Biology subjects

Acker, J.

Publications and source records attributed to Acker, J..

7 recordsLinked to original sources

bioRxiv2025

Phosphorylation enables allosteric control of a viral condensate

In many viruses, intrinsically disordered proteins (IDPs) drive the formation of replicative organelles essential for viral production. In species A rotaviruses, the disordered protein NSP5 forms condensates in cells via liquid-liquid phase separation (LLPS). Yet the sequence diversity of NSP5 raises the question of whether condensate formation is conserved across all strains and if distinct variants employ alternative mechanisms for nucleating phase separation. Using a machine learning approach, we demonstrate that NSP5 variants differ significantly in their propensity to phase-separate. We engineered a variant incorporating amino acid signatures from strains with low LLPS tendency, which failed to phase separate in vitro yet supported the formation of replicative condensates in recombinant viruses in cells. Low-tendency LLPS strains require phosphorylation of NSP5 to nucleate phase separation, whereas high-tendency strains do not, suggesting distinct nucleation mechanisms. Furthermore, hydrogen-deuterium exchange mass spectrometry revealed a phosphorylation-driven allosteric switch between binding sites on the high-propensity variant. These findings establish that phosphorylation plays a context-dependent role in the formation of replicative organelles across diverse rotaviruses.

biophysics↗

bioRxiv2025

Scaling relations of multi-component phase coexistence boundaries

Multicomponent phase-separating molecular systems play a key role as membraneless organelles in living cells. Phase diagrams are indispensable for probing the concentration-dependent behaviour of these condensates, yet their interpretation has remained largely qualitative due to the challenges of modelling complex, multicomponent interactions. Here, we present a generic framework for quantitative analysis of phase diagrams. We derive an analytical expression for the phase boundary normal vector, which reveals two distinct scaling regimes: one for associative phase separation, characterised by a power-law scaling analogous to mass-action kinetics that allows for direct extraction of dense phase solute molar ratios, and another for condensate dissolution, where an exponential scaling quantifies three-body repulsion effects triggered by the addition of a new component. We demonstrate the practical utility of our framework by applying it to a range of experimentally measured phase diagrams, including those for proteins such as NSP5, NSP2, FUS, Whi3, G3BP1, and antimicrobial peptides. Collectively, our work not only provides a clear quantitative interpretation of phase diagrams but also opens new avenues for the rigorous characterisation of multicomponent phase-separating systems.

biophysics↗

bioRxiv2023

Maf1 phosphorylation is regulated through the action of prefoldin-like Bud27 on PP4 phosphatase in Saccharomyces cerevisiae

Bud27 is a prefoldin-like protein that participates in transcriptional regulation mediated by the three RNA polymerases in Saccharomyces cerevisiae. Lack of Bud27 significantly affects RNA pol III transcription, although the involved mechanisms have not been characterized. Here we show that Bud27 regulates the phosphorylation state of the RNA pol III transcriptional repressor, Maf1, influences its nuclear localization, and likely its activity. We demonstrate that Bud27 is associated with the Maf1 main phosphatase PP4 in vivo, and that this interaction is required for proper Maf1 dephosphorylation. Lack of Bud27 decreases the interaction among PP4 and Maf1, Maf1 dephosphorylation, and its nuclear entry. Our data uncover a new nuclear function of Bud27, identify PP4 as a novel Bud27 interactor and demonstrate the effect of this prefoldin-like on the posttranslational regulation of Maf1. Finally, our data reveal a broader effect of Bud27 on PP4 activity by influencing, at least, the phosphorylation of Rad53.

molecular biology↗

bioRxiv2022

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition

BackgroundTransposable elements are ubiquitous and play a fundamental role in shaping genomes during evolution. Since excessive transposition can be mutagenic, mechanisms exist in the cells to keep these mobile elements under control. Although many cellular factors regulating the mobility of the retrovirus-like transposon Ty1 in Saccharomyces cerevisiae have been identified in genetic screens, only very few of them interact physically with Ty1 integrase (IN). ResultsHere, we perform a proteomic screen to establish Ty1 IN interactome. Among the 265 potential interacting partners, we focus our study on the conserved CK2 kinase. We confirm the interaction between IN and CK2, demonstrate that IN is a substrate of CK2 in vitro and identify the modified residues. We find that Ty1 IN is phosphorylated in vivo and that these modifications are dependent in part on CK2. No significant change in Ty1 retromobility could be observed when we introduce phospho-ablative mutations that prevent IN phosphorylation by CK2 in vitro. However, the absence of CK2 holoenzyme results in a strong stimulation of Ty1 retrotransposition, characterized by an increase in Ty1 mRNA and protein levels and a high accumulation of cDNA. ConclusionOur study highlights an important role of CK2 in the regulation of Ty1 retrotransposition. We provide the first evidence that Ty1 IN is post-translationally modified in vivo, as observed for retroviral INs, and demonstrate that CK2 strongly represses Ty1 mobility by inhibiting Ty1 transcription. The proteomic approach enabled the identification of many new Ty1 IN interacting partners, whose potential role in the control of Ty1 mobility will be interesting to study.

biochemistry↗

bioRxiv2022

Structural basis of Ty1 integrase tethering on RNA polymerase III for targeted DNA integration

The yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. Here, we report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Unexpectedly, IN1 binding associates with insertion of subunit C11 C-terminal Zn-ribbon into the Pol III funnel, which provides atomic evidence for a two-metal mechanism during RNA cleavage. Moreover, unstructured regions of subunits C53 and C37 reorganize close to C11, likely explaining the connection between the C37/C53 heterodimer and C11 during transcription reinitiation. Our results suggest that IN1 binding induces a Pol III configuration that favors chromatin residence, thus improving the likelihood of Ty1 integration.

molecular biology↗

bioRxiv2020

Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

RNA viruses induce formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation. We demonstrate that rotavirus proteins NSP5 and NSP2 undergo phase separation in vitro and form RNA-rich condensates in vivo that can be reversibly dissolved by aliphatic diols. During infection, these RNA-protein condensates became less dynamic and impervious to aliphatic diols, indicating a transition from a liquid to solid state. Some aspects of assembly of rotavirus replication factories mirror the formation of cytoplasmic ribonucleoprotein granules, while the selective enrichment of viral transcripts appears to be a unique feature of these condensates. Such complex RNA-protein condensates that underlie replication of RNA viruses represent an attractive target for developing novel therapeutic approaches.

biophysics↗

bioRxiv2019

The Ty1 integrase nuclear localization signal is necessary and sufficient for retrotransposon targeting to tRNA genes

Integration of transposable elements into the genome is mutagenic. Mechanisms that target integration into relatively safe locations and minimize deleterious consequences for cell fitness have emerged during evolution. In budding yeast, the integration of the Ty1 LTR retrotransposon upstream of RNA polymerase III (Pol III)-transcribed genes requires the interaction between the AC40 subunit of Pol III and Ty1 integrase (IN1). Here we show that the IN1-AC40 interaction involves a short linker sequence in the bipartite nuclear localization signal (bNLS) of IN1. Mutations in this sequence do not impact the frequency of Ty1 retromobility, instead they decrease the recruitment of IN1 to Pol III-transcribed genes and the subsequent integration of Ty1 at these loci. The replacement of Ty5 retrotransposon targeting sequence by the IN1 bNLS induces Ty5 integration into Pol III-transcribed genes. Therefore, the IN1 bNLS is both necessary and sufficient to confer integration site specificity on Ty1 and Ty5 retrotransposons.

molecular biology↗