Biology subjects

Bard, J. A. M.

Publications and source records attributed to Bard, J. A. M..

5 recordsLinked to original sources

bioRxiv2024

Transcriptome-wide mRNA condensation precedes stress granule formation and excludes stress-induced transcripts

Stress-induced mRNP condensation is conserved across eukaryotes, resulting in stress granule formation under intense stresses, yet the mRNA composition and function of these condensates remain unclear. Exposure of ribosome-free mRNA following stress is thought to cause condensation and stress granule formation through mRNA-sequence-dependent interactions, leading to disproportionate condensation of long mRNAs. Here we show that, in striking contrast, virtually all mRNAs condense in response to multiple stresses in budding yeast with minor length dependence and often without stress granule formation. New transcripts escape mRNP condensation, enabling their selective translation. Inhibiting translation initiation causes formation of mRNP condensates that are distinct from stress granules and P-bodies; these translation-initiation-inhibited condensates (TIICs) are omnipresent, even in unstressed cells. Stress-induced mRNAs are excluded from TIICs due to the timing of their expression, indicating determinants of escape that are independent of sequence. Together, our results reveal a previously undetected level of translation-linked molecular organization and stress-responsive regulation.

cell biology↗

bioRxiv2023

An adaptive biomolecular condensation response is conserved across environmentally divergent species

Cells must sense and respond to sudden maladaptive environmental changes--stresses--to survive and thrive. Across eukaryotes, stresses such as heat shock trigger conserved responses: growth arrest, a specific transcriptional response, and biomolecular condensation of protein and mRNA into structures known as stress granules under severe stress. The composition, formation mechanism, adaptive significance, and even evolutionary conservation of these condensed structures remain enigmatic. Here we provide an unprecedented view into stress-triggered condensation, its evolutionary conservation and tuning, and its integration into other well-studied aspects of the stress response. Using three morphologically near-identical budding yeast species adapted to different thermal environments and diverged by up to 100 million years, we show that proteome-scale biomolecular condensation is tuned to species-specific thermal niches, closely tracking corresponding growth and transcriptional responses. In each species, poly(A)-binding protein--a core marker of stress granules--condenses in isolation at species-specific temperatures, with conserved molecular features and conformational changes modulating condensation. From the ecological to the molecular scale, our results reveal previously unappreciated levels of evolutionary selection in the eukaryotic stress response, while establishing a rich, tractable system for further inquiry.

cell biology↗

bioRxiv2022

Adaptive preservation of orphan ribosomal proteins in chaperone-stirred condensates

Ribosome biogenesis is among the most resource-intensive cellular processes, with ribosomal proteins accounting for up to half of all newly synthesized proteins in eukaryotic cells. During stress, cells shut down ribosome biogenesis in part by halting rRNA synthesis, potentially leading to massive accumulation of aggregation-prone "orphan" ribosomal proteins (oRPs). Here we show that during heat shock in yeast and human cells, oRPs accumulate as reversible condensates at the nucleolar periphery recognized by the Hsp70 co-chaperone Sis1/DnaJB6. oRP condensates are liquid-like in cell-free lysate but solidify upon depletion of Sis1 or inhibition of Hsp70. When cells recover from heat shock, oRP condensates disperse in a Sis1-dependent manner, and their ribosomal protein constituents are incorporated into functional ribosomes in the cytosol, enabling cells to efficiently resume growth. One sentence summaryDuring stress, molecular chaperones preserve "orphan" ribosomal proteins (RPs) - RPs that are not bound to rRNA - in liquid-like condensates, maintaining the RPs in a usable form and enabling cells to efficiently resume growth upon recovery from stress.

cell biology↗

bioRxiv2021

Ubiquitin modulates 26S proteasome conformational dynamics and promotes substrate degradation

The 26S proteasome is the major ATP-dependent protease in eukaryotic cells, where it catalyzes the degradation of thousands of proteins for general homeostasis and the control of vital processes. It specifically recognizes appropriate substrates through attached ubiquitin chains and uses its ATPase motor for mechanical unfolding and translocation into a proteolytic chamber. Here, we used single-molecule Forster Resonance Energy Transfer (FRET) measurements to provide unprecedented insights into the mechanisms of selective substrate engagement, ATP-dependent degradation, and the regulation of these processes by ubiquitin chains. Our assays revealed the proteasome conformational dynamics and allowed monitoring individual substrates as they progress through the central channel during degradation. We found that rapid transitions between engagement- and processing-competent conformations of the proteasome control substrate access to the ATPase motor. Ubiquitin-chain binding functions as an allosteric regulator to slow these transitions, stabilize the engagement-competent state, and facilitate degradation initiation. The global conformational transitions cease upon substrate engagement, and except for apparent motor slips when encountering stably folded domains, the proteasome remains in processing-competent states for substrate translocation and unfolding, which is further accelerated by ubiquitin chains. Our studies revealed the dependence of ATP-dependent substrate degradation on the conformational dynamics of the proteasome and its allosteric regulation by ubiquitin chains, which ensure substrate selectivity and prioritization in a crowded cellular environment.

biochemistry↗

bioRxiv2021

Chaperones directly and efficiently disperse stress-triggered biomolecular condensates

Heat shock triggers formation of intracellular protein aggregates and induction of a molecular disaggregation system. Although this system (Hsp100/Hsp70/Hsp40 in most cellular life) can disperse aggregates of model misfolded proteins, its activity on these model substrates is puzzlingly weak, and its endogenous heat-induced substrates have largely eluded biochemical study. Recent work has revealed that several cases of apparent heat-induced aggregation instead reflect evolved, adaptive biomolecular condensation. In budding yeast Saccharomyces cerevisiae, the resulting condensates depend on molecular chaperones for timely dispersal in vivo, hinting that condensates may be major endogenous substrates of the disaggregation system. Here, we show that the yeast disaggregation system disperses heat-induced biomolecular condensates of poly(A)-binding protein (Pab1) orders of magnitude more rapidly than aggregates of the most commonly used model substrate, firefly luciferase. Pab1 condensate dispersal also differs from aggregate dispersal in its molecular requirements, showing no dependence on small heat-shock proteins and a strict requirement for type II Hsp40. Unlike luciferase, Pab1 is not fully threaded (and thus not fully unfolded) by the disaggregase Hsp104 during dispersal, which we show can contribute to the extreme differences in dispersal efficiency. The Hsp70-related disaggregase Hsp110 shows some Pab1 dispersal activity, a potentially important link to animal systems, which lack cytosolic Hsp104. Finally, we show that the long-observed dependence of the disaggregation system on excess Hsp70 stems from the precise mechanism of the disaggregation system, which depends on the presence of multiple, closely spaced Hsp70s for Hsp104 recruitment and activation. Our results establish heat-induced biomolecular condensates of Pab1 as a direct endogenous substrate of the disaggregation machinery which differs markedly from previously studied foreign substrates, opening a crucial new window into the native mechanistic behavior and biological roles of this ancient system.

biochemistry↗