Structure of the 16S rRNA pseudouridine synthase RsuA bound to uracil and UMP

In Escherichia coli, the pseudouridine synthase RsuA catalyzes formation of pseudouridine (psi) at position 516 in 16S rRNA during assembly of the 30S ribosomal subunit. We have determined the crystal structure of RsuA bound to uracil at 2.0 A resolution and to uridine 5'-monophosphate (UMP) at 2.65 A resolution. RsuA consists of an N-terminal domain connected by an extended linker to the central and C-terminal domains. Uracil and UMP bind in a cleft between the central and C-terminal domains near the catalytic residue Asp 102. The N-terminal domain shows structural similarity to the ribosomal protein S4. Despite only 15% amino acid identity, the other two domains are structurally similar to those of the tRNA-specific psi-synthase TruA, including the position of the catalytic Asp. Our results suggest that all four families of pseudouridine synthases share the same fold of their catalytic domain(s) and uracil-binding site.     

Characterization of the cloned full-length and a truncated human target of rapamycin: activity, specificity, and enzyme inhibition as studied by a high capacity assay

The mammalian target of rapamycin (mTOR/TOR) is implicated in cancer and other human disorders and thus an important target for therapeutic intervention. To study human TOR in vitro, we have produced in large scale both the full-length TOR (289 kDa) and a truncated TOR (132 kDa) from HEK293 cells. Both enzymes demonstrated a robust and specific catalytic activity towards the physiological substrate proteins, p70 S6 ribosomal protein kinase 1 (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphor-specific antibodies in Western blotting. We developed a high capacity dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) for analysis of kinetic parameters. The Michaelis constant (Km) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 microM, respectively. Dose-response and inhibition mechanisms of several known inhibitors, the rapamycin-FKBP12 complex, wortmannin and LY294002, were also studied in DELFIA. Our data indicate that TOR exhibits kinetic features of those shared by traditional serine/threonine kinases and demonstrate the feasibility for TOR enzyme screen in searching for new inhibitors.     

RECQ1 plays a distinct role in cellular response to oxidative DNA damage

RECQ1 is the most abundant RecQ homolog in humans but its functions have remained mostly elusive. Biochemically, RECQ1 displays distinct substrate specificities from WRN and BLM, indicating that these RecQ helicases likely perform non-overlapping functions. Our earlier work demonstrated that RECQ1-deficient cells display spontaneous genomic instability. We have obtained key evidence suggesting a unique role of RECQ1 in repair of oxidative DNA damage. We show that similar to WRN, RECQ1 associates with PARP-1 in nuclear extracts and exhibits direct protein interaction in vitro. Deficiency in WRN or BLM helicases have been shown to result in reduced homologous recombination and hyperactivation of PARP under basal condition. However, RECQ1-deficiency did not lead to PARP activation in undamaged cells and nor did it result in reduction in homologous recombination repair. In stark contrast to what is seen in WRN-deficiency, RECQ1-deficient cells hyperactivate PARP in a specific response to H₂O₂ treatment. RECQ1-deficient cells are more sensitive to oxidative DNA damage and exposure to oxidative stress results in a rapid and reversible recruitment of RECQ1 to chromatin. Chromatin localization of RECQ1 precedes WRN helicase, which has been shown to function in oxidative DNA damage repair. However, oxidative DNA damage-induced chromatin recruitment of these RecQ helicases is independent of PARP activity. As other RecQ helicases are known to interact with PARP-1, this study provides a paradigm to delineate specialized and redundant functions of RecQ homologs in repair of oxidative DNA damage.     

Multi‐modal adaptor‐clathrin contacts drive coated vesicle assembly

Clathrin‐coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated‐pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the β2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo‐EM structure of clathrin cages assembled in the presence of β2 hinge‐appendage (β2HA). We find that the β2‐appendage binds in at least two positions in the cage, demonstrating that multi‐modal binding is a fundamental property of clathrin‐AP2 interactions. In one position, β2‐appendage cross‐links two adjacent terminal domains from different triskelia. Functional analysis of β2HA‐clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin‐box motif on the hinge and the “sandwich site” on the appendage. We propose that β2‐appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.     

Amplified drought induced by climate change reduces seedling emergence and increases seedling mortality for two Mediterranean perennial herbs

Seedling recruitment is a bottleneck for population dynamics and range shift. The vital rates linked to recruitment by seed are impacted by amplified drought induced by climate change. In the Mediterranean region, autumn and winter seedling emergence and mortality may have strong impact on the overall seedling recruitment. However, studies focusing on the temporal dynamic of recruitment during these seasons are rare. This study was performed in a deciduous Mediterranean oak forest located in southern France and quantifies the impact of amplified drought conditions on autumn and winter seedling emergence and seedling mortality rates of two herbaceous plant species with meso‐Mediterranean and supra‐Mediterranean distribution (respectively, Silene italica and Silene nutans). Seedlings were followed from October 2019 to May 2020 in both undisturbed and disturbed plots where the litter and the aboveground biomass have been removed to create open microsites. Amplified drought conditions reduced seedling emergence and increased seedling mortality for both Silene species but these negative effects were dependent on soil disturbance conditions. Emergence of S. italica decreased only in undisturbed plots (−7%) whereas emergence of S. nutans decreased only in disturbed plots (−10%) under amplified drought conditions. The seedling mortality rate of S. italica was 51% higher under amplified drought conditions in undisturbed plots while that of S. nutans was 38% higher in disturbed plots. Aridification due to lower precipitation in the Mediterranean region will negatively impact the seedling recruitment of these two Silene species. Climate change effects on early vital rates may likely have major negative impacts on the overall population dynamic.     

Survival and migration patterns of naturally and hatchery-reared Atlantic salmon (Salmo salar) smolts in a Lake Ontario tributary using acoustic telemetry

1. Atlantic salmon (Salmo salar) smolts are often stocked into rivers to supplement natural reproduction, yet hatchery-reared fish have lower survival compared to wild conspecifics. However, few studies have assessed riverine migratory performance and survival differences in hatchery and wild smolts, or more specifically naturally reared smolts (hatchery fish released earlier as parr), particularly in rivers with weirs which may further reduce survival.
2. Using acoustic telemetry, including a subset of fish with novel transmitters that identify predation events, we assessed survival and migration patterns of hatchery- (2017: n = 32; 2018: n = 30) and naturally reared Atlantic salmon smolts (2017: n = 8; 2018: n = 30) in a Lake Ontario tributary with two weirs to better understand their ecology and assess the influence of environmental parameters on migration.
3. Naturally reared smolts were 13.9 times more likely to survive than hatchery-reared smolts and mark–recapture models indicated that weirs did not reduce survival for either group. Survival per km was lowest at the release site, indicating pre-migration mortality, and specifically high stocking-related mortality of hatchery-reared smolts. Speed and times of day fish migrated (i.e. migratory performance) did not vary by rearing group, suggesting that the high mortality of hatchery-reared smolts may be due to other factors related to hatchery and stocking operations. Overall mean (± SD) migration speed for smolts was 0.70 ± 0.39 km/hr and movements occurred significantly more frequently at night (18:00–06:00).
4. Smolts were detected in Lake Ontario after they left the river; however, the array in Lake Ontario was not conducive to providing much detail regarding movement patterns. There was no predation of the two predation tags detected in Lake Ontario, indicating that movements were made by smolts and not predators.
5. With ongoing restoration efforts of Atlantic salmon in Lake Ontario, it was important to understand the smolt migration patterns and success of the stocked fish. Our findings of similar migratory performance yet different relative survival of hatchery- and naturally reared smolts help inform management with regards to stocking strategies that could improve Atlantic salmon reintroduction success.

     

Growth, starvation and enzyme activity in white muscle of atlantic cod: at what point do muscle metabolic capacities change?

Muscle protein decreases only during prolonged starvation of Atlantic cod (Gadus morhua, Gadidae), but in the absence of protein renewal, muscle metabolic capacities may decrease before marked loss of muscle protein. This study aimed at elucidating the threshold at which decreases in growth and condition reduce muscle metabolic capacities, as well as identifying the indicators that best explain changes in metabolic capacities. To generate a wide spectrum of individual growth rates, condition factors and proximate compositions, cod showing different initial condition were fed or starved for different periods of time. The relationships between muscle proteins and metabolic enzyme activities (LDH and CCO) on one hand, and growth rate, condition factor, hepato- and gonadosomatic index and muscle and liver water and energy contents, on the other hand, were examined through linear regression models. Multiple linear regressions explained a large proportion of the observed variability in proteins and enzyme activities. Changes in LDH and CCO activities were not driven by changes in growth rate. Muscle water was the only significant correlate for both enzymes. Enzyme activities decreased as soon as muscle water began to rise. Increases in water content from 79 to 92% resulted in a 10-fold decrease in LDH and CCO activities.     

The effects of spacing on growth, morphology and biomass production and allocation in two hybrid poplar clones growing in the boreal region of Canada

Intra-clonal competition was studied in young hybrid poplar plantations to assess the effects of spacing on growth, biomass production and allocation, and morphological characteristics of above- and below-ground tree parts. Three spacings were used as whole-plots (1 × 1 m, 3 × 3 m and 5 × 5 m), with two hybrid poplar clones as subplots (BT747, Populus balsamifera L. × P. trichocarpa Torr. & Gray; MB915, P. maximowiczii A. Henry × P. balsamifera L.) in a split-plot design. After six growing seasons, diameter at breast height (dbh) increased by about 120% from the 1 × 1 m to the 5 × 5 m spacing for clone MB915, while there was no significant change in dbh for the other clone. The effect of spacing on height growth was opposite for the clones; it increased by about 175% from the narrowest to the widest spacing for clone MB915, while it decreased by about 27% for clone BT747. Estimates of above-ground biomass production after six growing seasons were significantly reduced with increasing spacing, with 29.6, 4.9 and 3.2 MgDM ha⁻¹ on average from the narrowest to the widest spacing. Branch traits and the vertical distribution of leaf area were the most affected by spacing for both clones, while live crown ratio and percentage of syllepsis did not change. Spacing also affected proportions of biomass allocated to stem, leaves, and branches, but allocation to roots did not change.