Fig. 2: NRBP1 and NRBP2 contrarily regulate ORF1p and L1 mRNA association.
From: Opposing roles of pseudokinases NRBP1 and NRBP2 in regulating L1 retrotransposition
a ORF1p (green) forms cytoplasmic foci co-localizing with DDX6 (magenta) in MCF-7 cells, and their formation is enhanced upon NRBP1 knockdown. Nuclei: blue. Yellow arrows indicate ORF1p foci. Scale bar 10 µm. n = 3 biological replicates with similar results. b NRBP2 (red) overexpression induces ORF1p (green) foci co-localizing with G3BP1 (magenta) and DDX6 (magenta) in MCF-7 cells. Nuclei: blue. Scale bar 10 µm. n = 2 biological replicates with similar results. c L1 mRNA is not enriched in ORF1p foci upon NRBP1 knockdown in MCF-7 cells. Immunofluorescence (ORF1p, green) and smFISH (L1 mRNA, magenta) are shown. Arrows (top) indicate partial co-localization of ORF1p and L1 mRNA; arrows (bottom) indicate ORF1p puncta lacking L1 mRNA enrichment. Nuclei: blue. Scale bar 10 µm. n = 3 biological replicates with similar results. d Line profile (yellow line in c, top) shows partial co-localization between L1 mRNA and ORF1p in shControl cells. Pearson’s r = 0.74. e Line profile (yellow line in c, bottom) reveals no enrichment of L1 mRNA in ORF1p foci upon NRBP1 knockdown. Pearson’s r = −0.23. f ORF1p RIP-qPCR quantifying endogenous ORF1p-associated L1 mRNA upon NRBP1 knockdown in MCF-7 cells. Primer pairs target L1 5′ UTR, ORF1, ORF2. n = 3 biological replicates. Data: mean ± SEM. Two-sided unpaired t-test; no multiple comparison adjustment. p = 6.25 × 10-6 (5′ UTR), 8.7 × 10-6 (ORF1), 3.6 × 10-7 (ORF2). * * * * p < 0.0001. g Western blot showing NRBP1 knockdown efficiency and ORF1p IP in (f). Uncropped blots in Source Data. h NRBP2 overexpression prevents the interaction between ORF1p and L1 mRNA. RIP-qPCR was carried out to quantify Flag-tagged ORF1p co-immunoprecipitated L1 mRNA levels in HeLa cells. n = 3 biological replicates. Data: mean ± SEM. Two-sided unpaired t-test; no multiple comparison correction. p = 6.83 × 10-8 (5′ UTR), 3.7 × 10-9 (ORF1), 6.41 × 10-7 (ORF2). * * * * p < 0.0001. i Western blot assesses efficiency of HA-NRBP2 transfection and ORF1p IP for the experiments in (h). Uncropped blots in Source Data.
