Solution structure of a tmRNA-binding protein,SmpB, from Thermus thermophilus

The molecular mechanisms that govern cell movement are the subject of intense study, as they impact biologically and medically important processes such as leukocyte chemotaxis and angiogenesis, among others. We demonstrate that leukocyte chemotaxis is prevented by the macrolide immunosuppressant rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR)/ribosomal p70-S6 kinase (p70S6K) pathway. Both neutrophil chemotaxis and chemokinesis elicited by granulocyte-macrophage colony-stimulating factor (GM-CSF) were strongly inhibited by rapamycin with an IC₅₀ of 0.3 nM. Inhibition, although at a higher dose, was also observed when the chemoattractant was interleukin-8. As for the mechanism, rapamycin targeted the increase of phosphorylation of p70S6K due to GM-CSF treatment, as demonstrated with specific anti-p70S6K immunoprecipitation and subsequent immunoblotting with anti-T⁴²¹/S⁴²⁴ antibodies. Rapamycin also inhibited GM-CSF-induced actin polymerization, a hallmark of leukocyte migration. The specificity of the effect of rapamycin was confirmed by the use of the structural analog FK506, which did not have a significant effect on chemotaxis but effectively rescued rapamycin-induced p70S6K inhibition. This was expected from a competitive effect of both molecules on FK506-binding proteins (FKBP). Additionally, GM-CSF-induced chemotaxis was completely (>90%) blocked by a combination of rapamycin and the MAPK kinase (MEK) inhibitor PD-98059. In summary, the results presented here indicate for the first time that rapamycin, at sub-nanomolar concentrations, inhibits GM-CSF-induced chemotaxis and chemokinesis. This serves to underscore the relevance of the mTOR/S6K pathway in neutrophil migration.     

Solution structure of an RNA fragment with the P7/P9.0 region and the 3'-terminal guanosine of the tetrahymena group I intron

In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 3' terminus of the intron (omegaG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/omegaG) including the GBS and omegaG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, omegaG is recognized by the formation of a base triple with the G264 x C311 base pair, and this recognition is stabilized by the stacking interaction between omegaG and C262. The bulged structure at A263 causes a large helical twist angle (40 +/- 80) between the G264 x C311 and C262 x G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a "Bulge-and-Twist" (BT) pocket. With another twist angle between the C262 x G312 and G413 x C313 base pairs (45 +/- 100), the axis of GBS/omegaG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 A resolution electron density map (Golden et al., Science, 1998, 282:345-358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA.     

Binding of ribosome recycling factor to ribosomes,comparison with tRNA

The prokaryotic post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Because of the structural similarity of RRF and tRNA, we compared the biochemical characteristics of RRF binding to ribosomes with that of tRNA. Unesterified tRNA inhibited the disassembly of the post-termination complex in a competitive manner with RRF, suggesting that RRF binds to the A-site. Approximately one molecule of ribosome-bound RRF was detected after isolation of the RRF-ribosome complex. RRF and unesterified tRNA similarly inhibited the binding of N-acetylphenylalanyl-tRNA to the P-site of non-programmed but not programmed ribosomes. Under the conditions in which unesterified tRNA binds to both the P- and E-sites of non-programmed ribosomes, RRF inhibited 50% of the tRNA binding, suggesting that RRF does not bind to the E-site. The results are consistent with the notion that a single RRF binds to the A- and P-sites in a somewhat analogous manner to the A/P-site bound peptidyl tRNA. The binding of RRF and tRNA to ribosomes was influenced by Mg(2+) and NH(4)(+) ions in a similar manner.     

Comparing strategies for selection of low-density SNPs for imputation-mediated genomic prediction in U. S. Holsteins

SNP chips are commonly used for genotyping animals in genomic selection but strategies for selecting low-density (LD) SNPs for imputation-mediated genomic selection have not been addressed adequately. The main purpose of the present study was to compare the performance of eight LD (6K) SNP panels, each selected by a different strategy exploiting a combination of three major factors: evenly-spaced SNPs, increased minor allele frequencies, and SNP-trait associations either for single traits independently or for all the three traits jointly. The imputation accuracies from 6K to 80K SNP genotypes were between 96.2 and 98.2%. Genomic prediction accuracies obtained using imputed 80K genotypes were between 0.817 and 0.821 for daughter pregnancy rate, between 0.838 and 0.844 for fat yield, and between 0.850 and 0.863 for milk yield. The two SNP panels optimized on the three major factors had the highest genomic prediction accuracy (0.821–0.863), and these accuracies were very close to those obtained using observed 80K genotypes (0.825–0.868). Further exploration of the underlying relationships showed that genomic prediction accuracies did not respond linearly to imputation accuracies, but were significantly affected by genotype (imputation) errors of SNPs in association with the traits to be predicted. SNPs optimal for map coverage and MAF were favorable for obtaining accurate imputation of genotypes whereas trait-associated SNPs improved genomic prediction accuracies. Thus, optimal LD SNP panels were the ones that combined both strengths. The present results have practical implications on the design of LD SNP chips for imputation-enabled genomic prediction.     

Conformational Changes in HIV-1 Reverse Transcriptase that Facilitate Its Maturation

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Evaluation of linkage disequilibrium in wheat with an L1-regularized sparse Markov network

Linkage disequilibrium (LD) is defined as a stochastic dependence between alleles at two or more loci. Although understanding LD is important in the study of the genetics of many species, little attention has been paid on how a covariance structure between many loci distributed across the genome should be represented. Given that biological systems at the cellular level often involve gene networks, it is appealing to evaluate LD from a network perspective, i.e., as a set of associated loci involved in a complex system. We applied a Markov network (MN) to study LD using data on 1,279 markers derived from 599 wheat inbred lines. The MN attempts to account for association between two markers, conditionally on the remaining markers in the network model. In this study, the recovery of the structure of a LD network was done through two variants of pseudo-likelihoods subject to an L1 penalty on the MN parameters. It is shown that, while the L1-regularized Markov network preserves features of a Bayesian network (BN), the nodes in the resulting networks have fewer links. The resulting sparse network, encoding conditional independencies, provides a clearer picture of association than marginal LD metrics, and a sparse graph eases interpretation markedly, since it includes a smaller number of edges than a BN. Thus, an L1-regularized sparse Markov network seems appealing for representing conditional LD with high-dimensional genomic data, where variables, e.g., single nucleotide polymorphism markers, are expected to be sparsely connected.     

Solution structure of a GAAG tetraloop in helix 6 of SRP RNA from Pyrococcus furiosus

The NMR structure of a 12-mer RNA derived from the helix 6 of SRP RNA from Pyrococcus furiosus, whose loop-closing base pair is U.G, was determined, and the structural and thermodynamic properties of the RNA were compared with those of a mutant RNA with the C:G closing base pair. Although the structures of the two RNAs are similar to each other and adopt the GNRR motif the conformational stabilities are significantly different to each other It was suggested that weaker stacking interaction of the GAAG loop with the U:G closing base pair in 12-mer RNA causes the lower conformational stability.