Biology subjects

Thomae, A. W.

Publications and source records attributed to Thomae, A. W..

6 recordsLinked to original sources

bioRxiv2026

Hybrid Drosophila cell lines show selective genome instabilities

Background: Post-zygotic reproductive isolation is thought to be caused by genomic conflict. However, the exact nature of these detrimental incompatibilities is largely unknown. To identify genomic regions that became incompatible during the formation of species and are therefore prone to get lost in hybrids, we established hybrid cell lines derived from an authentic interspecies cross between Drosophila melanogaster and Drosophila simulans. Results: All four independently derived lines selectively lost the D. melanogaster allele of the hybrid incompatibility gene Hmr, confirming its incompatibility with cellular viability. Moreover, each line underwent distinct but directionally biased erosion of additional D. melanogaster chromosomal regions, preferentially affecting the X chromosome, chromosome 4, and chromosome 3R. Loss of the D. melanogaster X was dosage compensated by upregulation of genes on the hemizygous D. simulans X, while D. melanogaster rDNA was paradoxically amplified across all lines. Nucleolar dominance was driven by relative rDNA copy number rather than species identity, overturning the prevailing view of fixed species-based dominance. Finally, the measurements of the species-specific proteome revealed species specific differences in the RNA to protein coefficient K_p as novel and yet undescribed sources of molecular conflict in hybrids. Conclusions: These findings reveal a hierarchical, constraint-driven mechanism of hybrid genome resolution.

evolutionary biology↗

bioRxiv2022

Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless

Unwinding RNA secondary structures by RNA helicases is essential for RNA metabolism. How the basic unwinding reaction of DExH-type helicases is regulated by their accessory domains is unresolved. Here, we combine structural and functional analyses to address this challenge for the prototypic DExH RNA helicase maleless (MLE) from Drosophila. We captured the helicase cycle of MLE with multiple structural snapshots. We discovered that initially, dsRBD2 flexibly samples substrate dsRNA and aligns it with the open helicase tunnel. Subsequently, dsRBD2 releases RNA and associates with the helicase core, leading to closure of the tunnel around ssRNA. Structure-based MLE mutations confirm the functional relevance of the structural model in cells. We propose a molecular model in which the dsRBD2 domain of MLE orchestrates large structural transitions that depend on substrate RNA but are independent of ATP. Our findings reveal the fundamental mechanics of dsRNA unwinding by DExH helicases with high general relevance for dosage compensation and specific implications for MLEs human orthologue DHX9/RHA mechanisms in disease.

molecular biology↗

bioRxiv2022

Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation

Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600-630) and two in the far red channel (633/641-680), supplemented by a single further redshifted fluorochrome (670/701-750) were depleted with 775 nm. To the best of our knowledge, these are the first published five color STED images. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.

cell biology↗

bioRxiv2021

The Integrity of the Speciation Core Complex is necessary for centromeric binding and reproductive isolation in Drosophila

Postzygotic isolation by genomic conflict is a major cause for the formation of species. Despite its importance, the molecular mechanisms that result in the lethality of interspecies hybrids are still largely unclear. The genus Drosophila, which contains over 1600 different species, is one of the best characterized model systems to study these questions. We showed in the past that the expression levels of the two hybrid incompatibility factors Hmr and Lhr diverged in the two closely related Drosophila species, D. melanogaster and D. simulans, resulting in an increased level of both proteins in interspecies hybrids. This overexpression leads to mitotic defects, a misregulation in the expression of transposable elements and a decreased fertility. In this work, we describe a distinct six subunit Speciation Core Complex (SCC) containing HMR and LHR and analyse the effect of Hmr mutations on complex function and integrity. Our experiments suggest that HMR acts as a bridging factor between centromeric chromatin and pericentromeric heterochromatin, which is required for both its physiological function and its ability to cause hybrid male lethality.

biochemistry↗

bioRxiv2018

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAK RNA binding motifs. NMR titrations combined with filter binding experiments document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

biochemistry↗

bioRxiv2018

Intricate structure of the interphase chromocenter revealed by the analysis of a factor involved in species formation

In higher eukaryotes centromeres often coalesce into a large intranuclear domain called the chromocenter. Chromocenters are important for the organization of pericentric heterochromatin and a disturbance of their formation results in an upregulation of repetitive elements and causes defects in chromosome segregation. Mutations in the gene encoding for the centromere associated Drosophila speciation factor HMR show very similar phenotypes suggesting a role of HMR in chromocenter architecture and function. We performed confocal and super resolution microscopy as well as proximity based biotinylation experiments of HMR and its associated protein HP1a to generate a molecular map of HMR and HP1a bound chromatin. Our work reveals an intricate internal structure of the centromeric chromatin region, which suggests a role of HMR in separating heterochromatin from centromeric chromatin.

biochemistry↗