Fig 1: lncRAP2 forms a complex with mRNA stability and translation regulators(A and B) lncRAP2 does not engage translating ribosomes. Tracks show signal from ribosome profiling, RNA, and RNA interactome sequencing studies of differentiated white adipocytes (A). Data are pooled from n = 2–3 replicates. Translatability, measured by ribosome release from open reading frames after encountering a stop codon, is quantified in (B).(C) Efficient and specific enrichment of mature lncRAP2 by hybridization-based purification. Exon-targeting probes effectively retrieve ~90% of cellular lncRAP2 RNA, quantified by qPCR, from differentiated 3T3-L1 adipocytes, whereas <1% was retrieved by intron-targeting probes, no probes, or probes targeting unrelated RNAs. RNase treatment eliminates lncRAP2 transcripts prior to purification. ***p <0.001 (t test).(D and E) ChIRP-MS identifies specific lncRAP2-binding proteins. The relative enrichment of high-confidence interactors captured by antisense purification of lncRAP2 in white adipocytes or other lncRNAs in other formaldehyde cross-linked cells is shown in (D). Unique peptide counts for specific lncRAP2 interactors are shown in (E).(F) Validation of lncRAP2 and Igf2bp2 direct interaction in mature white adipocytes from mouse (left) and human (right). Native immunoprecipitation of Igf2bp2 specifically captures >50% of lncRAP2, compared to <2% for Ddx47 and Exosc6 (specific interactors) or Serbp1 and HnrpU (broad interactors). *p <0.05 (t test).(G and H) Igf2bp2 depletion blocks adipogenesis. Shown are lipid accumulation (G) and relative expression of key adipocyte genes (H) in day 6 differentiated white adipocytes pretreated with control or Igf2bp2-targeting DsiRNAs.
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