Biology subjects

Jeske, M.

Publications and source records attributed to Jeske, M..

7 recordsLinked to original sources

bioRxiv2026

Localization-dependent activation of the DEAD-box ATPase Vasa by eLOTUS domains

DEAD-box RNA helicases remodel RNA structures in many cellular pathways, yet how their activity is spatially controlled in cells remains poorly understood. The Drosophila germline helicase Vasa functions in ovaries only when localized to cytoplasmic granules, a process mediated by eLOTUS-domain proteins. Here, we define the mechanism by which eLOTUS domains activate Vasa. Biochemical analyses reveal that Vasa alone is largely inactive. eLOTUS domains bind the open conformation of Vasa and promote formation of the closed RNA- and ATP-bound state by accelerating RNA engagement. This stimulation requires a positively charged intrinsically disordered sequence within eLOTUS that increases RNA association. Mutations in this element abolish Vasa stimulation while preserving binding. We demonstrate that Vasa activation is essential in vivo. Together, these findings reveal a localization-dependent mechanism for regulating the DEAD-box helicase Vasa, in which enzymatic activity is gated by a spatially restricted cofactor.

biochemistry↗

bioRxiv2025

Probing direct interactions between nuclear proteins in cells with nxReLo

Nearly all biological processes depend on protein-protein interactions (PPIs). While various methods exist to study these interactions, investigating those that involve nuclear proteins, including structurally complex proteins containing long disordered regions, remains a significant challenge. Here, we developed nxReLo, a simple and fast cell culture-based colocalization assay, designed to identify and characterize interactions between nuclear proteins. PIWI-interacting RNAs (piRNAs) safeguard germline genome integrity, and the PPIs that facilitate piRNA production are therefore essential for animal reproduction, yet remain incompletely understood. We used nxReLo to investigate interactions between members of the Drosophila melanogaster Rhino-Deadlock-Cutoff (RDC) complex and two associated components, Bootlegger and Moonshiner, a nuclear protein network required for piRNA expression. We demonstrate the utility of the nxReLo assay by systematically screening pairwise interactions within the RDC network and by assembling a multiprotein complex from multiple components. By combining nxReLo assays with AlphaFold structural prediction, we characterized the Cutoff-Deadlock and the Bootlegger-Deadlock complexes in detail, providing molecular and structural insights. Specifically, we refined the domains involved in the interaction and identified interface point mutations that interfered with complex formation, validating the predicted structures. In conclusion, nxReLo facilitates rapid and simple testing of direct interactions between nuclear proteins in a cellular context, which is particularly important when working with structurally challenging proteins or when established interaction assays prove unsuccessful.

biochemistry↗

bioRxiv2024

Recurrent innovation of protein-protein interactions in the Drosophila piRNA pathway

Despite being essential for fertility, genome defence pathway genes often evolve rapidly. However, little is known about the molecular basis of this adaptation. Here, we characterize the evolution of a protein interaction network within the PIWI-interacting small RNA (piRNA) genome defence pathway in Drosophila at unprecedented scale and evolutionary resolution. We uncover pervasive rapid evolution of a protein interaction network anchored at the Heterochromatin Protein 1 (HP1) paralog Rhino. Using complementary phylogenetic analysis, high-throughput yeast-two-hybrid matrix screening, and in vivo interaction analyses in cross-species transgenic flies, we characterized three distinct evolutionary protein interaction trajectories across [~]40 million years of Drosophila evolution. The data set covering 11 piRNA pathway proteins of five Drosophila species revealed several protein interactions that are fully conserved, indicating functional conservation despite overall rapid amino acid sequence change. Other interactions are preserved through co-evolution and were detected only between proteins within or from closely related species. We also identified sets of species-restricted protein interactions which, through rewiring of a Rhino-anchored transcription factor network, may preserve critical roles in enabling and adapting piRNA production from heterochromatic loci. In sum, our analyses dissected principles of interaction evolution in an adaptively evolving protein-protein interaction network uncovering evolutionary and functional insight into germline piRNA production across Drosophila species. Our work provides key experimental evidence in support of a model proposing that intermolecular interaction innovation is a major molecular mechanism of evolutionary adaptation in protein-coding genes.

molecular biology↗

bioRxiv2024

Molecular insight into the network of Drosophila cytoplasmic piRNA pathway proteins through a combination of systematic interaction screening and structural prediction

piRNA-bound PIWI proteins mediate the silencing of transposons at both the transcriptional and post-transcriptional levels, processes that are critical for genome integrity and fertility in animals. While numerous additional proteins are known to be essential for piRNA biogenesis and function in Drosophila and other animals, their molecular and mechanistic functions have remained largely unknown. To improve our molecular understanding of the Drosophila piRNA pathway, we used a cell culture-based protein-protein interaction assay called ReLo to perform a systematic pairwise interaction screen involving 22 factors operating in the cytoplasm, including PIWI proteins, Tudor domain-containing proteins (TDRDs), RNA helicases, and mitochondrial surface proteins. Through additional ReLo interaction testing and structural modeling using AlphaFold-Multimer, we have characterized six protein complexes at the molecular and structural levels. We believe that the results of this screen and our methodological approach are likely to guide future research into the molecular mechanisms underlying piRNA biogenesis and function.

biochemistry↗

bioRxiv2023

Structural basis for binding of Smaug to the GPCR Smoothened and to the germline inducer Oskar

Drosophila Smaug and its orthologs comprise a family of mRNA repressor proteins that exhibit various functions during animal development. Smaug proteins contain a characteristic RNA-binding sterile- motif (SAM) domain and a conserved but uncharacterized N-terminal domain (NTD). Here, we resolved the crystal structure of the NTD of the human SAM domain-containing protein 4A (SAMD4A, a.k.a. Smaug1) to 2.0 [A] resolution, which revealed its composition of a homodimerization D-subdomain and a subdomain with similarity to a PHAT domain. Furthermore, we show that Drosophila Smaug directly interacts with the Drosophila germline inducer Oskar and with the Hedgehog signaling transducer Smoothened through its D-PHAT domain. We determined the crystal structure of the D-PHAT domain of Smaug in complex with a Smoothened -helical peptide to 1.61 [A] resolution. The peptide binds within a groove that is formed by both the D- and PHAT subdomains. Structural modeling supported by experimental data suggested that an -helix within the disordered region of Oskar binds to the D-PHAT domain in a mode similar to Smoothened. Together, our data uncover the N-terminal D-PHAT domain of Smaug as peptide-binding domain.

biochemistry↗

bioRxiv2022

RNA binding proteins Smaug and Cup induce CCR4-NOT-dependent deadenylation of the nanos mRNA in a reconstituted system

Posttranscriptional regulation of the maternal nanos mRNA is essential for the development of the anterior - posterior axis of the Drosophila embryo. The nanos RNA is regulated by the protein Smaug. Binding to Smaug recognition elements (SREs) in the nanos 3-UTR, Smaug nucleates the assembly of a larger repressor complex including the eIF4E-T paralog Cup and five additional proteins. The Smaug-dependent complex represses translation of nanos and induces its deadenylation by the CCR4-NOT deadenylase. Here we report an in vitro reconstitution of the Drosophila CCR4-NOT complex and Smaug-dependent deadenylation. We find that Smaug by itself is sufficient to cause deadenylation by the Drosophila or human CCR4-NOT complexes in an SRE-dependent manner. CCR4-NOT subunits NOT10 and NOT11 are dispensable, but the NOT module, consisting of NOT2, NOT3 and the C-terminal part of NOT1, is required. Smaug interacts with the C-terminal domain of NOT3. Both catalytic subunits of CCR4-NOT contribute to Smaug-dependent deadenylation. Whereas the CCR4-NOT complex itself acts distributively, Smaug induces a processive behavior. The cytoplasmic poly(A) binding protein (PABPC) has but a minor effect on Smaug-dependent deadenylation. Among the additional constituents of the Smaug-dependent repressor complex, Cup also facilitates CCR4-NOT-dependent deadenylation, both independently and in cooperation with Smaug.

biochemistry↗

bioRxiv2022

ReLo: a simple colocalization assay to identify and characterize physical protein-protein interactions

The characterization of protein-protein interactions (PPIs) is fundamental for understanding biochemical processes. Many methods have been established to identify and study direct PPIs; however, the screening and investigation of PPIs involving large or poorly soluble proteins remain challenging. As a result, we developed ReLo, a simple, rapid, and versatile cell culture-based method for detecting and investigating interactions in a cellular context. Importantly, our data strongly suggest that with ReLo specifically direct binary PPIs are detected. By applying additional bridging experiments ReLo can also be used to determine the binding topology of subunits within multiprotein complexes. Moreover, ReLo has the potential to identify protein domains that mediate complex formation, screen for interfering point mutations, study interactions that depend on conformation or protein arginine methylation, and it is sensitive to drugs that mediate or interfere with an interaction. Taken together, ReLo is a simple and quick alternative for the study of PPIs particularly when established methods fail.

cell biology↗