Periodic Reporting for period 4 - sRNA-EMB (Small RNA regulation of the body plan and epigenome in Arabidopsis embryos)
Période du rapport: 2020-01-01 au 2020-06-30
After fertilization, the basic body plans of both plants and animals are established during early embryo development. However, despite the fundamental importance of this formative phase of the plant’s life to society's understanding of developmental biology and agricultural practices, the molecular mechanisms that generate the most basic cell-types in plants remain largely uncharacterized. Small RNAs are short non-coding RNAs that regulate gene expression in plants and animals. Although small RNAs are essential for proper gene regulation and cellular differentiation, little is known regarding their embryonic functions, especially in plants. Arabidopsis thaliana (Arabidopsis) is a highly suitable model system to study the regulatory roles of small RNAs because of the abundance of genetic resources and available (genome-wide) data. Moreover, Arabidopsis embryos undergo invariant division patterns and individual cells rapidly differentiate from each other to generate the most basic plant cell-types arranged in correct positions. Early Arabidopsis embryos are therefore morphologically simple structures composed of diverse cell types making them ideal for determining the influence of small RNAs on fundamental cellular differentiation and reprogramming events.
The objectives of the proposed research were designed to assess the regulatory roles of small RNAs in establishing the basic body plan in plant embryos. By utilizing a combination of conventional and novel next-generation sequencing technologies and genetic approaches, we have identified and characterized the small RNAs present in developing embryos including microRNAs (miRNAs) and small interfering RNAs (siRNAs). We found that multiple miRNAs mediate the cleavage and repression of at least 59 transcripts including 30 encoding transcription factors. Moreover, several of these miRNA-mediated repression of transcription factors are dynamic across cell types and individually required for the proper morphogenesis of several embryonic cell lineages. This indicates that in addition to transcriptional regulation, post-transcriptional regulation of transcription factors is critical for embryonic pattern formation in plants. Additionally, compared to other tissues we found that siRNAs are highly enriched in embryos where they direct the (re-)methylation of transposable elements in the new generation. siRNA-directed methylation of transposable elements helps ensure that they remain immobilized and thus preserves genomic integrity. Interestingly, we found that chromatin and sRNAs form a feedback loop to provide a balance between cellular growth and genome defense. In addition to generating biological insights, we have developed various genome-wide technologies suitable for a broad range of eukaryotic species including low-input profiling of small RNA populations and the 5’ ends of RNAs, synthetic RNAs for normalization of genome-wide small RNA sequencing data, and software to inspect the purity of RNA sequencing data, as well as transcriptome assembly. Altogether, the experiments funded by this action allowed the development of tools which in turn yielded insights into how small RNAs help establish the basic body plan and nascent epigenome of early embryos. Our work has helped clarify important questions in small RNA and reproductive biology, and will serve as a foundation for future research.
The objectives of the proposed research were designed to assess the regulatory roles of small RNAs in establishing the basic body plan in plant embryos. By utilizing a combination of conventional and novel next-generation sequencing technologies and genetic approaches, we have identified and characterized the small RNAs present in developing embryos including microRNAs (miRNAs) and small interfering RNAs (siRNAs). We found that multiple miRNAs mediate the cleavage and repression of at least 59 transcripts including 30 encoding transcription factors. Moreover, several of these miRNA-mediated repression of transcription factors are dynamic across cell types and individually required for the proper morphogenesis of several embryonic cell lineages. This indicates that in addition to transcriptional regulation, post-transcriptional regulation of transcription factors is critical for embryonic pattern formation in plants. Additionally, compared to other tissues we found that siRNAs are highly enriched in embryos where they direct the (re-)methylation of transposable elements in the new generation. siRNA-directed methylation of transposable elements helps ensure that they remain immobilized and thus preserves genomic integrity. Interestingly, we found that chromatin and sRNAs form a feedback loop to provide a balance between cellular growth and genome defense. In addition to generating biological insights, we have developed various genome-wide technologies suitable for a broad range of eukaryotic species including low-input profiling of small RNA populations and the 5’ ends of RNAs, synthetic RNAs for normalization of genome-wide small RNA sequencing data, and software to inspect the purity of RNA sequencing data, as well as transcriptome assembly. Altogether, the experiments funded by this action allowed the development of tools which in turn yielded insights into how small RNAs help establish the basic body plan and nascent epigenome of early embryos. Our work has helped clarify important questions in small RNA and reproductive biology, and will serve as a foundation for future research.
In general, we accomplished the originally proposed objectives of our ERC-funded project. At the beginning of this project, we developed a few biotechnology applications/tools that increased our ability to study genome-wide molecular biology phenomena in a quantitative manner from extremely low amounts of material. For instance, we developed exogenous small RNA spike-in oligonucleotides that allow for absolute normalization of small RNA sequencing (sRNA-Seq) data (Lutzmayer et al. [2017] Scientific Reports). These sRNA spike-ins facilitate comparisons of small RNA levels across different tissue types and genotypes, as well as genome-wide estimations of sRNA:mRNA stoichiometries. We also produced a statistical tool that revealed the presence of substantial RNA contamination from maternal tissues in nearly all published Arabidopsis endosperm and early embryo transcriptomes (Schon and Nodine [2017] Plant Cell). Not only is this a useful tool for the community to ensure the generation of accurate datasets, but we also found that maternal RNA contamination in previously published datasets had been repeatedly misinterpreted as epigenetic phenomena including the magnitude and maternal bias of imprinted genes in seeds. We also developed a method that enables 1) the identification of transcription start sites at single-nucleotide resolution from single-cell levels of total RNA and 2) small RNA-mediated transcript cleavage from ≥10,000-fold less total RNA compared to previous approaches (Schon et al. [2018] Genome Research). We generated a transcriptome resource of Arabidopsis embryos and found that embryo development can be categorized into four distinct phases (Hofmann et al. [2019] Plant Reproduction). With these and additional tools, we generated a genome-wide resource of small RNAs and their functional activities during Arabidopsis embryogenesis, developed methods for low-input small RNA-seq and small RNA in situ hybridizations, and demonstrated that microRNA-mediated repression of six transcription factors are required individually for proper division patterns of various embryonic cell lineages (Plotnikova et al. [2019] Plant Cell). More generally, this project demonstrated that post-transcriptional regulation of multiple transcription factors is critically important for the establishment of the plant body plan. Finally, we demonstrated that chromatin regulates small RNA expression in order to provide a cell-autonomous feedback loop to reconstitute pre-existing transposon methylation states during growth and development including those that ensure silencing of transposons in the future germ line (Papareddy et al. [2020] bioRxiv).
Molecular biology approaches are increasingly being applied to specific cell-types and even individual cells to more precisely understand complex gene regulatory mechanisms at the cellular level. The applications and tools that we are developing are therefore not only instrumental to our ability to accomplish our project's stated aims, but are generally helpful to research groups studying cell-type specific processes. For example, the sRNA spike-in method we developed can be used to improve the interpretation of small RNA-related data that are routinely generated and used in both basic and applied research. Therefore, this work may also have a broad impact extending from fundamental molecular biology research to clinical assessment of molecular markers related to disease-afflicted cell-types. For example, we have licensed our sRNA spike-in technology to a local Viennese biotech company for the development of biomedical diagnostic kits. Moreover, our statistical tool to detect contamination in seed transcriptome datasets had large implications in the field of epigenetic imprinting. In summary, due to the broad applicability of these and additional methods we developed, this ERC-funded project is clearly having an impact beyond the results originally expected from the originally stated aims.