Project description:To investigate gene specificity at the level of translation in both the human genome and viruses we devised a high-throughput bicistronic assay to quantify cap-independent translation. We uncover thousands of novel cap-independent translation sequences and provide insights on the landscape of translational regulation in both human and viruses. We find extensive translational elements in the 3â untranslated region (3âUTR) of human transcripts and the polyprotein region of un-capped RNA viruses. Through the characterization of regulatory elements underlying cap-independent translation activity we identify potential mechanisms of secondary structure, short sequence motif and base-pairing with the 18S rRNA. Furthermore, we systematically map the 18S rRNA regions for which reverse complementary enhance translation. Thus we provide insights into the mechanisms of translational control in humans and viruses. high-throughput bicistronic assay for obtaining cap-independent translation measurements of 55,000 fully designed sequences in parallel using fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq).
Project description:Sugarcane established industrial crop providing sugar, ethanol and biomass-derived electricity around the world. Cane sugar content is an important, breeding target, but its improvement remains very slow in many breeding programmes. Biotechnology strategies to improve sucrose accumulation made little progress at crop level, mainly due to the limited understanding of its regulation. MiRNAs regulate many metabolic processes in plants. However, their roles and target genes associated with sugarcane sucrose accumulation remains unknown. Here, we conducted high-throughput sequencing of transcriptome, small RNAs and degradome of leaves and stem of two sugarcane genotypes with contrasting sucrose content from the early to late stages of sucrose accumulation stages, which provided more insights into miRNA-associated gene regulation during sucrose accumulation. Transcriptome analysis identified 18,722 differentially expressed genes (DEGs) between both genotypes during sucrose accumulation. The major DEGs identified were involved in starch and sucrose metabolism, and photosynthesis etc. miRNA sequencing identified 563 known and 281 novel miRNAs from both genotypes during sucrose accumulation. Of these, 311 miRNAs were differentially expressed.752 targets of 368 miRNAs (609 targets for 260 known miRNAs and 168 targets for 108 novel miRNAs) were identified by degradom sequencing.Several known and novel miRNAs and their target genes associated with sugar metabolism, sugar transport and sucrose storage were identified in this study.This new insight into the complex network of sucrose accumulation in sugarcane will help identify candidate targets for sucrose improvement in sugarcane through molecular means.
Project description:To better prepare for future viral outbreaks, scalable and adaptable platforms to study emerging infections are essential. Understanding virus–host interactions, particularly the mechanisms of cell entry, is critical for developing effective therapeutics and vaccines. Current approaches often rely on live virus assays requiring high-containment facilities, limiting speed, scalability, and accessibility. As a proof-of-principle, we developed a novel screening platform—Ceudovitox—using pseudotyped viruses (PVs) bearing the chikungunya virus (CHIKV) envelope protein. These PVs were engineered to express herpes simplex virus-1 thymidine kinase, enabling selective killing of infected cells with ganciclovir. A heterogeneous CRISPR-Cas9 knockout cell pool was then screened using this "killer" PV system, allowing identification of CHIKV entry factors via next-generation sequencing.