Biology subjects

Plaschka, C.

Publications and source records attributed to Plaschka, C..

7 recordsLinked to original sources

bioRxiv2026

Super-resolved imaging of mRNA ultrastructure in cells

Messenger RNA (mRNA) is a central polymer of gene expression, whose ultrastructure and defining regulatory rules remain unclear. To visualize the ultrastructure of mRNA, we here develop Combi-PAINT, a powerful and generalizable method for combinatorial super-resolved DNA-PAINT multiplexing, which we combine with high-efficiency RNA labelling and MINFLUX microscopy. This approach enables the nanometer-precision tracing of mRNA in three dimensions in cells. We use Combi-PAINT to visualize multiple distinct mRNA species inside the human cell nucleus and cytoplasm, revealing their quantitative ultrastructures and transcript-specific molecular patterns. By bridging sequence specificity with nanometer resolution, we provide a new lens for studying the cellular life of mRNA.

biophysics↗

bioRxiv2025

Activator-promoter compatibility in mammals: a CpG-Island-specific co-activator directly bridges transcription factors to TFIID

Transcription from CpG island (CGI) promoters controls the expression of two-thirds of mammalian genes, yet despite their prevalence, it remains unknown whether CGI-specific co-activators with intrinsic specificity (i.e. compatibility) for these promoters exist or by what mechanisms they might function. Here, we perform proteome-wide functional screens to identify more than fifty transcriptional activators that are intrinsically specific to CGI promoters, establishing that promoter-class-specific activators are a widespread feature of mammalian gene regulation. Among these, we identify Host Cell Factor 1 (Hcfc1) as the founding member of CGI-specific co-activators. Hcfc1 is essential for the expression of thousands of CGI-promoter-driven genes and acquires CGI-specificity through a two-step mechanism: CGI-associated transcription factors recruit Hcfc1 through its Kelch domain, and Hcfc1 in turn directly engages the general transcription factor TFIID through a dedicated activation domain. The Hcfc1-TFIID interaction overcomes a key rate-limiting step for CGI promoter initiation, TFIID recruitment, thereby directly enabling transcription. Hcfc1 thus functions as a promoter-class-specific bridge between CGI-bound transcription factors and the general transcription machinery, analogous to Mediator but with intrinsic promoter specificity. Together, we uncover a dedicated activation pathway for CGI promoters, reveal a fundamental mechanistic difference in transcription activation between promoter classes in mammals, and establish co-activator-promoter compatibility as a core principle in mammalian gene regulation.

molecular biology↗

bioRxiv2025

Structure of cytoplasmic RNA polymerase II

RNA polymerase II (Pol II) must be assembled in the cytoplasm before it enters the nucleus, where it transcribes protein-coding genes. Although transcription by Pol II is intensively studied, how this central multi-subunit enzyme is made and the role of dedicated factors remains unclear. Here, we report the integrative structural analysis of a native human Pol II from the cytoplasm captured near the end of biogenesis. The complex contained Gdown1 and three biogenesis factors - RPAP2 and the critical small GTPases GPN1 and GPN3. Cryo-EM analysis of the complex revealed how Gdown1 and RPAP2 associate with Pol II and prevent the premature association of transcription factors. Further biochemical and cryo-EM analysis revealed how RPAP2 recruits GPN1-GPN3 to the complex, and how the assembly of the RPAP2-GPN1-GPN3 complex is controlled by GTP hydrolysis. The combined results uncover a network of interactions that chaperone cytoplasmic Pol II to prevent aberrant interactions, reveal a GTP-controlled switch during the final stages of Pol II biogenesis, and suggest a general mechanism for the action of GPN-loop GTPase family of enzymes.

biochemistry↗

bioRxiv2025

Molecular basis of polyadenylated RNA fate determination in the nucleus

Eukaryotic genomes generate a plethora of polyadenylated (pA+) RNAs1,2, that are packaged into ribonucleoprotein particles (RNPs). To ensure faithful gene expression, functional pA+ RNPs, including protein-coding RNPs, are exported to the cytoplasm, while transcripts within non-functional pA+ RNPs are degraded in the nucleus1-4. How cells distinguish these opposing fates remains unknown. The DExD-box ATPase UAP56/DDX39B is a central component of functional pA+ RNPs, promoting their docking to the nuclear pore complex (NPC)-anchored transcription and export complex 2 (TREX-2) (ref.5,6), which triggers transcript release from UAP56 to facilitate export (ref.7,8). Here, we uncover that the Poly(A) tail exosome targeting (PAXT) connection9 harbors its own TREX-2-like module, which releases pA+ RNAs from UAP56 for decay by the nuclear exosome. The core of this module consists of a LENG8-PCID2-SEM1 (LENG8-PS) trimer, which we show is structurally and functionally equivalent to the central GANP-PCID2-SEM1 (GANP-PS) trimer of TREX-2. Mutagenesis and transcriptomic data demonstrate that the nuclear fate of pA+ RNPs is governed by the contending actions of nucleoplasmic PAXT and NPC-associated TREX-2, which interpret RNA-bound UAP56 as a signal for RNA decay or export, respectively. As RNA targets of PAXT are generally short and intron-poor, we propose an overall model for pA+ RNP fate determination, whereby the distinct sub-nuclear localizations of PAXT and TREX-2 govern the degradation of short non-functional pA+ RNAs while allowing export of their longer and functional counterparts.

molecular biology↗

bioRxiv2024

A molecular switch orchestrates the nuclear export of human messenger RNA

The nuclear export of messenger RNA (mRNA) is a key step in eukaryotic gene expression (Kohler and Hurt, 2007). Despite recent insights into the packaging of newly transcribed mRNAs into ribonucleoprotein complexes (mRNPs) (Pacheco-Fiallos et al., 2023; Bonneau et al., 2023), the subsequent events that govern mRNA export are poorly understood. Here, we elucidate the molecular basis of human mRNA export licensing, which involves the remodeling of mRNP-bound transcription-export complexes (TREX), the formation of export-competent mRNPs, the docking of mRNPs at the nuclear pore complex (NPC), and the release of mRNPs at the NPC to initiate export. Our biochemical and structural data uncover the ATPase DDX39/UAP56 as a central molecular switch that directs mRNPs through the TREX and the NPC-anchored TREX-2 complexes using its ATPase and mRNA-binding cycle. Collectively, these findings establish a mechanistic framework for a general and conserved mRNA export pathway.

molecular biology↗

bioRxiv2023

Mutual homeostasis of charged proteins

Protein dosage is regulated to maintain cellular homeostasis and health. The dosage of proteins containing disordered low complexity domains (LCDs) must be particularly well-controlled to prevent aberrant disease, yet no mechanism to maintain homeostasis has been identified1, 2. Here we report a mutual homeostatic mechanism that controls the concentration of such proteins, termed interstasis, in which proteins with similar LCDs co-regulate their combined dosage through collective negative feedback. We focused on the mechanism that exploits the fundamental multivalency of GA-rich RNA regions that encode charged LCDs, including those with arginine-enriched mixed charge domains (R-MCDs). Modest variations in the abundance of an R-MCD protein change the properties of nuclear speckles, a protein-RNA condensate, selectively trapping multivalent GA-rich mRNAs to promote their nuclear retention. This interstasis depends on conserved codon biases, shared by amniotes, which enhance the multivalency of GA-rich regions encoding charged LCDs. The threshold of interstasis is modulated by CLK kinases, which affect the nuclear speckle localisation of proteins such as TRA2B, key binder of GA-rich RNAs. Notably, many classes of LCDs are encoded by RNA regions containing multivalency-enhancing codon biases, each preferentially bound by specific proteins, suggesting that interstasis might co-regulate many classes of functionally related LCD-containing proteins through dose-sensitivity of various types of protein-RNA condensates.

molecular biology↗

bioRxiv2021

The molecular principles of Piwi-mediated co-transcriptional silencing through the dimeric SFiNX complex

Nuclear Argonaute proteins, guided to nascent target RNAs by their bound small RNAs, elicit co-transcriptional silencing through heterochromatin formation at transposon insertions and repetitive genomic loci. The molecular mechanisms involved in this process are incompletely understood. Here, we propose that the SFiNX complex, a silencing mediator downstream of nuclear Piwi-piRNA complexes in Drosophila, enables co-transcriptional silencing via the formation of molecular condensates. Condensate formation is stimulated by nucleic acid binding and requires SFiNX dimerization, mediated by the dynein light chain protein, LC8/Cutup. LC8s function within SFiNX can be bypassed with a heterologous dimerization domain, suggesting that dimerization is a constitutive feature of SFiNX. Mutations preventing LC8-mediated SFiNX dimerization result in loss of condensate formation in vitro and inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. Formation of molecular condensates might be a general mechanism that underlies effective heterochromatin establishment at small RNA target loci in a co-transcriptional manner.

molecular biology↗