There are multiple RNA degradation mechanisms in eukaryotes, key among these is mRNA decapping, which requires the Dcp1-Dcp2 complex. Decapping is involved in various processes including nonsense-mediated decay (NMD), a process by which aberrant transcripts with a premature termination codon are targeted for translational repression and rapid decay. NMD is ubiquitous throughout eukaryotes and the key factors involved are highly conserved, although many differences have evolved. We investigated the role of Aspergillus nidulans decapping factors in NMD and found that they are not required, unlike Saccharomyces cerevisiae. Intriguingly, we also observed that the disruption of one of the decapping factors, Dcp1, leads to an aberrant ribosome profile. Importantly this was not shared by mutations disrupting Dcp2, the catalytic component of the decapping complex. The aberrant profile is associated with the accumulation of a high proportion of 25S rRNA degradation intermediates. We identified the location of three rRNA cleavage sites and show that a mutation targeted to disrupt the catalytic domain of Dcp2 partially suppresses the aberrant profile of Δdcp1 strains. This suggests that in the absence of Dcp1, cleaved ribosomal components accumulate and Dcp2 may be directly involved in mediating these cleavage events. We discuss the implications of this.
The role of post-transcriptional RNA modification is of growing interest. One example is the addition of non-templated uridine residues to the 3´ end of transcripts. In mammalian systems uridylation is integral to cell cycle control of histone mRNA levels. This regulatory mechanism is dependent on the nonsense mediated decay (NMD) component, Upf1, which promotes histone mRNA uridylation and degradation in response to the arrest of DNA synthesis. We have identified a similar system in Aspergillus nidulans, where Upf1 is required for the regulation of histone mRNA levels. However, other NMD components are also implicated, distinguishing it from the mammalian system. As in human cells, 3´ uridylation of histone mRNA is induced upon replication arrest. Disruption of this 3´ tagging has a significant but limited effect on histone transcript regulation, consistent with multiple mechanisms acting to regulate mRNA levels. Interestingly, 3´ end degraded transcripts are also subject to re-adenylation. Both mRNA pyrimidine tagging and re-adenylation are dependent on the same terminal-nucleotidyltransferases, CutA and CutB, and we show this is consistent with the in vitro activities of both enzymes. Based on these data we argue that mRNA 3´ tagging has diverse and distinct roles associated with transcript degradation, functionality and regulation.
Ganoderma boninense is known to be the causal agent for basal stem rot (BSR) affecting the oil palm industry worldwide thus cumulating to high economic losses every year. Several reports have shown that a compatible monokaryon pair needs to mate; producing dikaryotic mycelia to initiate the infection towards the oil palm. However, the molecular events occurs during mating process are not well understood. We performed transcriptome sequencing using Illumina RNA-seq technology and de novo assembly of the transcripts from monokaryon, mating junction and dikaryon mycelia of G. boninense. Raw reads from these three libraries were deposited in the NCBI database with accession number SRR1745787, SRR1745773 and SRR1745777, respectively.
The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at -12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.
Food waste (FW) minimization at the source by using food waste biodigester (FWBs) has a vast potential to lower down the impact of increasing organic fraction in municipal solid waste generation. To this end, this research sought to check the performance of locally isolated hydrolase-producing bacteria (HPB) to improve food waste biodegradation rate. Two under-explored HPB identified as Bacillus paralicheniformis GRA2 and Bacillus velezensis TAP5 were able to produce maximum amylase, cellulase, protease and lipase activities, and demonstrated a significant hydrolase synergy in co-culture fermentation. In vitro biodegradation analysis of both autoclaved and non-autoclaved FW revealed that the HPB inoculation was effective to degrade total solids (>62%), protein (>19%), total fat (>51), total sugar (>86%), reducing sugar (>38%) and starch (>50%) after 8-day incubation. All co-culture treatments were recorded superior to the respective monocultures and the uninoculated control. The results of FW biodegradation using batch-biodigester trial indicated that the 1500 mL and 1000 mL inoculum size of HPB inoculant reached a plateau on the 4th day, with gross biodegradation percentage (GBP) of >85% as compared to control (66.4%). The 1000 mL inoculum was sufficient to achieve the maximum GBP (>90%) of FW after an 8-day biodigestion in a FWB.
Glaciozyma antarctica PI12, is a psychrophilic yeast isolated from Antarctic sea. In this work, Expressed Sequence Tags (EST) from cells exposed to three different temperatures; 15 °C, 0 °C and -12 °C were generated to identify genes associated with cold adaptation. A total of 5376 clones from each library were randomly picked and sequenced. Comparative analyses from the resulting ESTs in each condition identified several groups of genes required for cold adaptation. Additionally, 319 unique transcripts that encoded uncharacterised functions were identified in the -12 °C library and are currently unique to G. antarctica. Gene expression analysis using RT-qPCR revealed two of the unknown genes to be up-regulated at -12 °C compared to 0 °C and 15 °C. These findings further contribute to the collective knowledge into G. antarctica cold adaptation and as a resource for understanding the ecological and physiological tolerance of psychrophilic microbes in general.
Lichen is a symbiotic organism that exists as a single composite body consisting of a mycobiont (fungus) and a photobiont (algae or a cyanobacterium). Many lichen species are considered as extremophiles due to their tolerance to radiation, desiccation, temperature and pollution. However, not all lichen species are tolerant to harsh environmental conditions as several species are sensitive for example to nitrogen, sulphur, acidity, heavy metals, halogens (e.g. fluoride) and ozone. Thus, to better understand why some lichens can withstand exposure to pollutants as opposed to those that are susceptible, we focused on the lichen species of Dirinaria known for their wide distribution in the tropics, subtropics and pantropical, and moderate tolerance to air pollution. Their moderate tolerance to air pollution affords them to thrive in good air quality environments as well as polluted air environments. Lichen samples of Dirinaria sp., UKM-J1 and UKM-K1, were respectively collected from two areas with different levels of air quality based on Air Pollutant Index or API (with index pollutant criteria of PM10, carbon monoxide, ozone, nitrogen dioxide and sulfur dioxide) in the outskirt of Jerantut (UKM-J1), a rural area in the middle of Peninsular Malaysia and the township of Klang (UKM-K1), in a busy area of the Klang Valley, Malaysia. API was monitored throughout 2012-2013 whereby the sample collection site in Klang showed markedly higher concentrations of pollutants in all the index pollutant criteria as compared to that of Jerantut. We performed transcriptome sequencing using Illumina RNA-seq technology and de novo assembly of the transcripts from the lichen samples. Raw reads from both libraries were deposited in the NCBI database with the accession number SRP138994.
Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 °C. The partially purified CbhI has a specific activity of 4.195 Umg-1 and a low Km value of 1.88 mM when p-nitrophenyl-β-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (kcat/Km) was 5.68 × 10-4 mM-1s-1, which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporium. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg-1), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (Ki) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.
Regular endurance training improves muscle oxidative capacity and reduces the risk of age-related disorders. Understanding the molecular networks underlying this phenomenon is crucial. Here, by exploiting the power of computational modeling, we show that endurance training induces profound changes in gene regulatory networks linking signaling and selective control of translation to energy metabolism and tissue remodeling. We discovered that knockdown of the mTOR-independent factor Eif6, which we predicted to be a key regulator of this process, affects mitochondrial respiration efficiency, ROS production, and exercise performance. Our work demonstrates the validity of a data-driven approach to understanding muscle homeostasis.
Extremely low temperatures present various challenges to life that include ice formation and effects on metabolic capacity. Psyhcrophilic microorganisms typically have an array of mechanisms to enable survival in cold temperatures. In this study, we sequenced and analysed the genome of a psychrophilic yeast isolated in the Antarctic region, Glaciozyma antarctica. The genome annotation identified 7857 protein coding sequences. From the genome sequence analysis we were able to identify genes that encoded for proteins known to be associated with cold survival, in addition to annotating genes that are unique to G. antarctica. For genes that are known to be involved in cold adaptation such as anti-freeze proteins (AFPs), our gene expression analysis revealed that they were differentially transcribed over time and in response to different temperatures. This indicated the presence of an array of adaptation systems that can respond to a changing but persistent cold environment. We were also able to validate the activity of all the AFPs annotated where the recombinant AFPs demonstrated anti-freeze capacity. This work is an important foundation for further collective exploration into psychrophilic microbiology where among other potential, the genes unique to this species may represent a pool of novel mechanisms for cold survival.