During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4 gamma I (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5’ UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5’ UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity, but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5’ UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

Pieris brassicae L. is a serious pest of cultivated crucifers in several parts of the world. Larvae of P. brassicae also feed prolifically on garden nasturtium (Tropaeolum majus L., of the family Tropaeolaceae). Proteolytic digestion was studied in larvae feeding on multiple hosts. Fourth instars were collected from cauliflower fields before transfer onto detached, aerial tissues of selected host plants in the lab. Variable levels of midgut proteases were detected in larvae fed on different hosts using protein substrates (casein and recombinant RBCL cloned from cauliflower) and diagnostic, synthetic substrates. Qualitative changes in midgut trypsin activities and quantitative changes in midgut chymotrypsin activities were implicated in physiological adaptation of larvae transferred to T. majus. Midgut proteolytic activities were inhibited to different extents by serine protease inhibitors, including putative trypsin inhibitors isolated from herbivore-attacked and herbivore-free leaves of cauliflower (CfTI) and T. majus (TpTI). Transfer of larvae to T. majus significantly influenced feeding parameters but not necessarily when transferred to different tissues of the same host. Results obtained are relevant for devising sustainable pest management strategies, including transgenic approaches using genes encoding plant protease inhibitors.

Barley Yellow Dwarf Virus (BYDV) is a positive strand RNA virus that lacks the canonical 5′ 7-methylguanosine cap and a 3′ poly-A tail. Instead, BYDV utilizes a cruciform cap independent translation element (CITE) in its 3′UTR RNA (BYDV-like CITE or BTE) that binds eukaryotic translation initiation factor (eIF) 4F and recruits 40S ribosomal subunits in the presence of active helicase factors (eIF4A, eIF4B, eIF4F and ATP). A long-range, 5-nucleotide, base-pairing kissing loop interaction between the 3′BTE and a 5′UTR stem-loop is necessary for translation to initiate. The 40S–eIF complex does not bind to the BYDV 5′UTR, suggesting the involvement of additional factors. We identified eIF3 as a component of the 3′BTE recruited complex using affinity-tagged 3′BTE RNA pull-down assays. Fluorescence anisotropy binding and gel shift assays showed that the 3′BTE and 5′UTR RNAs can simultaneously and non-competitively bind eIF3 in the presence of active helicase factors forming a single, macromolecular complex. Further, quantitative studies showed eIF3 increased recruitment of the 40S subunit by more than 25-fold. We propose a new role for eIF3, where eIF3 bridges BYDV’s UTRs, stabilizes the long-range 5′-3′ interaction, and facilitates recruitment of the 40S–eIF complex to the 5′UTR, leading to translation initiation.

Ethnopharmacological relevance Solanaceae plants have been used as traditional medicines in Mizoram, India. This warrants the presence of therapeutic compounds and various bioactive phytochemicals in these plants, and characterizing their structures could lead to a possible focus for drug development. Aim of the study Solanaceae plants are incredible sources of proteins and minerals; some even have high medicinal values which has been recognized traditionally. The present study was designed to explore and document the ethnobotany, phytochemical and mineral nutrient composition, antimicrobial properties, antioxidant potential and to identify functional groups from edible species of Solanaceae from Mizoram, India. Materials and methods Field surveys and samples collection was conducted from Aizawl District, Mizoram, India. All the studied samples were extracted using Soxhlet apparatus for the analysis of bioactive compounds. The total phenol, total flavonoid and total anthocyanin contents were determined using standard methods. The antioxidant activities were measured using DPPH free radical scavenging, APX, CAT and SOD activities. The proximate analyses and mineral contents were determined by standard methods. The antibacterial potential was determined using the agar well diffusion method, and the functional groups were analysed using FTIR. All the results were reported as the mean ± standard deviation. The linear regression coefficient (R 2 ) for total flavonoid and phenolic content with antioxidant activity was analysed using Graph Pad Prism Version 5. P-value < 0.05 was considered significant. Results The phytochemical screenings showed the presence of alkaloids, tannins, flavonoids, terpenoids and saponins in all the samples. The highest total phenolic content was found in Solanum anguivi Lam. (29.51 mg GAE/g), and Capsicum annuum L. contained the highest total flavonoids (35.15 ± 0.03 mg/g). Proteins and carbohydrates contents were found to be the highest in Solanum melongena L. (28.49 mg/g) and Physalis angulata L. ( 35.64 mg/g) respectively. Elemental analysis showed the presence of Calcium (Ca), Copper (Cu), Iron (Fe), Manganese (Mn), Zinc (Zn), Potassium (K), Magnesium (Mg) and Sodium (Na) in high proportion in all the studied samples. All the plant extracts showed effective antibacterial activities against Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa . The Fourier Transformed Infra-Red Spectroscopy (FTIR) spectra revealed multiple functional groups in these plants species which could be used to identify bioactive compounds that can be subsequently utilized as herbal remedies for various ailments. Conclusion Our findings suggest that a considera...

Gene fragments encoding the large subunit (LS) of Rubisco (RBCL) were cloned from various species of host plants of phytophagous Lepidoptera and expressed as recombinant proteins in Escherichia coli. Recombinant RBCLs were compared among each other along with casein and native Rubisco as proteinaceous substrates for measuring total midgut protease activities of fourth instar larvae of Helicoverpa armigera feeding on casein, Pieris brassicae feeding on cauliflower, and Antheraea assamensis feeding on Litsea monopetala and Persea bombycina. Cognate rRBCL (from the pertinent host plant species) substrates performed similar to noncognate rRBCL reflecting the conserved nature of encoding genes and the versatile use of these recombinant proteins. Casein and recombinant RBCL generally outperformed native Rubisco as substrates, except where inclusion of a reducing agent in the enzyme assay likely unfolded the plant proteins. Levels of total midgut protease activities detected in A. assamensis larvae feeding on two primary host species were similar, suggesting that the suite(s) of digestive enzymes in these insects could hydrolyze a plant protein efficiently. Protease activities detected in the presence of protease inhibitors and the reducing agent dithiothreitol (DTT) suggested that recombinant RBCL was a suitable protein substrate for studying insect proteases using in vitro enzyme assays and substrate zymography.