title: 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.
authors.affiliation: Department of Cardiology, Boston Children's Hospital, Boston, MA 02115.
authors.forename: Pingzhu
authors.initials: P
authors.lastname: Zhou
authors.forename: Yijing
authors.initials: Y
authors.lastname: Zhang
authors.forename: Qing
authors.initials: Q
authors.lastname: Ma
authors.forename: Fei
authors.initials: F
authors.lastname: Gu
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Day
authors.forename: Aibin
authors.initials: A
authors.lastname: He
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Sean M
authors.initials: SM
authors.lastname: Stevens
authors.forename: Daniel
authors.initials: D
authors.lastname: Romo
authors.forename: William T
authors.initials: WT
authors.lastname: Pu
mesh_terms: D000483:Alleles; D000818:Animals; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D004452:Echocardiography; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D051379:Mice; D012335:RNA, Ribosomal / Q000302:isolation & purification*; D022661:RNA, Untranslated / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism; D059467:Transcriptome / Q000235:genetics*
chemical_list: D017931:DNA Primers; C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D012335:RNA, Ribosomal; D022661:RNA, Untranslated; D012269:Ribosomal Proteins; C000630690:Rpl10 protein, mouse; D049452:Green Fluorescent Proteins
keywords: heart; pressure overload