Biology subjects

Längst, G.

Publications and source records attributed to Längst, G..

5 recordsLinked to original sources

bioRxiv2026

The SWI/SNF subunit SWI3B functions with the m6A writer complex to establish embryo patterning in Arabidopsis

N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes and is essential for Arabidopsis embryogenesis. However, how m6A mRNA methylation is coordinated with other regulatory pathways during development including embryogenesis remains largely unknown. Here, we report the SWI/SNF chromatin-remodeling subunit SWI3B as a bona fide interactor of the m6A methyltransferase MTA. Like m6A writer mutants, SWI3B is required for early embryo development. We demonstrate that the interaction between MTA and SWI3B is required for MTA function during embryogenesis. MTA and SWI3B are both required to establish the correct expression pattern of WOX8 and proper auxin maxima during early embryogenesis. Transcriptome analysis of isolated embryos from mta, swi3b, and fip37 mutants identified a shared set of upregulated transcripts, including STM as well as several NAC and ERF transcription factors that are normally absent or expressed at very low levels during early embryogenesis. Embryo-specific overexpression of ANAC087 and ERF114 genes phenocopied early embryonic defects observed in mta and swi3b mutants, indicating that their ectopic expression contributes to the observed developmental phenotype. Moreover, SWI3B and MTA are both required for m6A deposition on specific developmental transcripts. Together, our findings uncover a mechanism by which chromatin remodeling and m6A-mediated RNA regulation cooperate to suppress the precocious stability of key developmental regulators, thereby contributing to the establishment of the transcriptional program required for early embryo patterning in Arabidopsis. HighlightsO_LIThe SWI/SNF subunit SWI3B is a functional interactor of the m6A methyltransferase MTA during Arabidopsis embryogenesis C_LIO_LISWI3B and MTA cooperate to establish embryo patterning, WOX8 expression, and auxin maxima C_LIO_LIMTA and SWI3B suppress precocious expression of STM, ANAC087 and ERF114 transcription factors that disrupt early embryo development C_LIO_LISWI3B links chromatin-associated regulation with m6A-mediated control of transcript stability C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/744595v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1056f76org.highwire.dtl.DTLVardef@1d8b9d6org.highwire.dtl.DTLVardef@190918corg.highwire.dtl.DTLVardef@439ce1_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2023

The RALF Signaling Pathway Regulates Cell Wall Integrity during Pollen Tube Growth in Maize

Autocrine signaling pathways regulated by RAPID ALKALINIZATION FACTORs (RALFs) control cell wall integrity during pollen tube germination and growth in Arabidopsis. To investigate the role of pollen-specific RALFs in another plant species, we combined gene expression data with phylogenetic and biochemical studies to identify candidate orthologs in maize. We show that Clade IB ZmRALF2/3 mutants, but not that of Clade III ZmRALF1/5 caused cell wall instability in the sub-apical region of the growing pollen tube. ZmRALF2/3 are mainly located to the cell wall and are partially able to complement the pollen germination defect of their Arabidopsis orthologs AtRALF4/19. Mutations in ZmRALF2/3 compromise pectin distribution pattern leading to altered cell wall thickness, hyperphosphorylation of ZmPEX cell wall proteins and pollen tube burst. Clade IB, but not Clade III ZmRALFs are capable to interact with pollen-specific CrRLK1L receptor kinases ZmFERL4/7/9 and GPI-anchored co-receptors ZmLLG1/2 at similar binding affinities. In contrast, binding affinity to ZmPEX2/4 cell wall proteins is about five times higher. Based on these data, we now propose a dosage-dependent model showing how Clade IB RALFs act as extracellular sensors to regulate cell wall integrity and thickness during pollen tube growth in plants. One sentence summaryPollen-specific RALFs interact at different binding affinities with receptor kinases, GPI-anchored proteins and cell wall proteins to regulate cell wall integrity during pollen tube growth in maize.

developmental biology↗

bioRxiv2022

Impact of single mutations on binding kinetics oftriplex forming oligos revealed by fluorescence proximitysensing in heliX(R) biosensor

The sequence specific association of RNA with DNA via formation of RNA/DNA triple helices is relevant for regulation of gene expression, repair mechanisms, and chromatin organization. Formation of such RNA/DNA triplexes requires poly-purine sequences, which allow for Hoogsteen base-paring in addition to Watson-Crick pairing in the major groove of DNA. Binding of triplex forming oligos (TFOs) is sequence-specific and understanding sequence dependencies is key for the development of targeted tools for gene therapy. Here, we highlight a direct approach for determining binding kinetics and binding constants for TFOs using the state-of-the-art heliX(R)biosensor tool. With this, we provide key insights into the binding kinetics of RNA and DNA TFOs to a triplex targeting site (TTS)-containing DNA double helix measured in real-time. Dependent on the introduced base, point mutations in one position of a triplex forming oligo (TFO) can change the dissociation constant (KD) by several orders of magnitude or just by one log, affecting primarily the dissociation rate. Furthermore, we demonstrate that the heliX(R)biosensor assay is also well-suited for detection of rather weak triplex formation. The weakest binding we could identify was 140 M, for a TFO, which other studies considered as non-binding.

biophysics↗

bioRxiv2019

RNA polymerase I passage through nucleosomes depends on its lobe binding subunits

RNA polymerase I (Pol I) is a highly efficient enzyme specialized to synthesize most of the ribosomal RNA. After nucleosome deposition at each round of replication the Pol I transcription machinery has to deal with nucleosomal barriers. It was suggested that Pol I-associated factors facilitate chromatin transcription, but it is not known whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here we used in vitro transcription assays to study purified Pol I of the yeast S. cerevisiae and Pol I mutants in comparison to Pol II and Pol III to pass a nucleosome. Under identical conditions, purified Pol I and Pol III, but not Pol II, were able to transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. The contribution of Pol I specific subunit domains to efficient passage through nucleosomes in context with transcription rate and processivity is discussed.

biochemistry↗

bioRxiv2018

RNA polymerase I transcription fidelity, speed and processivity depend on the interplay of its lobe binding subunits

Eukaryotic RNA polymerases I and III (Pol I and III) consist of core subunits, which are conserved in RNA polymerase II (Pol II). Additionally, Pol I and III have specific subunits, associating with the so-called lobe structure first described within Pol II. In Pol I of the yeast S. cerevisiae, these are Rpa34.5, and the N-terminal domains of Rpa49 and Rpa12.2, here referred to as the lobe-binding module (lb-module). We analyzed functions of the lb-module in a defined in vitro transcription system. Cooperation between lb-module components influenced transcription fidelity, elongation speed, and release of stalled Pol I complexes to continue elongation. Interestingly, lb-module containing Pol I and III, but not Pol II, were able to transcribe nucleosomal templates. Our data suggest, how the Pol I specific subunits may contribute to accurate and processive transcription of ribosomal RNA genes.

biochemistry↗