Biology subjects

Chiang, B.-J.

Publications and source records attributed to Chiang, B.-J..

3 recordsLinked to original sources

bioRxiv2024

Massively Parallel Polyribosome Profiling Reveals Translation Defects of Human Disease-Relevant UTR Mutations

The untranslated regions (UTRs) of mRNAs harbor regulatory elements influencing translation efficiency. Although 3.7% of disease-relevant human mutations occur in UTRs, their exact role in pathogenesis remains unclear. Through metagene analysis, we mapped pathogenic UTR mutations to regions near coding sequences, with a focus on the upstream open reading frame (uORF) initiation site. Subsequently, we utilized massively parallel poly(ribo)some profiling to compare the ribosome associations of 6,555 pairs of wildtype and mutant UTR fragments. We identified 46 UTR variants that altered polysome profiles, with enrichment in pathogenic mutations. Both univariate analysis and the elastic net regression model highlighted the significance of motifs of short repeated sequences, including SRSF2 binding sites, as mutation hotspots that lead to aberrant translation. Furthermore, these polysome-shifting mutations exhibited considerable impact on RNA secondary structures, particularly for upstream AUG-containing 5 UTRs. Integrating these features, our model achieved high accuracy (AUROC > 0.8) in predicting polysome-shifting mutations in the test dataset. Additionally, several lines of evidence indicate that changes in uORF usage underlie the translation deficiency arising from these mutations. Illustrating this, we demonstrate that a pathogenic mutation in the IRF6 5 UTR suppresses translation of the primary open reading frame by creating a uORF. Remarkably, site- directed ADAR editing of the mutant mRNA rescued this translation deficiency. Overall, our study provides insights into the molecular mechanisms of UTR mutations and their links to clinical impacts through translation defects. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/589132v2_ufig1.gif" ALT="Figure 1000"> View larger version (50K): org.highwire.dtl.DTLVardef@57217corg.highwire.dtl.DTLVardef@575410org.highwire.dtl.DTLVardef@15013bforg.highwire.dtl.DTLVardef@408840_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Development of a tightly regulated copper-inducible transient gene expression system in Nicotiana benthamiana incorporating suicide exon and Cre recombinase

Chemical-inducible gene expression systems have been frequently used to regulate gene expression for functional genomics in various plant species. However, a convenient chemical-inducible system that can tightly regulate transgene expression in Nicotiana benthamiana is still missing. In this study, we developed a tightly regulated copper-inducible system that can be used to regulate transgene expression and perform cell death assays in N. benthamiana. We tested several chemical-inducible systems using Agrobacterium-mediated transient expression and found that the copper-inducible system showed the least concerns of leakiness issues. Using the MoClo-based synthetic biology approach, we optimized the design of the copper-inducible system and incorporated the use of the suicide exon HyP5SM/OsL5 and Cre/LoxP as additional regulatory elements to enhance the tightness of the regulation. This new design allowed us to tightly control the hypersensitive cell death induced by several tested NLRs and their matching AVRs, and it can also be easily applied to regulate the expression of other transgenes in transient expression assays. Our findings provide new approaches for both fundamental and translational studies in plant functional genomics.

plant biology↗

bioRxiv2023

Functional divergence shaped the network architecture of plant immune receptors

In solanaceous plants, several sensor NLRs and their helper NLRs, known as NRC, form a complex network to confer immunity against pathogens. While the sensor NLRs and downstream NRC helpers display diverse genetic compatibility, the evolution and molecular basis of the complex network structure remained elusive. Here we demonstrated that functional divergence of NRC3 variants has shaped the genetic architecture of the NLR network. Natural NRC3 variants form three allelic groups displaying distinct compatibilities with sensor NLRs. Ancestral sequence reconstruction and analyses of natural and chimeric variants identified six key amino acids involved in sensor-helper compatibility, with two residues critical for subfunctionalization. Co-functioning Rpi-blb2 and NRC3 variants showed stronger transient interactions upon effector detection, with NRC3 membrane-associated complexes forming subsequently. Our findings reveal how mutations in helper NLRs, particularly NRC3, have driven the evolution of their transient interactions with sensor NLRs, leading to subfunctionalization and contributing significantly to the complexity of the NRC network in plant immunity. TeaserHelper NLR subfunctionalization alters transient interactions with sensor NLRs, enhancing plant immune system complexity.

plant biology↗