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Post-transcriptional regulation of RNA degradation in early zebrafish development

Periodic Reporting for period 2 - DecodeDegRNA (Post-transcriptional regulation of RNA degradation in early zebrafish development)

Reporting period: 2021-09-01 to 2023-02-28

Gene expression is a tightly controlled process. Cells in living organisms express certain genes at an exact time and place, by tightly controlling RNA molecules from their creation by transcription to their final degradation. This regulation lies at the heart of fundamental biological processes, such as the development of an embryo. Curiously, there is a significant lack of systematic understanding of how RNA degradation is regulated, including shortage of knowledge about the molecular mechanisms involved and their functional and physiological implications. Transcriptional silencing makes early embryos an ideal system with which to study RNA degradation.
The goal of this project is to uncover mechanisms and functions of RNA degradation in early development using zebrafish embryos as an in vivo model system. We aim to develop a systematic and predictive understanding of RNA degradation: technologies to globally measure it on a genome-scale, the molecular mechanisms involved, its functional and physiological implications during early embryogenesis and models to decode and predict it. The developed methodology and models will find broad application in RNA degradation studies in other organisms and diverse biological contexts, ranging from disease mechanisms to engineering of RNA- protein interactions.
Aim 1.1: A comprehensive catalog of embryonic cis-regulatory elements.
1. collected time-series samples for mRNA stability analysis with different UTR-Seq reporters.
2. optimized a protocol to measure changes to polyA tail length of UTR-Seq reporters, a process that occurs upstream to mRNA stability.

Aim 1.2: A high-resolution atlas of RNA regulatory dynamics during early embryogenesis.
1. Optimizing protocols for pulsed labeling of RNA in zebrafish embryos
2. Collecting samples for a continuous labeling time-course dataset

Aim 1.3: Decoding rules for in vivo degradation of native mRNAs.
1. Preliminary development and testing of higher order models based on deep learning tools

Aim 2.1: Monitoring mRNA degradation at single-cell resolution
1. Optimizing a single-cell metabolic labeling assay in live embryos using the drop-Seq platform
2. Application of modeling tools to infer zygotic vs. maternal RNA quantities in single cells
3. Elicit critical information on gene regulation as it unfolds over both time and space, which is obscured in standard single-cell analyses.

Aim 2.2: Mapping cis-signal activities at single-cell resolution
1. Analyzed embryos injected with UTR-Seq reporters via three alternative single-cell sequencing platforms: (1) drop-Seq, (2) in-drops and (3) commercial 10X chromium.

Aim 3.1: Functional studies of trans-regulators of mRNA stability
1. Designing and applying a CRISPR/cas9 based mutagenesis approach to target 16 candidate RNA binding proteins (RBPs) from 6 different families.
2. Generated 6 stable null mutants, 5 isolated heterozygous mutants, and screening founders for final 5 candidates.
3. Cloned 4 candidate RBP genes for in-vitro mRNA synthesis and gain-of-function analysis.

Aim 3.3: Uncovering molecular outcomes of maternal mRNA degradation
1. developed an analysis pipeline to classify maternal genes and analyze their degradation patterns in early embryos, by exploiting exon and intron expression in ribosomal depleted RNA-Seq samples.
2. Implementing a comparative genetic analysis to uncover a physiological role for a timely mRNA degradation.
3. Optimized an assay for over-expression of a stable versions of unstable maternal mRNAs and testing its physiological implications on development.
4. Analyzed function and developmental roles for changes in mRNA stability of 3 candidate genes: maternal Buckyball (Buc), 4 core Histone mRNAs and zygotic tbxta.
This project aims to systematically decipher the roles and mechanisms of post-transcriptional RNA degradation within embryonic gene expression programs. Its successful implementation will address important questions in developmental biology and regulation of RNA degradation.

Aim 1: This aim will generate two large datasets: (1) catalogues of cis-elements that regulate mRNA degradation across different positions, lengths and modifications, and (2) a high-resolution atlas of RNA regulatory dynamics during early embryogenesis. Based on this data, we will (1) build novel computational tools to systematically map mRNA degradation profiles and their cis-regulatory determinants, (2) develop a predictive model of genomic information within native mRNA sequences and define regulatory rules of maternal mRNA degradation and (3) provide design tools to predict the impact of genetic variations on mRNA stability and expression patterns in early embryos, and determine the effects of variants that are associated with disease or positive selection and to design synthetic mRNAs with pre-defined stabilities.

Aim 2: This aim will develop a technology to monitor the regulation of mRNA stability in early embryogenesis at single-cell resolution on a genome-scale. The technology is based on single-cell resolution metabolic labeling and reporter assays, in combination with novel computational tools for the analysis of those datasets to map both mRNA degradation rates and cis-element activities within single cells and cell-types of developing zebrafish embryos. We will use this data to investigate key questions on mRNA stability and its roles during development: (1) Decode differential mRNA stabilities and cis-element activities between cell-types and spatially restricted embryonic cell populations. (2) Determine cell-to-cell variability of mRNA stabilities, its stochastic variation and its functional roles in regulation of cellular population. (3) Provide an improved resolution and a refined cis-regulatory code for decoding mRNA degradation patterns within specific developmental cell-types in early embryos. (4) Decode regulation of mRNA stability within developmental gene expression programs that define and maintain cellular states.

Aim 3: This aim will generate comprehensive gene network models of the maternal mRNA decay programs in zebrafish embryos, a key regulatory event in early embryogenesis that is shared in all animals. These gene network models will describe known regulatory interactions, predict novel regulators and reveal missing components. We will use these models to (1) characterize the molecular mechanisms, functions and physiological roles of maternal RNA degradation (2) implement and apply a screening strategy for maternal mRNAs, and uncover physiological roles for their timely degradation during early embryogenesis.
These outcomes have important practical implications on understanding disease mechanisms, drug discovery, and therapeutics. The resulting technology will provide principled and unbiased tools to decode regulation of RNA stability across many systems and processes, ranging from disease mechanisms to the engineering of RNA-protein interactions.