Biology subjects

Chao, J. A.

Publications and source records attributed to Chao, J. A..

4 recordsLinked to original sources

bioRxiv2025

Quantitative comparison of methodologies for translation site imaging in living cells

Single-molecule imaging of translation sites in living cells has enabled the dynamics of protein synthesis to be investigated with high spatial and temporal resolution. These methodologies utilize the interaction between a multimerized epitope tag and its cognate fluorescent nanobody to detect nascent polypeptides as they emerge from the ribosome. Here, we present a systematic comparison of current methodologies and determine that the ALFA-tag reduces perturbations of mRNA expression and increases the fluorescent signal of translation sites.

biophysics↗

bioRxiv2021

Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1a.

Endoplasmic reticulum (ER) to nucleus homeostatic signalling, known as the unfolded protein response (UPR), relies on the non-canonical splicing of XBP1 mRNA. The molecular switch that initiates splicing is the oligomerization of the ER stress sensor and UPR endonuclease IRE1. While IRE1 can form large clusters that have been proposed to function as XBP1 processing centers on the ER, the actual oligomeric state of active IRE1 complexes as well as the targeting mechanism that recruits XBP1 to IRE1 oligomers, remain unknown. Here, we used a single molecule imaging approach to directly monitor the recruitment of individual XBP1 transcripts to the ER surface. We confirmed that stable ER association of unspliced XBP1 mRNA is established through HR2-dependent targeting and relies on active translation. In addition, we show that IRE1-catalyzed splicing mobilizes XBP1 mRNA from the ER membrane in response to ER stress. Surprisingly, we find that XBP1 transcripts are not recruited into large IRE1 clusters, which only assemble upon overexpression of fluorescently-tagged IRE1 during ER stress. Our findings support a model where ribosome-engaged, ER-poised XBP1 mRNA is processed by functional IRE1 assemblies that are homogenously distributed throughout the ER membrane.

cell biology↗

bioRxiv2019

The structural basis for RNA selectivity by the IMP family of RNA binding proteins

The Igf2 mRNA binding proteins (ZBP1/IMP1, IMP2, IMP3) are highly conserved post-transcriptional regulators of RNA stability, localization and translation. They play important roles in cell migration, neural development, metabolism and cancer cell survival. The knockout phenotypes of individual IMP proteins suggest that each family member regulates a unique pool of RNAs, yet evidence and an underlying mechanism for this is lacking. Here, we combine SELEX and NMR spectroscopy to demonstrate that the major RNA binding domains of the two most distantly related IMPs (ZBP1 and IMP2) bind to different consensus sequences and regulate targets consistent with their knockout phenotypes and roles in disease. We find that the targeting specificity of each IMP is determined by few amino acids in their variable loops. As variable loops often differ amongst KH domain paralogs, we hypothesize that this is a general mechanism for evolving specificity and regulation of the transcriptome.

biochemistry↗

bioRxiv2018

Single-molecule imaging of mRNA localization and regulation during the integrated stress response

Biological phase transitions form membrane-less organelles that generate distinct cellular environments. How molecules are partitioned between these compartments and the surrounding cellular space and the functional consequence of this localization is not well understood. Here, we report the localization of mRNA to stress granules(SGs) and processing bodies(PBs), which are distinct biomolecular condensates, and its effect on translation and mRNA degradation during the integrated stress response. Using single mRNA imaging in living human cells, we find that the interactions of mRNAs with SGs and PBs have different dynamics and that specific RNA binding proteins can anchor mRNAs within these compartments. During recovery from stress, mRNAs that were within SGs and PBs are translated and degraded at similar rates as their cytosolic counterparts.

molecular biology↗