Biology subjects

Khong, A.

Publications and source records attributed to Khong, A..

7 recordsLinked to original sources

bioRxiv2025

RNA structure directs RNA partitioning and is actively disrupted inside stress granules to enable cellular recovery

RNA structures play important roles in liquid-liquid phase separation. However, how it is regulated during stress response and stress granule formation is still under studied. Here, we performed in vivo RNA structure probing before and after sodium arsenite treatment, and in stress granules. While RNAs generally become more double-stranded upon stress, they maintain their single-strandedness inside stress granules. We showed that RNA single-strandedness enables increased inclusion inside stress granules and that stress granule-enriched RNAs form fewer intra- and intermolecular RNA-RNA interactions. Additionally, several RNA binding proteins including SRSF1 are enriched in differential structure regions. eCLIP analysis revealed that SRSF1 binds to single-stranded regions along RNAs, and increased SRSF1 binding enabled better inclusion of RNAs in stress granules, whereas depletion of SRSF1 decreased stress granule formation under mild oxidative stress. We also observed the active unwinding of RNAs inside stress granules regulated by helicases, including DDX3X, and showed that inhibition of DDX3X results in slower dissolution of stress granules during recovery. Our study reveals the existence of multiple mechanisms to maintain RNA single-strandedness inside stress granules and to allow reversibility of stress granule formation, highlighting the importance of regulating RNA structure to enable cellular plasticity and stress response.

molecular biology↗

bioRxiv2022

Building bridges from genome to physiology using machine learning and Drosophila experimental evolution

Drosophila experimental evolution, with its well-defined selection protocols, has long supplied useful genetic material for the analysis of functional physiology. While there is a long tradition of interpreting the effects of large-effect mutants physiologically, in the genomic era identifying and interpreting gene-to-phenotype relationships has been challenging, with many labs not resolving how physiological traits are affected by multiple genes throughout the genome. Drosophila experimental evolution has demonstrated that multiple phenotypes change due to the evolution of many loci across the genome, creating the scientific challenge of sifting out differentiated but noncausal loci for individual characters. The fused lasso additive model method (FLAM) allows us to infer some of the differentiated loci that have relatively greater causal effects on the differentiation of specific phenotypes. The experimental material used in the present study comes from 50 populations that have been selected for different life-histories and levels of stress resistance. Differentiation of cardiac robustness, starvation resistance, desiccation resistance, lipid content, glycogen content, water content, and body masses was assayed among 40 to 50 of these experimentally-evolved populations. Through FLAM, we combined physiological analysis from eight parameters with whole-body pooled-seq genomic data to identify potentially causally linked genomic regions. We have identified approximately 1,900 significantly differentiated 50 kb genomic windows among our 50 populations, with 161 of those identified genomic regions highly likely to have a causal effect connecting specific genome sites to specific physiological characters.

physiology↗

bioRxiv2022

Stress granules promote chemoresistance by triggering cellular quiescence

Cells respond to cellular stress by forming stress granules, molecular condensates containing non-translating messenger ribonucleoproteins. Stress granules form during chemotherapy and promote cell survival and chemoresistance, although the mechanism of this effect is not understood. We provide several lines of evidence that stress granules enhance cell survival by promoting cellular quiescence. First, we see a correlation between spontaneous stress granule formation and cell-cycle exit under non-stress conditions. Second, cells deficient in proteins required for stress granule formation (G3BP1/2) are less likely to exit the cell cycle under non-stress, stress, and chemotherapeutic conditions. Third, rescuing stress granule formation in G3BP1/2 knockout cells restores the fraction of cycling cells to wild-type levels. Finally, cells with enhanced stress granule formation (ddx6 knockout cells) show an increased propensity to exit the cell cycle. These results suggest that stress granules are important regulators of cellular quiescence, which could enable the identification of new anti-chemoresistance therapies that target stress granules.

molecular biology↗

bioRxiv2021

Limited effects of m6A modification on mRNA partitioning into stress granules

Recent studies have argued that the m6A modification of mRNAs promotes mRNA recruitment to stress granules through the interaction with YTHDF proteins (Anders et al., 2018; Ries et al., 2019). However, mRNAs that contain multiple m6A modified sites partition similarly into stress granules in both wild-type and m6A-deficient cells by single-molecule FISH suggesting m6A modifications play a minor role in mRNA partitioning into stress granules. Moreover, multiple linear regression analysis suggests m6A modification plays a minimal role in stress granule recruitment. Finally, the artificial tethering of 25 YTHDF proteins on reporter mRNAs leads to only a modest increase in mRNA partitioning to stress granules. These results indicate m6A modification makes a small, but measurable, contribution to recruiting specific mRNAs to stress granules.

molecular biology↗

bioRxiv2019

Modulation of RNA condensation by the eIF4A RNA helicase

Stress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules form in part through intermolecular RNA-RNA interactions, although the process of RNA condensation is poorly understood. In vitro, we demonstrate that RNA is effectively recruited to the surfaces of RNA or RNP condensates. We demonstrate that the DEAD-box protein eIF4A reduces RNA condensation in vitro and limits stress granule formation in cells. This defines a purpose for eIF4A to limit intermolecular RNA-RNA interactions in cells, thereby allowing for proper RNP function. These results establish an important role for DEAD-box proteins as ATP-dependent RNA chaperones that can limit the intermolecular condensation and entanglement of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates. eTOC BlurbStress granules are formed in part by the process of RNA condensation, which is mediated by and promotes trans RNA-RNA interactions. The essential DEAD-box protein and translation initiation factor eIF4A limits stress granule formation by reducing RNA condensation through its function as an ATP-dependent RNA binding protein, behaving analogously to how protein chaperones like HSP70 combat protein aggregates. HighlightsO_LIRNA condensates promote intermolecular RNA-RNA interactions at their surfaces C_LIO_LIeIF4A limits the recruitment of RNAs to stress granules in cells C_LIO_LIeIF4A reduces the nucleation of stress granules in cells C_LIO_LIRecombinant eIF4A1 inhibits the condensation of RNA in vitro in an ATP-dependent manner C_LI

biochemistry↗

bioRxiv2018

mRNP architecture in translating and stress conditions reveals an ordered pathway of mRNP compaction

Stress granules (SGs) are non-translating mRNP assemblies that form during stress. Herein, we use multiple smFISH probes for specific mRNAs to examine their SG recruitment and spatial organization. We observed that ribosome run-off is required for SG entry with long ORF mRNAs being delayed in SG accumulation, revealing SG transcriptome changes over time. Moreover, mRNAs are ~20X compacted from an expected linear length when translating and compact ~2 fold further in a stepwise manner beginning at the 5 end during ribosome run-off. Surprisingly, the 5 and 3 ends of the examined mRNAs were separated in non-stress conditions, but in non-translating conditions, the ends of AHNAK and DYNC1H1 mRNAs become close, suggesting the closed-loop model of mRNPs preferentially forms on non-translating mRNAs. These results suggest translation inhibition triggers a mRNP reorganization that brings ends closer, which has implications for the regulation of mRNA stability and translation by 3 UTR elements and the poly(A) tail.

molecular biology↗

bioRxiv2018

Imaging of single mRNA translation repression reveals diverse interactions with mRNP granules

During cellular stress mRNAs exit translation and accumulate in stress granules and P-bodies, although the dynamics of these interactions remain unclear. We imaged in real-time single mRNAs, their translational output, and mRNA-granule interactions during stress. We observed single mRNAs interacting with stress granules and P-bodies, with mRNAs moving bidirectionality between them. While translating mRNAs only interact with RNP granules dynamically, non-translating mRNAs can form stable associations that rigidly immobilize the mRNA within the granule. Imaging thousands of individual mRNA-granule interactions showed the probability of stable association increases with both mRNA length and granule size. Therefore, the recruitment of mRNAs to RNP granules involves both highly dynamic and stable interactions, influenced by several parameters, demonstrating a new layer of complexity in mRNA regulation during stress.\n\nOne Sentence SummarymRNAs interact with stress granules and P-bodies in stable and dynamic manners influenced by ribosome association, mRNA length, and granule size.

molecular biology↗