Fig. 1: NRBP1 and NRBP2 interact with L1-encoded ORF1p and contrarily regulate L1 retrotransposition.

a Scatter plots showing proteins significantly enriched (fold change ≥ 5, p < 0.05) in NRBP1/2 immunoprecipitation (IP) vs. IgG in MCF-7 cells. NRBP1 and NRBP2 are shown in red. Previously known NRBP1 interactors are labeled in blue. ORF1p is marked in orange. Statistical significance for protein enrichment was assessed using a one-sided two-sample Student’s t-test, with a valid value filter applied to at least two out of three replicates. No multiple testing correction was applied. b Endogenous ORF1p is co-immunoprecipitated with NRBP1 and/or NRBP2. An antibody recognizing both NRBP1 and NRBP2 was used to pull down endogenous NRBP1 and NRBP2 in MCF-7 cells. Co-precipitated ORF1p was detected by using ORF1p antibody. n = 3 biological replicates with similar results. Uncropped blots in Source Data. c Endogenous ORF1p is co-immunoprecipitated with both NRBP1 and NRBP2. MCF-7 cells were transfected with either Myc-Flag-tagged NRBP1 or Myc-Flag-tagged NRBP2. The cell lysates were incubated with Flag antibody and the immunoprecipitated proteins were detected by Western blot. n = 2 biological replicates with similar results. Uncropped blots in Source Data. d, e NRBP1 (d) and NRBP2 (e) are co-immunoprecipitated with ORF1p with or without RNA in HeLa cells. RNasin and RNase A were used to protect or digest RNA in the cell lysate. n = 3 biological replicates with similar results. Uncropped blots in Source Data. f-m, NRBP1 activates and NRBP2 inhibits L1 retrotransposition. Representative colony formation assays are shown in (f), (h), (j) and (l). Quantification and representative Western blots are shown in (g), (i), (k), and (m). Data: mean ± SEM; two-sided unpaired t-test without multiple comparison adjustment. n = 4 (f, g), 3 (h, i, l, m) biological replicates. Due to the small increase and high variability in L1 activity upon NRBP2 knockdown, seven biological replicates were performed for the assay in (j) and (k). Retrotransposition activity was normalized to pcDNA3.1 control to account for possible cytotoxic effects of NRBP1/2 manipulation.