Biology subjects

Jansen, R.-P.

Publications and source records attributed to Jansen, R.-P..

6 recordsLinked to original sources

bioRxiv2026

Proximity Labelling Reveals Spatial Organisation of Mitochondrial Protein Import Complexes

Mitochondrial protein import and local translation at the mitochondrial outer membrane (MOM) require coordinated interactions between protein translocases and RNA-associated factors, yet the molecular organisation of these interactions remains largely unresolved. Here, we used APEX2-mediated proximity labelling to define the proximal proteomes associated with the cytosolic face of the TOM and SAM complexes by fusing APEX2 to TOMM22 and MTX2, respectively. Quantitative mass spectrometry identified known TOM/SAM-associated proteins together with multiple RNA-binding proteins (RBPs), supporting the emerging role of the MOM in localised translation and other RNA-related processes. Among identified RBPs, the exonuclease EXD2 was consistently enriched in APEX2-TOMM22 and MTX2-APEX2 datasets and remained associated upon puromycin treatment, indicating a translation-independent interaction. Together, our findings provide insights into the molecular organisation of mitochondrial import sites and identify EXD2 as a TOM/SAM-associated factor at the MOM.

biochemistry↗

bioRxiv2026

DRUG TARGET IDENTIFICATION VIA A CONDITIONALLY STABILIZED TurboID ENZYME

Different small-molecule drugs targeting the same protein can produce divergent clinical outcomes through poorly characterized interactome changes. Existing proximity labeling approaches for target identification suffer from background biotinylation independent of small-molecule recruitment, obscuring true drug targets and their binding partners. Here, we incorporate a destabilizing domain (DD) into the biotin targeting chimera (BioTAC) framework to create ddBioTAC, wherein the proximity labeling enzyme TurboID is selectively stabilized only upon binding of a bifunctional targeting molecule. Using the bromodomain-targeting molecule NICE-01 in HeLa cells, we demonstrate that, in the absence of the bifunctional targeting molecule the destabilized TurboID enzyme (TurboID-DD) exhibits reduced protein levels and biotinylation activity compared to the control TurboID-FKBP (FK506-binding protein), while recovering comparable activity upon NICE-01 treatment. This results in an eightfold improvement in specific enrichment of the known target bromodomain containing protein 4 (BRD4) and its interactors, including MED1 and EF1D. Proteome-wide mass spectrometry confirms that ddBioTAC more accurately discriminates drug targets and proximal interactors from non-specific background, advancing unbiased drug-induced interactome profiling.

biochemistry↗

bioRxiv2024

Proximity labeling reveals differential interaction partners of the human mitochondrial import receptor proteins TOMM20 and TOMM70

Import of most mitochondrial proteins requires that their precursor proteins are bound by the (peripheral) receptor proteins TOM20, TOM22, and TOM70. For budding yeast TOM20 and TOM70, there is evidence of specific yet overlapping substrate recognition, but no such data is available for metazoan cells. Using APEX2-based proximity labeling, we thus created association profiles for human TOMM20 and TOMM70 in HeLa cells. We particularly focused on their interaction with RNA-binding proteins (RBPs) since there is evidence for RNA association with the mitochondrial outer membrane (MOM) and local translation at the mitochondrial surface, but these processes are poorly understood. Our results show a preferred association of several RBPs and translation factors with TOMM20 over TOMM70. These include SYNJBP2, a previously identified membrane-bound RBP that binds and protects mRNAs encoding mitochondrial proteins. Translational inhibition by puromycin resulted in an even increased association of these RBPs with TOMM20 compared to TOMM70, suggesting that TOMM20 but not TOMM70 might play a role in preserving cellular hemostasis during translation stress by retaining protective RBPs and translation-related proteins at the MOM.

biochemistry↗

bioRxiv2024

A consensus motif in ASH1 and further transcripts unifies several RNA motifs required for interaction with the She2p/She3p transport machinery and mRNA localization in yeast

Asymmetric localization of the ASH1 transcript is a central step in the regulation of mating type switching in Saccharomyces cerevisae and a paradigm for localized mRNAs specifically recognized by the She2p/3p transport machinery. Four RNA elements in ASH1 are known to mediate bud localization, but it remained unclear what a consensus motif of all four She2p/3p recognition sites (SRS) might look like. In this study, we derive the SRS consensus motif, which is characterized by an L-shaped, double-stranded RNA conformation with two cytosine-containing sequence motifs at conserved positions and a central asymmetric bulge. It is noteworthy that the spatial arrangement of these features can be retained, despite their altered order within the nucleotide sequence. We termed these variations "configurations" and confirmed in biochemical studies that SRS function in ASH1 elements is preserved when changing configurations. We tested other known and predicted SRS elements in target transcripts of She2p/3p by biochemical and biophysical studies and confirmed the conserved SRS function even in switched configuration. Consistently, we present the SRS instance collection, which includes further predictions of SRS instances of transcript targets of the She2p/3p translocation pathway.

cell biology↗

bioRxiv2019

Local translation of yeast ERG4 mRNA at the endoplasmic reticulum requires the brefeldin A resistance protein Bfr1

Brefeldin A resistance factor 1 (Bfr1p) is a non-essential RNA-binding protein and multi-copy suppressor of brefeldin A sensitivity in Saccharomyces cerevisiae. Deletion of BFR1 leads to multiple defects, including altered cell shape and size, change in ploidy, induction of P-bodies and chromosomal mis-segregation. Bfr1p has been shown to associate with polysomes, binds to several hundred mRNAs, and can target some of them to P-bodies. Although this implies a role of Bfr1p in translational control of mRNAs, its molecular function remains elusive. In the present study, we show that mutations in RNA-binding residues of Bfr1p impede its RNA-dependent co-localization with ER, yet do not mimic the known cellular defects seen upon BFR1 deletion. However, a Bfr1 RNA-binding mutant is impaired in binding to ERG4 mRNA which encodes an enzyme required for the final step of ergosterol biosynthesis. Consistently, bfr1{Delta} strains show a strong reduction in Erg4p protein levels. Polysome profiling of bfr1{Delta} or bfr1 mutant strains reveals a strong shift of ERG4 mRNA to polysomes, consistent with a function of Bfr1p in elongation. Collectively, our data reveal that Bfr1 has at least two separable functions: one in RNA-binding and elongation during translation, in particular at the ER membrane, and one in ploidy control or chromosome segregation.

cell biology↗

bioRxiv2018

β-actin mRNA interactome mapping by proximity biotinylation

The molecular function and fate of mRNAs are controlled by RNA binding proteins (RBPs). However, identification of the interacting proteome of a specific mRNA in vivo is still very challenging. Based on the widely-used RNA tagging with MS2 aptamers for RNA visualization, we developed a novel RNA proximity biotinylation (RNA-BioID) method by tethering biotin ligase (BirA*) via MS2 coat protein (MCP) at the 3UTR of endogenously MS2 tagged {beta}-actin mRNA in mouse embryonic fibroblasts (MEFs). We demonstrate the dynamics of the {beta}-actin mRNA interactome by characterizing its changes upon serum-induced localization of the mRNA. Apart from the previously known interactors, we identified over 60 additional {beta}-actin associated RBPs by RNA-BioID, among them the KH-domain containing protein FUBP3/MARTA2 has shown to be required for {beta}-actin mRNA localization. This protein binds to the 3-untranslated region of {beta}-actin mRNA, is essential for {beta}-actin mRNA localization but does not interact with the characterized {beta}-actin zipcode element. RNA-BioID provides a tool to identify new mRNA interactors and to study the dynamic view of the interacting proteome of endogenous mRNAs in space and time.

cell biology↗