Interrogating translational efficiency and lineage-specific transcriptomes using ribosome affinity purification

Abstract

Transcriptional profiling is a useful strategy to study development and disease. Approaches to isolate RNA from specific cell types, or from specific cellular compartments, would extend the power of this strategy. Previous work has shown that isolation of genetically tagged ribosomes (translating ribosome affinity purification; TRAP) is an effective means to isolate ribosome-bound RNA selectively from transgene-expressing cells. However, widespread application of this technology has been limited by available transgenic mouse lines. Here we characterize a TRAP allele (Rosa26 fsTRAP ) that makes this approach more widely accessible. We show that endothelium-specific activation of Rosa26 fsTRAP identifies endothelial cell-enriched transcripts, and that cardiomyocyte-restricted TRAP is a useful means to identify genes that are differentially expressed in cardiomyocytes in a disease model. Furthermore, we show that TRAP is an effective means for studying translational regulation, and that several nuclear-encoded mitochondrial genes are under strong translational control. Our analysis of ribosome-bound transcripts also shows that a subset of long intergenic noncoding RNAs are weakly ribosome-bound, but that the majority of noncoding RNAs, including most long intergenic noncoding RNAs, are ribosome-bound to the same extent as coding transcripts. Together, these data show that the TRAP strategy and the Rosa26 fsTRAP allele will be useful tools to probe cell type-specific transcriptomes, study translational regulation, and probe ribosome binding of noncoding RNAs.heart | pressure overload G enome-wide and unbiased measurement of RNA transcript levels using microarrays and RNA-seq (1) has powered fundamental advances in biology over the past decade. However, when used to study tissues composed of multiple cell types, RNA expression profiling faces two fundamental limitations. First, whole-tissue transcript levels represent the average of the distinct cell lineages in the tissue, and this averaging process can result in loss of important information or misassignment of gene expression changes in one type of cell to another. Second, RNA profiling measures transcript abundance, but translational regulation is also an important determinant of gene expression (2, 3).To overcome these limitations, approaches have been developed to isolate RNAs from selected cell types and/or selected transcript fractions (4-7). Often these approaches involve tissue dissociation followed by FACS, but this is slow and the dissociation procedure itself likely alters expression profiles. Translating ribosome affinity purification (TRAP) permits isolation of transcripts from selected cell types of intact tissues, without dissociation (4). In this approach, ribosomes of selected tissues are genetically labeled by transgenic expression of GFP fused to L10a, an integral component of the 60S ribosomal subunit. To collect RNA from the transgene-expressing subpopulation of a tissue, whole-cell lysates are prepared under conditions that stabilize ribosomes...

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