Discovery of small molecules that target a tertiary-structured RNA.

There is growing interest in therapeutic intervention that targets disease-relevant RNAs using small molecules. While there have been some successes in RNA-targeted small-molecule discovery, a deeper understanding of structure-activity relationships in pursuing these targets has remained elusive. One of the best-studied tertiary-structured RNAs is the theophylline aptamer, which binds theophylline with high affinity and selectivity. Although not a drug target, this aptamer has had many applications, especially pertaining to genetic control circuits. Heretofore, no compound has been shown to bind the theophylline aptamer with greater affinity than theophylline itself. However, by carrying out a high-throughput screen of low-molecular-weight compounds, several unique hits were identified that are chemically distinct from theophylline and bind with up to 340-fold greater affinity. Multiple atomic-resolution X-ray crystal structures were determined to investigate the binding mode of theophylline and four of the best hits. These structures reveal both the rigidity of the theophylline aptamer binding pocket and the opportunity for other ligands to bind more tightly in this pocket by forming additional hydrogen-bonding interactions. These results give encouragement that the same approaches to drug discovery that have been applied so successfully to proteins can also be applied to RNAs.

How Job Demands and Resources Relate to Experiences of Bullying and Negative Acts among University Employees.

This article addresses a gap in the work psychology literature regarding psychosocial working conditions and bullying among staff in academic organizations. We examine the influences, institutional demands, and resources attached to given academic positions, such as how the level of social support and cooperation influence the level of experienced negative acts at work and bullying in different work groups in an academic work environment. We also examine whether some professions or positions in an academic organization are more vulnerable due to organizational structure, perceived and experienced resources, and demands to bullying or experiencing more negative acts at work. A common division of different employees in the university sector is between administrative/technical staff and scientific personnel. Our hypothesis in this study is that there are significant differences among these two groups regarding levels of experienced social support and cooperation, as well as levels of experienced negative acts at work. We postulate that differences in job demands and resources lead to significantly different levels of self-reported bullying for the two main groups of personnel. We expect scientific personnel to be more exposed to negative acts at work and bullying due to differences in the demands and resources associated with these positions.

The relationships between consumer liking, sensory and chemical attributes of Vitis vinifera L. cv. Pinotage wines elaborated with different Oenococcus oeni starter cultures.

BACKGROUND: Malolactic fermentation (MLF) mediated by lactic acid bacteria (LAB) has been shown to modulate chemical and sensory attributes of wine. This study investigated the relation between consumer liking, chemical and sensory attributes of Vitis vinifera L. cv. Pinotage wines that were made over two vintages by four different lactic acid Oenococcus oeni starter cultures as well as a control treatment where MLF was prevented. RESULTS: Descriptive analysis showed that the sensory attributes buttery, caramel, vegetative flavour, fruity and nutty aroma differed significantly between the wines. These effects on the wines were not the same for the two vintages tested. Preference mapping results showed that the sensory attributes influenced the average consumer liking. The main chemical and sensory correlations found for MLF-treated wines were related to 2,3-butanedione (diacetyl) with the buttery character and various esters with fruity aromas. CONCLUSION: Although the direct effect of the bacterial starter cultures on wine sensory attributes is difficult to establish, and subject to variation over vintage, the present work suggests that the contribution of LAB starter cultures to wine sensory attributes can influence consumer liking. Selection of an MLF starter culture can thus potentially be used to develop specific wine styles.

Biochemical characterization of the Caenorhabditis elegans FBF.CPB-1 translational regulation complex identifies conserved protein interaction hotspots.

Caenorhabditis elegans CPB-1 (cytoplasmic polyadenylation element binding protein homolog-1) and FBF (fem-3 mRNA binding factor) are evolutionary conserved regulators of mRNA translation that belong to the CPEB (cytoplasmic polyadenylation element binding) and PUF (Pumilio and FBF) protein families, respectively. In hermaphrodite worms, CPB-1 and FBF control key steps during germline development, including stem cell maintenance and sex determination. While CPB-1 and FBF are known to interact, the molecular basis and function of the CPB-1⋅FBF complex are not known. The surface of CPB-1 that interacts with FBF was localized using in vivo and in vitro methods to a 10-residue region at the N-terminus of the protein and these residues are present in the FBF-binding protein GLD-3 (germline development defective-3). PUF proteins are characterized by the presence of eight α-helical repeats (PUF repeats) arranged side by side in an elongated structure. Critical residues for CPB-1 binding are found in the extended loop that connects PUF repeats 7 and 8. The same FBF residues also mediate binding to GLD-3, indicating a conserved binding mode between different protein partners. CPB-1 binding was competitive with GLD-3, suggestive of mutual exclusivity in vivo. RNA binding measurements demonstrated that CPB-1 alters the affinity of FBF for specific RNA sequences, implying a functional model where the coregulatory protein CPB-1 modulates FBF target selection.

A protein.protein interaction platform involved in recruitment of GLD-3 to the FBF.fem-3 mRNA complex.

The Pumilio and FBF (PUF) family of RNA-binding proteins interacts with protein partners to post-transcriptionally regulate mRNAs in eukaryotes. The interaction between PUF family member fem-3 binding factor (FBF) and germline development defective-3 (GLD-3) protein promotes spermatogenesis in Caenorhabditis elegans by increasing expression of the fem-3 mRNA. Defined here in these studies is the molecular basis for this critical interaction. A 10-amino-acid region within GLD-3 is required for FBF binding, while a 7-amino-acid loop in FBF between PUF repeats 7 and 8 is necessary for GLD-3 binding. These short sequences are conserved, as other FBF-binding proteins bear sequences similar to those in GLD-3 and other C. elegans PUF proteins contain sequences similar to those in FBF. The FBF-binding region of GLD-3 forms a ternary complex with FBF on the point mutation element (PME) in the fem-3 3' untranslated region, and formation of this GLD-3⋅FBF complex does not impact the RNA-binding activity of FBF. These data raise the possibility of alternative models involving the formation of a GLD-3⋅FBF⋅RNA complex in the regulation of germline mRNAs.

Cooperativity in RNA-protein interactions: global analysis of RNA binding specificity.

The control and function of RNA are governed by the specificity of RNA binding proteins. Here, we describe a method for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes. The method yields affinities for a vast array of RNAs in a single experiment, including both low- and high-affinity sites. It is reproducible and accurate. Using this approach,we analyzed members of the PUF (Pumilio and FBF) family of eukaryotic mRNA regulators. Our data identify effects of a specific protein partner on PUF-RNA interactions, reveal subsets of target sites not previously detected, and demonstrate that designer PUF proteins can precisely alter specificity. The approach described here is, in principle, broadly applicable for analysis of any molecule that binds RNA, including proteins, nucleic acids, and small molecules.

Identification of a conserved interface between PUF and CPEB proteins.

Members of the PUF (Pumilio and FBF) and CPEB (cytoplasmic polyadenylation element-binding) protein families collaborate to regulate mRNA expression throughout eukaryotes. Here, we focus on the physical interactions between members of these two families, concentrating on Caenorhabditis elegans FBF-2 and CPB-1. To localize the site of interaction on FBF-2, we identified conserved amino acids within C. elegans PUF proteins. Deletion of an extended loop containing several conserved residues abolished binding to CPB-1. We analyzed alanine substitutions at 13 individual amino acids in FBF-2, each identified via its conservation. Multiple single point mutations disrupted binding to CPB-1 but not to RNA. Position Tyr-479 was particularly critical as multiple substitutions to other amino acids at this position did not restore binding. The complex of FBF-2 and CPB-1 repressed translation of an mRNA containing an FBF binding element. Repression required both proteins and was disrupted by FBF-2 alleles that failed to bind CPB-1 or RNA. The equivalent loop in human PUM2 is required for binding to human CPEB3 in vitro, although the primary sequences of the human and C. elegans PUF proteins have diverged in that region. Our findings define a key region in PUF/CPEB interactions and imply a conserved platform through which PUF proteins interact with their protein partners.

The structure of the NXF2/NXT1 heterodimeric complex reveals the combined specificity and versatility of the NTF2-like fold.

NXF1-like members of the NXF (nuclear export factor) family orchestrate bulk nuclear export of mRNA, while functionally distinct NXF variant proteins carry out separate substrate-specific and tissue-specific RNA regulation. Metazoan organisms possess at least one NXF1-like gene and one or more NXF variant genes. Heterodimerization of both proteins with the NXT (NTF2-related export) protein is central to NXF family function; however, given the multiplicity of NXF/NXT complexes, the specificity and mechanism of heterodimerization remain unclear. Here, we report the structural and functional analyses of the Caenorhabditis elegans NXF variant ceNXF2 bound to ceNXT1. Contacts crucial for NXF/NXT heterodimer stability and specificity, including a probable site for phosphoregulation, have been identified. The ceNXF2 NTF2 domain bears at least two nucleoporin (Nup) binding pockets necessary for the colocalization of ceNXF2/ceNXT1 at the nuclear envelope. Unexpectedly, one Nup binding pocket is formed at the heterodimer interface of the ceNXF2/ceNXT1 complex, demonstrating that NXT binding directly regulates NXF function.

The structure of Aquifex aeolicus ribosomal protein S8 reveals a unique subdomain that contributes to an extremely tight association with 16S rRNA.

The assembly of ribonucleoprotein complexes occurs under a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. The ribosomal protein S8 from Aquifex aeolicus (AS8) is unique in that there is a 41-residue insertion in the consensus S8 sequence. In addition, AS8 exhibits an unusually high affinity for the 16S ribosomal RNA, characterized by a picomolar dissociation constant that is approximately 26,000-fold tighter than the equivalent interaction from Escherichia coli. Deletion analysis demonstrated that binding to the minimal site on helix 21 occurred at the same nanomolar affinity found for other bacterial species. The additional affinity required the presence of a three-helix junction between helices 20, 21, and 22. The crystal structure of AS8 was solved, revealing the helix-loop-helix geometry of the unique AS8 insertion region, while the core of the molecule is conserved with known S8 structures. The AS8 structure was modeled onto the structure of the 30S ribosomal subunit from E. coli, suggesting the possibility that the unique subdomain provides additional backbone and side-chain contacts between the protein and an unpaired base within the three-way junction of helices 20, 21, and 22. Point mutations in the protein insertion subdomain resulted in a significantly reduced RNA binding affinity with respect to wild-type AS8. These results indicate that the AS8-specific subdomain provides additional interactions with the three-way junction that contribute to the extremely tight binding to ribosomal RNA.

Consumers liking of private labels. An evaluation of intrinsic and extrinsic orange juice cues.

The objective of this article is to compare product quality and brand choice for private labels (PL) and national brands (NB). Over the past two decades, PL have gained larger and larger share of grocery sales, and nowadays PL play a crucial part in the European food retail sector. Since it is stated that most PL have moved on from being mostly low cost me-too products to become also premium products, we want to investigate if objective and perceived quality of PL fits the quality of NB. Four hypotheses are stated and tested on orange juice data from Norway. A trained sensory panel and consumers (n=105) evaluated six juice samples that vary according to three factors. These factors were (1) Brand (PL and NB), (2) Treatment (Gentle heat treatment and Pasteurized) and (3) Pulp (with and without). Principal component analysis, two-way ANOVA, and PLS regression were conducted, and the results indicate that variation in quality exists both among PL and NB, there is a large discrepancy between blind liking and brand choice, and that consumers with a positive attitude towards PL are more likely to choose a PL instead of a NB.

Protein-induced conformational changes of RNA during the assembly of human signal recognition particle.

The human signal recognition particle (SRP) is a large RNA-protein complex that targets secretory and membrane proteins to the endoplasmic reticulum membrane. The S domain of SRP is composed of roughly half of the 7SL RNA and four proteins (SRP19, SRP54, and the SRP68/72 heterodimer). In order to understand how the binding of proteins induces conformational changes of RNA and affects subsequent binding of other protein subunits, we have performed chemical and enzymatic probing of all S domain assembly intermediates. Ethylation interference experiments show that phosphate groups in helices 5, 6 and 7 that are essential for the binding of SRP68/72 are all on the same face of the RNA. Hydroxyl radical footprinting and dimethylsulphate (DMS) modifications show that SRP68/72 brings the lower part of helices 6 and 8 closer. SRP68/72 binding also protects the SRP54 binding site (helix 8 asymmetric loop) from chemical modification and RNase cleavage, whereas, in the presence of both SRP19 and SRP68/72, the long strand of helix 8 asymmetric loop becomes readily accessible to chemical and enzymatic probes. These results indicate that the RNA platform observed in the crystal structure of the SRP19-SRP54M-RNA complex already exists in the presence of SRP68/72 and SRP19. Therefore, SRP68/72, together with SRP19, rearranges the 7SL RNA in an SRP54 binding competent state.

Identification of the RNA binding regions of SRP68/72 and SRP72 by systematic mutagenesis of human SRP RNA.

Within the large domain of the human signal recognition particle (SRP), 18 mutant SRP RNAs were constructed to disrupt Watson-Crick and G-U basepairs in helices 5, 6 and 8. Using a double-filter assay, the competitive binding of the mutant RNAs to purified human SRP68/72 or to a 7.4 kDa RNA-binding fragment of SRP72 (72frg) was measured. Binding of SRP68/72 was impaired by several mutations in the large domain with the most pronounced effects caused by changes in helix 5 (residues 222-231) and helix 8 (residues 176-191 and 202-214). Binding of the 72frg was diminished prominently by altering helix 5, in particular residues 120-128, and was unaffected by deleting helices 6 and 8. Deleting helix 8 diminished binding of SRP68/72 to a greater extent than deleting helix 6. The data suggest that nucleotide residues throughout most of the large SRP domain are directly and/or indirectly engaged in the binding of SRP68. In contrast, SRP72 binds only to a portion of the 5ef region.

Structure, function and evolution of the signal recognition particle.

The signal recognition particle (SRP) is a ribonucleoprotein particle essential for the targeting of signal peptide-bearing proteins to the prokaryotic plasma membrane or the eukaryotic endoplasmic reticulum membrane for secretion or membrane insertion. SRP binds to the signal peptide emerging from the exit site of the ribosome and forms a ribosome nascent chain (RNC)-SRP complex. The RNC-SRP complex then docks in a GTP-dependent manner with a membrane-anchored SRP receptor and the protein is translocated across or integrated into the membrane through a channel called the translocon. Recently considerable progress has been made in understanding the architecture and function of SRP.