Dynamics of RNA modification by a multi-site-specific tRNA methyltransferase

In most organisms, the widely conserved 1-methyl-adenosine58 (m¹A₅₈) tRNA modification is catalyzed by an S-adenosyl-L-methionine (SAM)-dependent, site-specific enzyme TrmI. In archaea, TrmI also methylates the adjacent adenine 57, m¹A₅₇ being an obligatory intermediate of 1-methyl-inosine57 formation. To study this multi-site specificity, we used three oligoribonucleotide substrates of Pyrococcus abyssi TrmI (_PabTrmI) containing a fluorescent 2-aminopurine (2-AP) at the two target positions and followed the RNA binding kinetics and methylation reactions by stopped-flow and mass spectrometry. _PabTrmI did not modify 2-AP but methylated the adjacent target adenine. 2-AP seriously impaired the methylation of A₅₇ but not A₅₈, confirming that _PabTrmI methylates efficiently the first adenine of the A₅₇A₅₈A₅₉ sequence. _PabTrmI binding provoked a rapid increase of fluorescence, attributed to base unstacking in the environment of 2-AP. Then, a slow decrease was observed only with 2-AP at position 57 and SAM, suggesting that m¹A₅₈ formation triggers RNA release. A model of the protein–tRNA complex shows both target adenines in proximity of SAM and emphasizes no major tRNA conformational change except base flipping during the reaction. The solvent accessibility of the SAM pocket is not affected by the tRNA, thereby enabling S-adenosyl-L-homocysteine to be replaced by SAM without prior release of monomethylated tRNA.     

Abiotic constraints and biotic resistance control the establishment success and abundance of invasive <Emphasis Type="Italic">Humulus japonicus</Emphasis> in riparian habitats

Dispersal, abiotic and biotic constraints are all involved in explaining the success of invasive plants but how these factors influence the different life stages of an invader remains poorly known. Focusing on highly invaded riparian habitats we asked: (1) how do propagule pressure, resource availability and resident vegetation influence the success of the invasive Asian vine Humulus japonicus at different stages of its life cycle (i.e. seedling, vegetative and flowering) (2) what is the influence of increasing resource availability on the performance and trait plasticity of H. japonicus compared to a functionally similar co-occurring native species? To answer the first question we performed a repeated field survey along the Gardon River (S France) with detailed measurements of distance to the riverbed, soil characteristics, light availability, and resident vegetation cover. To answer the second question we used a greenhouse experiment to compare the biomass and three functional traits of H. japonicus and Galium aparine along a gradient of increasing water and nitrogen availability. Initial germination success was only determined by abiotic constraints, while the role of biotic resistance increased for later stages with establishment success favoured by the interaction of low resident vegetation cover and high soil fertility, and final integrated success favoured by high light availability. H. japonicus performed better and showed higher plasticity in plant height than G. aparine under increased resource availability while their biomass did not differ in the lower part of the resource gradient. Our study demonstrates that by combining field and experimental studies and analysing responses at different life stages we can gain a more complete understanding of how ecological filters shape successful invasions in the course of the life cycle.     

Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington’s Disease

1. Download high-res image (192KB)
2. Download full-size image

     

Comparison of redox and ligand binding behaviour of yeast and bovine cytochrome c oxidases using FTIR spectroscopy

Redox and CO photolysis FTIR spectra of yeast cytochrome c oxidase WT and mutants are compared to those from bovine and P. denitrificans CcOs in order to establish common functional features. All display changes that can be assigned to their E242 (bovine numbering) equivalent and to weakly H-bonded water molecules. The additional redox-sensitive band reported at 1736?cm^?1 in bovine CcO and previously assigned to D51 is absent from yeast CcO and couldn't be restored by introduction of a D residue at the equivalent position of the yeast protein. Redox spectra of yeast CcO also show much smaller changes in the amide I region, which may relate to structural differences in the region around D51 and the subunit I/II interface.     

The influence of population structure on gene expression and flowering time variation in the ubiquitous weed Capsella bursa‐pastoris (Brassicaceae)

Population structure is a potential problem when testing for adaptive phenotypic differences among populations. The observed phenotypic differences among populations can simply be due to genetic drift, and if the genetic distance between them is not considered, the differentiation may be falsely interpreted as adaptive. Conversely, adaptive and demographic processes might have been tightly associated and correcting for the population structure may lead to false negatives. Here, we evaluated this problem in the cosmopolitan weed Capsella bursa‐pastoris. We used RNA‐Seq to analyse gene expression differences among 24 accessions, which belonged to a much larger group that had been previously characterized for flowering time and circadian rhythm and were genotyped using genotyping‐by‐sequencing (GBS) technique. We found that clustering of accessions for gene expression retrieved the same three clusters that were obtained with GBS data previously, namely Europe, the Middle East and Asia. Moreover, the three groups were also differentiated for both flowering time and circadian rhythm variation. Correction for population genetic structure when analysing differential gene expression analysis removed all differences among the three groups. This may suggest that most differences are neutral and simply reflect population history. However, geographical variation in flowering time and circadian rhythm indicated that the distribution of adaptive traits might be confounded by population structure. To bypass this confounding effect, we compared gene expression differentiation between flowering ecotypes within the genetic groups. Among the differentially expressed genes, FLOWERING LOCUS C was the strongest candidate for local adaptation in regulation of flowering time.     

Inhibitor studies on non-photochemical plastoquinone reduction and H2 photoproduction in Chlamydomonas reinhardtii

In the absence of PSII, non-photochemical reduction of plastoquinones (PQs) occurs following NADH or NADPH addition in thylakoid membranes of the green alga Chlamydomonas reinhardtii. The nature of the enzyme involved in this reaction has been investigated in vitro by measuring chlorophyll fluorescence increase in anoxia and light-dependent O₂ uptake in the presence of methyl viologen. Based on the insensitivity of these reactions to rotenone, a type-I NADH dehydrogenase (NDH-1) inhibitor, and their sensitivity to flavoenzyme inhibitors and thiol blocking agents, we conclude to the involvement of a type-II NADH dehydrogenase (NDH-2) in PQ reduction. Intact Chlamydomonas cells placed in anoxia have the property to produce H₂ in the light by a Fe-hydrogenase which uses reduced ferredoxin as an electron donor. H₂ production also occurs in the absence of PSII thanks to the existence of a non-photochemical pathway of PQ reduction. From inhibitors effects, we suggest the involvement of a plastidial NDH-2 in PSII-independent H₂ production in Chlamydomonas. These results are discussed in relation to the absence of ndh genes in Chlamydomonas plastid genome and to the existence of 7 ORFs homologous to type-II NDHs in its nuclear genome.