifying the relevant in vivo substrates. Genetic methods provide a way of addressing these questions, but their application requires understanding the nuances of how different types of mutations can affect protein kinases. The goal of this article is to provide a brief introductory primer into these issues using examples from yeast MAPK cascades and to motivate future systematic genetic analysis focusing on individual residues of protein kinases.