Biology subjects

Laubinger, S.

Publications and source records attributed to Laubinger, S..

7 recordsLinked to original sources

bioRxiv2025

The spliceosome component U1-70k loads nascent transcripts onto FVE/SGS3 via co-condensation to embark on RNA silencing

Despite biochemical framework of RNA silencing has been studied well, how nascent transcripts enter RNA silencing remains poorly understood. Here we recovered U1-70k, a canonical spliceosome factor, as a novel component in posttranscriptional gene silencing (PTGS). We show that U1-70k interacts with SGS3 and FVE, two key components that supply RNA substrate for RNA-dependent RNA polymerase 6 (RDR6) to generate dsRNA. U1-70k promotes the loading of nascent RNA into the SGS3/FVE complex in the nucleus, which is subsequently translocated onto RDR6 in the cytosol. Importantly, U1-70k contains C-terminal low complexity (LC) domain and displays liquid-liquid phase separation (LLPS) pattern. U1-70k forms co-condensate with SGS3, another intrinsically disordered protein (IDP). The co-condensation is critical for PTGS as U1-70k LC variants display reduced SGS3 co-condensation, loss of RNA loading onto SGS3, and impaired RNAi function. Taken together, U1-70k channels nascent RNA to SGS3/FVE via its LLPS action to enter PTGS pathway.

molecular biology↗

bioRxiv2023

The Arabidopsis U1 snRNP regulates mRNA 3'-end processing

The removal of introns by the spliceosome is a key gene regulatory mechanism in eukaryotes, with the U1 snRNP subunit of the spliceosome playing a crucial role in the early stages of splicing. Studies in metazoans show that the U1 snRNP also conducts splicing-independent functions, but the lack of genetic tools and knowledge about U1 snRNP-associated proteins have limited the study of such splicing-independent functions in plants. Here, we describe an RNA-centric approach that identified more than 200 proteins associated with the Arabidopsis U1 snRNP, among them mRNA cleavage and polyadenylation factors. The loss of U1 core components is linked to premature cleavage and polyadenylation within gene bodies and alternative polyadenylation site selection in 3-UTRs. Overall, our work provides a comprehensive view of U1 snRNP interactors and reveals novel functions in regulating mRNA 3-end processing in Arabidopsis, thus establishing the groundwork for a better understanding of non-canonical functions of plant U1 snRNPs.

plant biology↗

bioRxiv2021

A high resolution single molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

BackgroundAccurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single molecule long read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation or incomplete cDNA synthesis. ResultsWe present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 160k transcripts - twice that of the best current Arabidopsis transcriptome and including over 1,500 novel genes. 79% of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We developed novel methods to determine splice junctions and transcription start and end sites accurately. Mis- match profiles around splice junctions provided a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identified high confidence transcription start/end sites and removed fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provided higher resolution of transcript expression profiling and identified cold- and light-induced differential transcription start and polyadenylation site usage. ConclusionsAtRTD3 is the most comprehensive Arabidopsis transcriptome currently available. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single molecule sequencing analysis from any species.

plant biology↗

bioRxiv2021

Transcriptome sequence reveals candidate genes involving in the post-harvest hardening of trifoliate yam Dioscorea dumetorum

Storage ability of D. dumetorum is restricted by a severe phenomenon of post-harvest hardening which starts 72h after harvest and renders tubers inedible. Previous work has only focused on the biochemistry changes affecting the PHH on D. dumetorum. To the best of our knowledge nobody has identified candidate genes responsible for hardness on D. dumetorum. Here, transcriptome analysis of D. dumetorum tubers was performed, 4 months after emergence (4MAE), after harvest (AH), 3 days AH (3DAH) and 14 days AH (14DAH) on four accessions using RNA-Seq. In total between AH and 3DAH, 165, 199,128 and 61 differentially expressed genes (DEGs) were detected in Bayangam 2, Fonkouankem 1, Bangou 1 and Ibo sweet 3 respectively. Functional analysis of DEGs revealed that genes encoding for cellulose synthase A, xylan O-acetyltransferase chlorophyll a/b binding protein 1,2,3,4 and transcription factor MYBP were found predominantly and significantly up-regulated 3DAH, implying that genes were potentially involved in the post-harvest hardening. A hypothetical mechanism of this phenomenon and its regulation has been proposed. These findings provide the first comprehensive insights into genes expression in yam tubers after harvest and valuable information for molecular breeding against the post-harvest hardening. A hypothetical mechanism of this phenomenon and its regulation has been proposed. These findings provide the first comprehensive insights into genes expression in yam tubers after harvest and valuable information for molecular breeding against the post-harvest hardening.

plant biology↗

bioRxiv2020

Independent parental contributions initiate zygote polarization in Arabidopsis thaliana

Embryogenesis of flowering plants is initiated by polarization of the zygote, a prerequisite for correct axis formation in the embryo. Zygote polarity and the decision between embryonic and non-embryonic development in the daughter cells is controlled by a MITOGEN-ACTIVATING PROTEIN (MAP) kinase signaling pathway including the MAPKK kinase YODA (YDA). Upstream of YDA act two members of the BRASINOSTEROID SIGNALING KINASE (BSK) family, BSK1 and BSK2. These membrane-associated proteins serve as signaling relay linking receptor kinase complexes with intracellular signaling cascades. The receptor kinases acting upstream of BSK1 and BSK2 in the zygote, however, have so far not been identified. Instead, YDA can in part be activated by the non-canonical BSK family member SHORT SUSPENSOR (SSP) that is contributed by the sperm cell during fertilization. Here, we show that the receptor kinase ERECTA plays a crucial role in zygote polarization as maternally contributed part of the embryonic YDA pathway. SSP on the other hand provides an independent, paternal input to YDA activation, outlining a Y-shaped pathway. We conclude that two independent parental contributions initiate zygote polarization and control suspensor formation and embryonic development.

plant biology↗

bioRxiv2019

Metabolic labeling of RNAs uncovers hidden features and dynamics of the Arabidopsis thaliana transcriptome

Transcriptome analysis by RNA sequencing (RNA-seq) has become an indispensable core research tool in modern plant biology. Virtually all RNA-seq studies provide a snapshot of the steady-state transcriptome, which contains valuable information about RNA populations at a given time, but lacks information about the dynamics of RNA synthesis and degradation. Only a few specialized sequencing techniques, such as global run-on sequencing (GRO-seq), have been applied in plants and provide information about RNA synthesis rates. Here, we demonstrate that RNA labeling with a modified, non-toxic uridine analog, 5-ethynyl uridine (5-EU), in Arabidopsis thaliana seedlings provides insight into the dynamic nature of a plant transcriptome. Pulse-labeling with 5-EU allowed the detection and analysis of nascent and unstable RNAs, of RNA processing intermediates generated by splicing, and of chloroplast RNAs. We also conducted pulse-chase experiments with 5-EU, which allowed us to determine RNA stabilities without the need for chemical inhibition of transcription using compounds such as actinomycin and cordycepin. Genome-wide analysis of RNA stabilities by 5-EU pulse-chase experiments revealed that this inhibitor-free RNA stability measurement results in RNA half-lives much shorter than those reported after chemical inhibition of transcription. In summary, our results show that the Arabidopsis nascent transcriptome contains unstable RNAs and RNA processing intermediates, and suggest that half-lives of plant RNAs are largely overestimated. Our results lay the ground for an easy and affordable nascent transcriptome analysis and inhibitor-free analysis of RNA stabilities in plants.

plant biology↗

bioRxiv2017

Arabidopsis U1 snRNP subunit LUC7 functions in alternative splicing and preferential removal of terminal introns

Introns are removed by the spliceosome, a large macromolecular complex composed of five ribonucleoprotein subcomplexes (U snRNP). The U1 snRNP, which binds to 5 splice sites, plays an essential role in early steps of the splicing reaction. Here, we show that Arabidopsis LUC7 proteins, which are encoded by a three-member gene family in Arabidopsis, are important for plant development and stress resistance. We show that LUC7 are U1 snRNP accessory proteins by RNA immunoprecipitation experiments and LUC7 protein complex purifications. Transcriptome analyses revealed that LUC7 proteins are not only important for constitutive splicing, but also affects hundreds of alternative splicing events. Interestingly, LUC7 proteins specifically promote splicing of a subset of terminal introns. Splicing of LUC7-dependent introns is a prerequisite for nuclear export and some splicing events are modulated by stress in a LUC7-dependent manner. Taken together our results highlight the importance of the U1 snRNP component LUC7 in splicing regulation and suggest a previously unrecognized role of a U1 snRNP accessory factor in terminal intron splicing.

plant biology↗