An in vitro system to silence mitochondrial gene expression.

The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an in vitro system to silence translation in purified mitochondria. In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.

Discovery of a Regulatory Subunit of the Yeast Fatty Acid Synthase.

Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a γ subunit of the 2.6 megadalton α6-ß6S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The γ subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The γ subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS.

Structural insights into human co-transcriptional capping.

Co-transcriptional capping of the nascent pre-mRNA 5' end prevents degradation of RNA polymerase (Pol) II transcripts and suppresses the innate immune response. Here, we provide mechanistic insights into the three major steps of human co-transcriptional pre-mRNA capping based on six different cryoelectron microscopy (cryo-EM) structures. The human mRNA capping enzyme, RNGTT, first docks to the Pol II stalk to position its triphosphatase domain near the RNA exit site. The capping enzyme then moves onto the Pol II surface, and its guanylyltransferase receives the pre-mRNA 5'-diphosphate end. Addition of a GMP moiety can occur when the RNA is ∼22 nt long, sufficient to reach the active site of the guanylyltransferase. For subsequent cap(1) methylation, the methyltransferase CMTR1 binds the Pol II stalk and can receive RNA after it is grown to ∼29 nt in length. The observed rearrangements of capping factors on the Pol II surface may be triggered by the completion of catalytic reaction steps and are accommodated by domain movements in the elongation factor DRB sensitivity-inducing factor (DSIF).

Structure of activated transcription complex Pol II-DSIF-PAF-SPT6.

Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Homo sapiens DSIF, PAF and SPT6 was determined at 3.1 Å resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminal-domain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation.

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex.

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2-GTP-Met-tRNAiMet and eIF3. The 'open' 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The 'closed' form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

Structural basis for c-di-AMP-dependent regulation of the bacterial stringent response by receptor protein DarB.

The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as potential targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress conditions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3'3'-cGAMP, and AMP. We show that DarB forms a homodimer with a parallel, head-to-head assembly of the monomers. We also confirm the DarB dimer binds two cyclic dinucleotide molecules or two AMP molecules; only one adenine of bound c-di-AMP is specifically recognized by DarB, while the second protrudes out of the donut-shaped protein. This enables DarB to bind also 3'3'-cGAMP, as only the adenine fits in the active site. In absence of c-di-AMP, DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abolishes this interaction. Furthermore, the DarB crystal structures reveal no conformational changes upon c-di-AMP binding, leading us to conclude the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. We thus derived a structural model of the DarB-Rel complex via in silico docking, which was validated with mass spectrometric analysis of the chemically crosslinked DarB-Rel complex and mutagenesis studies. We suggest, based on the predicted complex structure, a mechanism of stringent response regulation by c-di-AMP.

The impact of wildlife and environmental factors on hantavirus infection in the host and its translation into human risk.

Identifying factors that drive infection dynamics in reservoir host populations is essential in understanding human risk from wildlife-originated zoonoses. We studied zoonotic Puumala orthohantavirus (PUUV) in the host, the bank vole (Myodes glareolus), populations in relation to the host population, rodent and predator community and environment-related factors and whether these processes are translated into human infection incidence. We used 5-year rodent trapping and bank vole PUUV serology data collected from 30 sites located in 24 municipalities in Finland. We found that PUUV seroprevalence in the host was negatively associated with the abundance of red foxes, but this process did not translate into human disease incidence, which showed no association with PUUV seroprevalence. The abundance of weasels, the proportion of juvenile bank voles in the host populations and rodent species diversity were negatively associated with the abundance index of PUUV positive bank voles, which, in turn, showed a positive association with human disease incidence. Our results suggest certain predators, a high proportion of young bank vole individuals, and a diverse rodent community, may reduce PUUV risk for humans through their negative impacts on the abundance of infected bank voles.

A Cross-linking Mass Spectrometry Approach Defines Protein Interactions in Yeast Mitochondria.

Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.

Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes.

Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria.

The paradox of nest reuse: early breeding benefits reproduction, but nest reuse increases nest predation risk.

Many animals build new nests every breeding season instead of saving time by reusing old ones. One hypothesis is that nest reuse leads to increased predation risk if predators memorize nest locations and revisit these sites. Here we examine patterns in the prevalence of facultative nest reuse. Further, we relate nest reuse and timing of breeding to nest predation risk, clutch size and nestling survival. We analyse 1570 breeding attempts of the Eurasian sparrowhawk (Accipiter nisus) from Denmark (1977-1997) and from two sites in Norway (1985-2017). The probability of reuse varied between study areas, increased in replacement clutches, and was lower in adults compared to 1-year-old breeders. Pairs reusing nests laid their first egg on average 2.6 ± 1.0 SE days later than those building new nests, suggesting they are compensating for an already late breeding schedule. Indeed, reuse increased nest predation risk, but we discovered no other productive effects of reuse. In non-predated nests, late breeders had both smaller clutches and lower nestling survival. We propose that nest predation is a contributing driver to the behaviour of building a new nest each year, whereas nest reuse is a strategy to compensate for delayed onset of breeding, mainly used by inexperienced males.

Characterization of Cerebrospinal Fluid via Data-Independent Acquisition Mass Spectrometry.

Cerebrospinal fluid (CSF) is in direct contact with the brain and serves as a valuable specimen to examine diseases of the central nervous system through analyzing its components. These include the analysis of metabolites, cells as well as proteins. For identifying new suitable diagnostic protein biomarkers bottom-up data-dependent acquisition (DDA) mass spectrometry-based approaches are most popular. Drawbacks of this method are stochastic and irreproducible precursor ion selection. Recently, data-independent acquisition (DIA) emerged as an alternative method. It overcomes several limitations of DDA, since it combines the benefits of DDA and targeted methods like selected reaction monitoring (SRM). We established a DIA method for in-depth proteome analysis of CSF. For this, four spectral libraries were generated with samples from native CSF ( n = 5), CSF fractionation (15 in total) and substantia nigra fractionation (54 in total) and applied to three CSF DIA replicates. The DDA and DIA methods for CSF were conducted with the same nanoLC parameters using a 180 min gradient. Compared to a conventional DDA method, our DIA approach increased the number of identified protein groups from 648 identifications in DDA to 1574 in DIA using a comprehensive spectral library generated with DDA measurements from five native CSF and 54 substantia nigra fractions. We also could show that a sample specific spectral library generated from native CSF only increased the identification reproducibility from three DIA replicates to 90% (77% with a DDA method). Moreover, by utilizing a substantia nigra specific spectral library for CSF DIA, over 60 brain-originated proteins could be identified compared to only 11 with DDA. In conclusion, the here presented optimized DIA method substantially outperforms DDA and could develop into a powerful tool for biomarker discovery in CSF. Data are available via ProteomeXchange with the identifiers PXD010698, PXD010708, PXD010690, PXD010705, and PXD009624.

To breed or not to breed: drivers of intermittent breeding in a seabird under increasing predation risk and male bias.

Intermittent breeding may be adaptive for long-lived species subjected to large accessory reproductive costs, but it may also reflect reduced adaptation to the environment, reducing population growth. Nevertheless, environmental influences on breeding propensity, particularly that of predation risk, remain poorly understood and difficult to study, because non-breeders are typically not identified. Female eiders Somateria mollissima from the Baltic Sea provide an excellent testbed, because nesting females have been exposed to intensifying predation and growing male bias that may increase female harassment. We based our study on long-term data (14 years) on females captured and marked at the nest, and females individually identified at sea irrespective of capture status. We hypothesized that breeding propensity decreases with increasing predation risk and male bias, and increases with breeder age. Consistent with our hypotheses, females nesting on islands with higher nest predation risk were more likely to skip breeding, and breeding probability increased with age. In contrast, the steep temporal decline in breeding propensity could not be reliably attributed to annual adult sex ratio or to the abundance of white-tailed sea eagles (Haliaeetus albicilla), the main predator on females, at the nearby Hanko Bird Observatory. Breeding probability showed significant consistent individual variation. Our results demonstrate that spatiotemporal variation in predation risk affects the decision to breed and high incidence of non-breeding was associated with low fledging success. The increased frequency of intermittent breeding in this declining population should be explicitly considered in demographic models, and emphasis placed on understanding the preconditions for successful reproduction.

Velocity of density shifts in Finnish landbird species depends on their migration ecology and body mass.

A multitude of studies confirm that species have changed their distribution ranges towards higher elevations and towards the poles, as has been predicted by climate change forecasts. However, there is large interspecific variation in the velocity of range shifts. From a conservation perspective, it is important to understand which factors explain variation in the speed and the extent of range shifts, as these might be related to the species' extinction risk. Here, we study shifts in the mean latitude of occurrence, as weighted by population density, in different groups of landbirds using 40 years of line transect data from Finland. Our results show that the velocity of such density shifts differed among migration strategies and increased with decreasing body size of species, while breeding habitat had no influence. The slower velocity of large species could be related to their longer generation time and lower per capita reproduction that can decrease the dispersal ability compared to smaller species. In contrast to some earlier studies of range margin shifts, resident birds and partial migrants showed faster range shifts, while fully migratory species were moving more slowly. The results suggest that migratory species, especially long-distance migrants, which often show decreasing population trends, might also have problems in adjusting their distribution ranges to keep pace with global warming.

Advantage of specialism: reproductive output is related to prey choice in a small raptor.

Predatory species' usage of different prey types is affected by both prey availability and selectivity. The diet during the breeding season may affect the reproductive success of individual pairs. We studied the prey use of a small reversed size-dimorphic raptor, the Eurasian sparrowhawk Accipiter nisus, with respect to prey weight on two organizational levels. Using 13 years of data from southern Norway, we related reproductive output of individual breeding events to prey size taken. Further, we assessed the regional variation in prey usage between five Fennoscandian populations. This was done by fitting optimum-type functions to the prey species' numbers or relative predation risks. Pairs that successfully completed the season with more fledglings displayed less variation in prey size, suggesting a possible adaptive benefit of diet specialism, or possibly a correlative effect due to higher prey availability or lower female hunting effort. This finding contrasts with earlier raptor studies, which have suggested benefits of dietary (and hence nutritional) diversity. Indeed, our results might be limited to nutritionally substitutable prey items. We also found a tendency suggesting that older females raised more fledglings than 1-year-old females. In the population-level analysis, we found that optimum-type functions with constant width and spatially variable average best described the relationship between relative predation risk and log weight. This can reflect local conditions, such as prey availability. Our findings and new methodological tools could apply to a broader spectrum of predators. They also highlight the role of viewing usage or choice of prey at several spatial scales.

Anthropogenic bottom-up and top-down impacts on boreal breeding waterbirds.

Wetland habitats are changing under multiple anthropogenic pressures. Nutrient leakage and pollution modify physico-chemical state of wetlands and affect the ecosystem through bottom-up processes, while alien predators affect the ecosystems in a top-down manner. Boreal wetlands are important breeding areas for several waterbird species, the abundances of which potentially reflect both bottom-up and top-down ecosystem processes. Here, we use long-term national monitoring data gathered from c. 130 waterbird breeding sites in Finland from the 1980s to the 2020s. We hypothesised that the physico-chemical state of the waters and increasing alien predator abundance both play a role in steering the waterbird population trends. We set out to test this hypothesis by relating population changes of 17 waterbird species to changes in water chemistry and to regional alien predator indices while allowing species-specific effects to vary with foraging niche (dabblers, invertivore divers, piscivorous divers, herbivores), nesting site, female mass and habitat (oligotrophic, eutrophic). We found niche and nesting site-specific, habitat-dependent changes in waterbird numbers. While the associations with higher phosphorus levels and browning water were in overall positive at the oligotrophic lakes, the numbers of invertivore and piscivore diving ducks were most strongly negatively associated with higher phosphorus levels and browning water at the eutrophic lakes. Furthermore, increased pH levels benefitted piscivores. Invertivore diving duck species nesting on the wetlands had declined most on sites with high alien predator indices. Large herbivorous species and species preferring oligotrophic lakes seem to be successful. We conclude that the large-scale breeding waterbird decline in Finland is closely connected to both bottom-up and top-down processes, where negative associations are emphasised especially at eutrophic lakes. Niche-, nest site- and habitat-specific management actions are required to conserve declining waterbird populations. Managing wetlands on catchments level together with alien predator control may provide important approaches to future wetland management.

A roadmap for ribosome assembly in human mitochondria.

Mitochondria contain dedicated ribosomes (mitoribosomes), which synthesize the mitochondrial-encoded core components of the oxidative phosphorylation complexes. The RNA and protein components of mitoribosomes are encoded on two different genomes (mitochondrial and nuclear) and are assembled into functional complexes with the help of dedicated factors inside the organelle. Defects in mitoribosome biogenesis are associated with severe human diseases, yet the molecular pathway of mitoribosome assembly remains poorly understood. Here, we applied a multidisciplinary approach combining biochemical isolation and analysis of native mitoribosomal assembly complexes with quantitative mass spectrometry and mathematical modeling to reconstitute the entire assembly pathway of the human mitoribosome. We show that, in contrast to its bacterial and cytosolic counterparts, human mitoribosome biogenesis involves the formation of ribosomal protein-only modules, which then assemble on the appropriate ribosomal RNA moiety in a coordinated fashion. The presence of excess protein-only modules primed for assembly rationalizes how mitochondria cope with the challenge of forming a protein-rich ribonucleoprotein complex of dual genetic origin. This study provides a comprehensive roadmap of mitoribosome biogenesis, from very early to late maturation steps, and highlights the evolutionary divergence from its bacterial ancestor.

Coupling in goshawk and grouse population dynamics in Finland.

Different prey species can vary in their significance to a particular predator. In the simplest case, the total available density or biomass of a guild of several prey species might be most relevant to the predator, but behavioural and ecological traits of different prey species can alter the picture. We studied the population dynamics of a predator-prey setting in Finland by fitting first-order log-linear vector autoregressive models to long-term count data from active breeding sites of the northern goshawk (Accipiter gentilis; 1986-2009), and to three of its main prey species (1983-2010): hazel grouse (Bonasa bonasia), black grouse (Tetrao tetrix) and capercaillie (T. urogallus), which belong to the same forest grouse guild and show synchronous fluctuations. Our focus was on modelling the relative significance of prey species and estimating the tightness of predator-prey coupling in order to explain the observed population dynamics, simultaneously accounting for effects of density dependence, winter severity and spatial correlation. We established nine competing candidate models, where different combinations of grouse species affect goshawk dynamics with lags of 1-3 years. Effects of goshawk on grouse were investigated using one model for each grouse species. The most parsimonious model for goshawk indicated separate density effects of hazel grouse and black grouse, and different effects with lags of 1 and 3 years. Capercaillie showed no effects on goshawk populations, while the effect of goshawk on grouse was clearly negative only in capercaillie. Winter severity had significant adverse effects on goshawk and hazel grouse populations. In combination, large-scale goshawk-grouse population dynamics are coupled, but there are no clear mutual effects for any of the individual guild members. In a broader context, our study suggests that pooling data on closely related, synchronously fluctuating prey species can result in the loss of relevant information, rather than increased model parsimony.

Impact of climate change and prey abundance on nesting success of a top predator, the goshawk.

Contemporary research has documented a large number of shifts in spring phenology and changes in distribution range although the average spring temperatures have increased by only 0.3-0.6 °C over the past 100 years. Generally, earlier breeding birds have larger clutch sizes, and the advancing spring could thus potentially increase breeding success. Shifts in spring phenology can, however, be crucial for bird reproduction, and mistiming the breeding event may even have negative consequences for population development. Our aim was to explore how weather and prey abundance relates to the breeding performance of a north European top predator, the northern goshawk Accipiter gentilis. Our nationwide dataset from Finland, spanning the period 1989-2004, shows that ambient weather has a greater impact on the timing and success of breeding than the density of grouse Tetraonidae, the main prey of goshawks. Higher early spring temperatures were associated with advancing hatching date of goshawks. Correspondingly, grouse density and temperature during laying and brooding were positively associated with brood size, while precipitation showed a negative connection. Applying our models to a future scenario of climate warming, combined with a 50 % reduction in grouse density, suggests that average breeding dates will advance only 2.5 days and average breeding success would remain the same. Notably, breeding success was not spatially equal throughout Finland, as northern and eastern populations suffered most from declining grouse densities. The observed pattern is thus the opposite to what is expected from a population situated at the northern edge of its distribution range, and thus may help to understand why populations may not increase at the northern edge of their thermal distribution due to climate change.

Mixed effects of a national protected area network on terrestrial and freshwater biodiversity.

Protected areas are considered fundamental to counter biodiversity loss. However, evidence for their effectiveness in averting local extinctions remains scarce and taxonomically biased. We employ a robust counterfactual multi-taxon approach to compare occupancy patterns of 638 species, including birds (150), mammals (23), plants (39) and phytoplankton (426) between protected and unprotected sites across four decades in Finland. We find mixed impacts of protected areas, with only a small proportion of species explicitly benefiting from protection-mainly through slower rates of decline inside protected areas. The benefits of protection are enhanced for larger protected areas and are traceable to when the sites were protected, but are mostly unrelated to species conservation status or traits (size, climatic niche and threat status). Our results suggest that the current protected area network can partly contribute to slow down declines in occupancy rates, but alone will not suffice to halt the biodiversity crisis. Efforts aimed at improving coverage, connectivity and management will be key to enhance the effectiveness of protected areas towards bending the curve of biodiversity loss.