Structure of the LSm657 complex: an assembly intermediate of the LSm1-7 and LSm2-8 rings

The nuclear LSm2-8 (like Sm) complex and the cytoplasmic LSm1-7 complex play a central role in mRNA splicing and degradation, respectively. The LSm proteins are related to the spliceosomal Sm proteins that form a heteroheptameric ring around small nuclear RNA. The assembly process of the heptameric Sm complex is well established and involves several smaller Sm assembly intermediates. The assembly of the LSm complex, however, is less well studied. Here, we solved the 2.5 Å-resolution structure of the LSm assembly intermediate that contains LSm5, LSm6, and LSm7. The three monomers display the canonical Sm fold and arrange into a hexameric LSm657-657 ring. We show that the order of the LSm proteins within the ring is consistent with the order of the related SmE, SmF, and SmG proteins in the heptameric Sm ring. Nonetheless, differences in RNA binding pockets prevent the prediction of the nucleotide binding preferences of the LSm complexes. Using high-resolution NMR spectroscopy, we confirm that LSm5, LSm6, and LSm7 also assemble into a  60-kDa hexameric ring in solution. With a combination of pull-down and NMR experiments, we show that the LSm657 complex can incorporate LSm23 in order to assemble further towards native LSm rings. Interestingly, we find that the NMR spectra of the LSm57, LSm657-657, and LSm23-657 complexes differ significantly, suggesting that the angles between the LSm building blocks change depending on the ring size of the complex. In summary, our results identify LSm657 as a plastic and functional building block on the assembly route towards the LSm1-7 and LSm2-8 complexes.     

Fast selection of miRNA candidates based on large-scale pre-computed MFE sets of randomized sequences

Background

Small RNAs are important regulators of genome function, yet their prediction in genomes is still a major computational challenge. Statistical analyses of pre-miRNA sequences indicated that their 2D structure tends to have a minimal free energy (MFE) significantly lower than MFE values of equivalently randomized sequences with the same nucleotide composition, in contrast to other classes of non-coding RNA. The computation of many MFEs is, however, too intensive to allow for genome-wide screenings.

Results

Using a local grid infrastructure, MFE distributions of random sequences were pre-calculated on a large scale. These distributions follow a normal distribution and can be used to determine the MFE distribution for any given sequence composition by interpolation. It allows on-the-fly calculation of the normal distribution for any candidate sequence composition.

Conclusion

The speedup achieved makes genome-wide screening with this characteristic of a pre-miRNA sequence practical. Although this particular property alone will not be able to distinguish miRNAs from other sequences sufficiently discriminative, the MFE-based P-value should be added to the parameters of choice to be included in the selection of potential miRNA candidates for experimental verification.     

Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer

Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This "Q Exactive" instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an "enhanced Fourier Transformation" algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top 10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate- a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities.     

Active feeding of downstream migrating juvenile pink salmon (Oncorhynchus gorbuscha) revealed in a large Barents Sea river using diet and stable isotope analysis

The recent, rapid spreading of non-native pink salmon Oncorhynchus gorbuscha in the North Atlantic area has raised concerns about their possible negative impacts on native salmonid species. Potential interactions include competition for food resources during the short freshwater phase of juvenile O. gorbuscha, but little is known about their feeding behavior in the newly occupied North Atlantic rivers. Using stable isotope and stomach content analyses, patterns of freshwater feeding of non-native O. gorbuscha fry were studied in a large Fennoscandian river, the Teno, that discharges to the Barents Sea. Changes in stable isotope values (δ¹³C, δ¹⁵N, δ³⁴S) and stomach contents from the period of emergence (April to mid-May) to estuarine entry (late May/June) were examined and provided both temporally integrated and short-term indicators of freshwater feeding dependency. In addition, the occurrence of juvenile O. gorbuscha and changes in their length and weight during their emergence/migration period were investigated. Juvenile O. gorbuscha were at the spawning grounds from April through to mid-May with abundance peaking in mid-May. Fish moved to the estuary by late May and their abundance decreased toward June, and their body size increased concurrently. Stomach analyses indicated no feeding activity in April–early May in the spawning areas, but the stomach fullness indices increased markedly in fish sampled in the estuary in May and June. The most important prey items in stomachs were Chironomidae and Ephemeroptera larvae. Significant changes in all analysed stable isotopes were detected among sample periods, with a peak in mid-May and June showing significantly lower values than other sample periods. A change from the higher values reflective of parental marine feeding to the lower values reflective of freshwater feeding indicated active in-river feeding by juveniles during the study period. The documented active freshwater feeding of non-native juvenile O. gorbuscha suggests potential resource competition with native fluvial fishes, particularly salmonids.     

Genetic structure of the rare and endangered moss <Emphasis Type="Italic">Campylopus oerstedianus</Emphasis> (Dicranaceae) in Europe

The genetic structure, diversity and phylogeography of the moss species Campylopus oerstedianus in Europe was studied, based on the ITS region of the nrDNA of nine selected European populations. Although this species is only known in sterile stage, long-distance dispersal and gene flow among populations seem to be present within Europe. High levels of genetic differentiation between the investigated Greek population and the western European populations indicate a long lasting isolation. The populations from France and Switzerland root together and supposedly have a common origin. From the French populations, the one from the Pyrenees is the most basal one and the populations in the Massif Central and the Vosges Mts can be derived from it. This indicates relatively recent dispersal of the species from the Mediterranean to the north, in spite of the lack of sporophytes and the fact that the type of the dispersed propagules and their vectors are not obvious.     

Characterization of endogenous plasmids from Lactobacillus salivarius UCC118

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli.     

Assembly of two transgenes in an artificial chromatin domain gives highly coordinated expression in tobacco

The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation. We here present the first direct experimental support for this model in plants. Two reporter genes, the E. coli beta-glucuronidase gene and the firefly luciferase gene, driven by different promoters, were placed between copies of the chicken lysozyme A element, a member of the matrix-associated region (MAR) group of chromatin boundary elements, and introduced in tobacco (Nicotiana tabacum). In plants carrying A elements, quantitative enzyme activities and mRNA levels of both genes show high correlations compared to control plants. The A element thus creates an artificial chromatin domain that yields coordinated expression. Surprisingly, enzyme activities correlated poorly with their respective mRNA levels. We hypothesize that this indicates the occurrence of "error pipelines" in data generation: systematic errors of a given analytical method will point in the same direction and cancel out in correlation analysis, resulting in better correlations. In combining different methods of analysis, however, such errors do not cancel out and as a result relevant correlations can be masked. Such error pipelines will have to be taken into account when different types of (e.g., whole-genome) data sets are combined in quantitative analyses.     

A Self-Excising Cre Recombinase Allows Efficient Recombination of Multiple Ectopic Heterospecific Lox Sites in Transgenic Tobacco

To study the impact of different DNA configurations on the stability of transgene expression, a variant of the cre gene was developed. This variant allows for the highly efficient in planta removal of its own loxP-flanked coding sequence as well as other DNAs flanked by ectopic heterospecific lox sites, either lox511 or lox2272 or both, in trans. The plant intron-containing cre gene, cre ^INT , was configured in such a way that self-excision generated an intact hygromycin resistance selectable marker gene. In this combination, all selected transformants showed highly efficient excision. Plants obtained showed no indication of any chimerism, indicating a cell autonomous nature of the hygromycin selection during transformation and regeneration. The highly efficient concomitant removal of wildtype and heterospecific lox site-flanked DNA demonstrated that upon retransformation with the self-excising cre ^INT , sufficient amounts of Cre enzyme were produced prior to its removal. Plants obtained with cre ^INT showed much less frequently the Cre-associated phenomenon of reduced fertility than plants obtained with a continuous presence of Cre recombinase. The cre ^INT system has therefore advantages over systems with a continuously present Cre. The cre ^INT system was successfully used for removal of two chromatin boundary elements from transgene cassettes in tobacco. Analysis of plants with and without boundary elements on the same chromosomal location will contribute to a better evaluation of the role of such elements in the regulation of transgene expression in plants.     

Sequence polymorphisms cause many false cis eQTLs

Many investigations have reported the successful mapping of quantitative trait loci (QTLs) for gene expression phenotypes (eQTLs). Local eQTLs, where expression phenotypes map to the genes themselves, are of especially great interest, because they are direct candidates for previously mapped physiological QTLs. Here we show that many mapped local eQTLs in genetical genomics experiments do not reflect actual expression differences caused by sequence polymorphisms in cis-acting factors changing mRNA levels. Instead they indicate hybridization differences caused by sequence polymorphisms in the mRNA region that is targeted by the microarray probes. Many such polymorphisms can be detected by a sensitive and novel statistical approach that takes the individual probe signals into account. Applying this approach to recent mouse and human eQTL data, we demonstrate that indeed many local eQTLs are falsely reported as "cis-acting" or "cis" and can be successfully detected and eliminated with this approach.