Translational profiling through biotinylation of tagged ribosomes in zebrafish

Abstract

Heterogeneity within a population of cells of the same type is a common theme in metazoan biology. Dissecting complex developmental and physiological processes crucially relies on our ability to probe the expression profile of these cell subpopulations. Current strategies rely on cell enrichment based on sequential or simultaneous use of multiple intersecting markers starting from a heterogeneous cell suspension. The extensive tissue manipulations required to generate single-cell suspensions, as well as the complexity of the required equipment, inherently complicate these approaches. Here, we propose an alternative methodology based on a genetically encoded system in the model organism Danio rerio (zebrafish). In transgenic fish, we take advantage of the combinatorial biotin transfer system, where polysome-associated mRNAs are selectively recovered from cells expressing both a tagged ribosomal subunit, Rpl10a, and the bacterial biotin ligase BirA. We have applied this technique to skeletal muscle development and identified new genes with interesting temporal expression patterns. Through this work we have thus developed additional tools for highly specific gene expression profiling.

Robust Identification of Developmentally Active Endothelial Enhancers in Zebrafish Using FANS-Assisted ATAC-Seq

“…However, this approach requires generation of numerous transgenic lines and relies on ChIP-Seq, which is more technically demanding and requires more cells than ATAC-Seq. While such “Biotagging” approaches have begun to be applied in zebrafish (Housley et al, 2014; Trinh et al, 2017), FANS-assisted ATAC-Seq is less technically demanding and can be applied to available zebrafish transgenic lines using a small number of cells.…”

Ribosome profiling reveals the what, when, where and how of protein synthesis

“…Additionally, it can be combined with approaches to look at different polysome fractions 55,69 or the translating ribosome affinity capture (TRAP) 70–76 [G] strategy. Together these techniques could make it possible to explore regulated localized translation in specific neuronal subtypes and in response to learning programs.…”

Biotagging of Specific Cell Populations in Zebrafish Reveals Gene Regulatory Logic Encoded in the Nuclear Transcriptome

“…In vivo biotinylation of Avi-tagged Rpl10 protein in zebrafish embryos can purify ribosomes via the translating ribosome affinity purification (TRAP) method (Heiman et al., 2008) for translational profiling (Housley et al., 2014). A full understanding of the RNA landscape and its regulation would require profiles of both subcellular compartments.…”