Biology subjects

Joosten, J.

Publications and source records attributed to Joosten, J..

5 recordsLinked to original sources

bioRxiv2022

Dynamics and composition of small heat shock protein condensates and aggregates

Small heat shock proteins (sHSPs) are essential ATP-independent chaperones that protect the cellular proteome during stress. These proteins assemble into polydisperse oligomeric structures, the composition of which dramatically affects their chaperone activity. The biomolecular consequences of variations in sHSP ratios, especially inside living cells, remain elusive. Here, we study the consequences of altering the relative expression levels of HspB2 and HspB3. These chaperones are partners in a hetero-oligomeric complex, and genetic mutations that abolish their mutual interaction are associated with myopathic disorders. HspB2 displays three distinct phenotypes when co-expressed with HspB3 at varying ratios. Expression of HspB2 alone lead to formation of liquid nuclear condensates, while shifting the stoichiometry towards HspB3 resulted in the formation of large solid-like aggregates. Only cells co-expressing HspB2 with a limited amount of HspB3 showed a homogeneous nuclear distribution of HspB2. Strikingly, both condensates and aggregates were reversible, as shifting the HspB2:HspB3 balance in situ resulted in dissolution of these structures. To uncover the molecular composition of HspB2 condensates and aggregates, we used APEX-mediated proximity labelling. Most proteins interact transiently with the condensates and were neither enriched nor depleted. In contrast, we found that HspB2:HspB3 aggregates sequestered several disordered proteins among which autophagy factors, suggesting that the cell is actively attempting to clear these aggregates. This study presents a striking example of how changes in the relative expression levels of interacting proteins affects their phase behavior. Our approach can be a useful tool to study the role of protein stoichiometry in other biomolecular condensates. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/519563v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@bf5787org.highwire.dtl.DTLVardef@b7a18org.highwire.dtl.DTLVardef@17cd64corg.highwire.dtl.DTLVardef@d9392a_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Small heat shock protein hetero-oligomerization affects their chaperone function - The HspB2:HspB3 expression ratio determines phase separation and aggregation - HspB2 condensates and HspB2:HspB3 aggregates are fully reversible - Proximity labelling unveils autophagy factor recruitment to HspB2:HspB3 aggregates - Stoichiometry-dependant regulation of phase behaviour may be widespread in biology

molecular biology↗

bioRxiv2020

A regulatory network of a piRNA and lncRNA initiates responder and trailer piRNA formation during embryonic development of (i)Aedes(/i) mosquitoes

PIWI-interacting (pi)RNAs are small silencing RNAs that are crucial for the defense against transposable elements in germline tissues of animals. In Aedes aegypti mosquitoes, the piRNA pathway also contributes to gene regulation in somatic tissues, illustrating additional roles for piRNAs and PIWI proteins besides transposon repression. Here, we identify a highly abundant endogenous piRNA (propiR1) that associates with both Piwi4 and Piwi5. PropiR1-mediated target silencing requires base pairing in the seed region with supplemental base pairing at the piRNA 3 end. Yet, propiR1 represses a limited set of targets, among which the lncRNA AAEL027353 (lnc027353). Slicing of lnc027353 initiates production of responder and trailer piRNAs from the cleavage fragment. Expression of propiR1 commences early during embryonic development and mediates degradation of maternally provided lnc027353. Both propiR1 and its lncRNA target are conserved in the closely related Aedes albopictus mosquito, underscoring the importance of this regulatory network for mosquito development.

molecular biology↗

bioRxiv2020

An ancient satellite repeat controls gene expression and embryonic development in Aedes aegypti through a highly conserved piRNA

Tandem repeat elements such as the highly diverse class of satellite repeats occupy large parts of eukaryotic chromosomes. Most occur at (peri)centromeric and (sub)telomeric regions and have been implicated in chromosome organization, stabilization, and segregation1. Others are located more dispersed throughout the genome, but their functions remained largely enigmatic. Satellite repeats in euchromatic regions were hypothesized to regulate gene expression in cis by modulation of the local heterochromatin, or in trans via repeat-derived transcripts2,3. Yet, due to a lack of experimental models, gene regulatory potential of satellite repeats remains largely unexplored. Here we show that, in the vector mosquito Aedes aegypti, a satellite repeat promotes sequence-specific gene silencing via the expression of two abundant PIWI-interacting RNAs (piRNAs). Strikingly, whereas satellite repeats and piRNA sequences generally evolve extremely fast4-6, this locus was conserved for approximately 200 million years, suggesting a central function in mosquito biology. Tandem repeat-derived piRNA production commenced shortly after egg-laying and inactivation of the most abundant of the two piRNAs in early embryos resulted in an arrest of embryonic development. Transcriptional profiling in these embryos revealed the failure to degrade maternally provided transcripts that are normally cleared during maternal-to-zygotic transition. Our results reveal a novel mechanism in which satellite repeats regulate global gene expression in trans via piRNA-mediated gene silencing, which is fundamental to embryonic development. These findings highlight the regulatory potential of this enigmatic class of repeats.

molecular biology↗

bioRxiv2018

Induction and suppression of NF-κB signalling by a DNA virus of Drosophila

Interactions between the insect immune system and RNA viruses have been best studied in Drosophila, where RNA interference, NF-O_SCPLOWKC_SCPLOWB and JAK-STAT pathways underlie antiviral immunity. In response to these immune mechanisms, insect viruses have convergently evolved suppressors of RNA interference that act by diverse mechanisms to permit viral replication. However, interactions between the insect immune system and DNA viruses have received less attention, primarily because few Drosophila-infecting DNA virus isolates are available. Here, we use a recently-isolated DNA virus of Drosophila melanogaster, Kallithea virus, to probe known antiviral immune responses and virus evasion tactics in the context of DNA virus infection. We find that fly mutants for RNA interference and Immune deficiency (Imd), but not Toll, pathways are more susceptible to Kallithea virus infection. We identify the Kallithea virus-encoded protein gp83 as a potent inhibitor of Toll signalling, strongly suggesting that Toll mediates antiviral responses during Kallithea virus infection, but that it is suppressed by the virus. Further, we find that Kallithea gp83 inhibits Toll signalling either through NF-O_SCPLOWKC_SCPLOWB transcription factor regulation, or transcriptionally. Together, these results provide a broad description of known antiviral pathways in the context of DNA virus infection and identify the first Toll pathway inhibitor in a Drosophila virus, extending the known diversity of insect virus-encoded immune inhibitors.

microbiology↗

bioRxiv2018

The Tudor protein Veneno assembles the ping-pong amplification complex that produces viral piRNAs in Aedes mosquitoes

TUDOR-domain containing proteins facilitate PIWI interacting (pi)RNA biogenesis in Drosophila melanogaster and other model organisms. In Aedes aegypti mosquitoes, a somatically active piRNA pathway generates piRNAs from viral RNA during acute infection with cytoplasmic RNA viruses. Viral piRNA biogenesis requires ping-pong amplification by the PIWI proteins Ago3 and Piwi5. We hypothesized that Tudor proteins are required for viral piRNA production and performed a knockdown screen targeting all Ae. aegypti Tudor genes. Knockdown of several Tudor genes resulted in reduced viral piRNA levels, with silencing of AAEL012437 having the strongest effect. This protein, which we named Veneno, associates directly with Ago3 in an sDMA-dependent manner and localizes in cytoplasmic foci reminiscent of piRNA processing granules of Drosophila. Veneno-interactome analyses reveal a network of co-factors including the orthologs of the Drosophila piRNA pathway components Vasa and Yb, which in turn interacts directly with Piwi5. We propose that Veneno assembles a multi-protein complex for ping-pong dependent piRNA production from exogenous viral RNA.

microbiology↗