The 2'-O-ribose methyltransferase for cap 1 of spliced leader RNA and U1 small nuclear RNA in Trypanosoma brucei

mRNA cap 1 2'-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2'-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2'-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2'-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3'-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.     

Knowledge and Perceptions of Highly Pathogenic Avian Influenza (HPAI) among Poultry Traders in Live Bird Markets in Bali and Lombok,Indonesia

Highly Pathogenic Avian Influenza (HPAI) has been prevalent in Indonesia since 2003 causing major losses to poultry production and human deaths. Live bird markets are considered high risk areas due to the density of large numbers of mixed poultry species of unknown disease status. Understanding trader knowledge and perceptions of HPAI and biosecurity is critical to reducing transmission risk and controlling the disease. An interview-administered survey was conducted at 17 live bird markets on the islands of Bali and Lombok in 2008 and 2009. A total of 413 live poultry traders were interviewed. Respondents were mostly male (89%) with a mean age of 45 years (range: 19–81). The main source of AI information was TV (78%), although personal communication was also identified to be an important source, particularly among female traders (60%) and respondents from Bali (43%). More than half (58%) of live poultry traders interviewed knew that infected birds can transmit HPAI viruses but were generally unaware that viruses can be introduced to markets by fomites. Cleaning cages and disposing of sick and dead birds were recognized as the most important steps to prevent the spread of disease by respondents. Two thirds (n = 277) of respondents were unwilling to report sudden or suspicious bird deaths to authorities. Bali vendors perceive biosecurity to be of higher importance than Lombok vendors and are more willing to improve biosecurity within markets than traders in Lombok. Collectors and traders selling large numbers (>214) of poultry, or selling both chickens and ducks, have better knowledge of HPAI transmission and prevention than vendors or traders selling smaller quantities or only one species of poultry. Education was strongly associated with better knowledge but did not influence positive reporting behavior. Our study reveals that most live poultry traders have limited knowledge of HPAI transmission and prevention and are generally reluctant to report bird deaths. Greater efforts are needed to engage local government, market managers and traders in education and awareness programs, regulatory measures and incentive mechanisms. Understanding and evaluating the social responses to such an integrated approach could lead to more effective HPAI prevention and control.     

Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing

Background  

Despite tremendous progress in understanding the mechanisms of constitutive and alternative splicing, an important and widespread step along the gene expression pathway, our ability to deliberately regulate gene expression at this step remains rudimentary. The present study was performed to investigate whether a theophylline-dependent "splice switch" that sequesters the branchpoint sequence (BPS) within RNA-theophylline complex can regulate alternative splicing.     

Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.     

Life table analysis of the green rice leafhopper,Nephotettix virescens (distant) (Hemiptera: Cicadellidae), and efficient vector of rice tungro disease in asynchronous rice fields in Indonesia

Population dynamics of Nephotettix virescens was studied in 17 paddy fields transplanted at intervals of about 1 month in 1988–1990. The adult density was highest either in the immigrant or the 1st generation and sharply decreased to the 2nd generation. The survival rate of the 1st generation was lowest in the transition season when areal population density increased. Key factor analysis revealed that the nymphal and adult mortality of the 1st generation (kn) was the principal source of population fluctuations. No significant correaltion was found between kn and natural enemy density, natural enemy density/healthy egg density, or the precipitation during the nymphal period. On these bases adult emigration was suspected to be the key factor. Areal population build-up of N. virescens in the transition season was considered to occur as a result of increasing immigration to young stages of rice.     

Genomic signatures of host‐associated divergence and adaptation in a coral‐eating snail,Coralliophila violacea (Kiener, 1836)

The fluid nature of the ocean, combined with planktonic dispersal of marine larvae, lowers physical barriers to gene flow. However, divergence can still occur despite gene flow if strong selection acts on populations occupying different ecological niches. Here, we examined the population genomics of an ectoparasitic snail, Coralliophila violacea (Kiener 1836), that specializes on Porites corals in the Indo‐Pacific. Previous genetic analyses revealed two sympatric lineages associated with different coral hosts. In this study, we examined the mechanisms promoting and maintaining the snails’ adaptation to their coral hosts. Genome‐wide single nucleotide polymorphism (SNP) data from type II restriction site‐associated DNA (2b‐RAD) sequencing revealed two differentiated clusters of C. violacea that were largely concordant with coral host, consistent with previous genetic results. However, the presence of some admixed genotypes indicates gene flow from one lineage to the other. Combined, these results suggest that differentiation between host‐associated lineages of C. violacea is occurring in the face of ongoing gene flow, requiring strong selection. Indeed, 2.7% of all SNP loci were outlier loci (73/2,718), indicative of divergence with gene flow, driven by adaptation of each C. violacea lineage to their specific coral hosts.     

HepG2 cells expressing microRNA miR-122 support the entire hepatitis C virus life cycle

The liver-specific microRNA miR-122 is required for efficient hepatitis C virus (HCV) RNA replication both in cell culture and in vivo. In addition, nonhepatic cells have been rendered more efficient at supporting this stage of the HCV life cycle by miR-122 expression. This study investigated how miR-122 influences HCV replication in the miR-122-deficient HepG2 cell line. Expression of this microRNA in HepG2 cells permitted efficient HCV RNA replication and infectious virion production. When a missing HCV receptor is also expressed, these cells efficiently support viral entry and thus the entire HCV life cycle.     

Projection of the future EU forest CO2 sink as affected by recent bioenergy policies using two advanced forest management models

Forests of the European Union (EU) have been intensively managed for decades, and they have formed a significant sink for carbon dioxide (CO₂) from the atmosphere over the past 50 years. The reasons for this behavior are multiple, among them are: forest aging, area expansion, increasing plant productivity due to environmental changes of many kinds, and, most importantly, the growth rates of European forest having been higher than harvest rates. EU countries have agreed to reduce total emissions of GHG by 20% in 2020 compared to 1990, excluding the forest sink. A relevant question for climate policy is: how long will the current sink of EU forests be maintained in the near future? And could it be affected by other mitigation measures such as bioenergy? In this article we assess tradeoffs of bioenergy use and carbon sequestration at large scale and describe results of the comparison of two advanced forest management models that are used to project CO₂ emissions and removals from EU forests until 2030. EFISCEN, a detailed statistical matrix model and G4M, a geographically explicit economic forestry model, use scenarios of future harvest rates and forest growth information to estimate the future carbon balance of forest biomass. Two scenarios were assessed: the EU baseline scenario and the EU reference scenario (including additional bioenergy and climate policies). Our projections suggest a significant decline of the sink until 2030 in the baseline scenario of about 25–40% (or 65–125 Mt CO₂) compared to the models’ 2010 estimate. Including additional bioenergy targets of EU member states has an effect on the development of this sink, which is not accounted in the EU emission reduction target. A sensitivity analysis was performed on the role of future wood demand and proved the importance of this driver for the future sink development.     

An artificial riboswitch for controlling pre-mRNA splicing

Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3' splice site (3' ss) region of a model pre-mRNA (AdML-Theo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3' ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdML-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant.