Fig 1: RPSAP52 interacts with IGF2BP2 and HNRNPQ and influences proliferative pathways in MCF10A cells. a RNA pull-down assay to detect RPSAP52-associated proteins. In vitro synthesized full-length RPSAP52 transcript (including the alternative exon) or control sequences (the antisense transcript and the unrelated Uc.160+ RNA) were tested. The proteins retrieved were analyzed by SDS-PAGE and the band of ~70 kDa indicated by the arrow was identified by MS as containing IGF2BP2 and HNRNPQ. b Western blot showing the association between RPSAP52 RNA and IGF2BP2 and HNRNPQ proteins. Different truncated fragments of RPSAP52 RNA (as shown in the upper diagram) were incubated in the presence of MCF10A total protein extracts and the pulled-down material was subject to western blot with specific antibodies. Total extract from the MCF10A cell lines was used as input control, and a reaction without RNA (beads) as negative control. c Stable knockdown of RPSAP52 results in upregulation of let-7 family of miRNAs. Total RNA from MCF10A clones constitutively expressing two different shRNAs against RPSAP52 (sh1 or sh4) was analyzed by RT-qPCR to assess HMGA2 mRNA, RPSAP52 transcripts and let-7 miRNAs levels. Graphs represent the mean ±SD of three independent RNA extractions. Two-tailed student t-test were used (*P < 0.05, **P < 0.01, ns = not significant). d Western blot to analyze IGF2BP2, IGF1R, and RAS protein levels upon stable knockdown of RPSAP52 transcripts. e Transient transfection of MCF10A cells with locked nucleic acid (LNA)-based antisense oligonucleotides (ASOs) gapmers targeting HMGA2 mRNA (HMGA2), exon1 (RPSAP52 Ex) or the first intron (RPSAP52 RL1 and RL2) of RPSAP52 transcript. Expression levels of HMGA2, RPSAP52 and let-7 were measured by RT-qPCR. Graphs represent the mean ±SD of seven independent replicates (for HMGA2 and RPSAP52) or three replicates (for let-7). Two-tailed student t-test were used (*P < 0.05, **P < 0.01, ***P < 0.001). f Western blot to analyze IGF2BP2 and RAS protein levels upon transient knockdown of RPSAP52 transcripts. g Western blot to analyze IGF2BP2 and RAS protein levels upon transient overexpression of LIN28B protein in the background of RPSAP52 depletion. Source data are provided as a Source Data file
Fig 2: RPSAP52 is abundantly expressed in sarcoma and regulates the LIN28B/let-7 balance. a Upper graph: Pearson coefficient between RPSAP52 expression levels and CGI methylation in the TCGA sarcoma cohort indicates a negative correlation. Lower graph: Pearson’s index indicates a weaker association between RPSAP52 and HMGA2 expression levels in the same cohort. b Upper graphs: RT-qPCR analysis to estimate HMGA2 and RPSAP52 expression levels in a panel of Ewing’s sarcoma and rhabdomyosarcoma cell lines. Expression is relative to GUSB mRNA levels. Graphs represent the mean ±SD of three independent RNA extractions. Lower panel: semi-quantitative RT-PCR analysis of expression in the same cell lines. The two RPSAP52 isoforms are indicated, and the higher-migrating band depicted by an asterisk contains an additional exonic sequence (encompassing coordinates chr12:66,169,917–66,170,002 (hg19)), detected in those cells lines with the highest expression of RPSAP52. c RNA pull-down assays confirm the interaction of RPSAP52 with IGF2BP2 and HNRNPQ in A673 cell extracts. Different truncated fragments of RPSAP52 were assayed as indicated, and the band identified by MS and corresponding to IGF2BP2 and HNRNPQ is indicated on the protein gel (left). Western blot to test the association between RPSAP52 RNA and IGF2BP2 and HNRNPQ proteins (middle panel). The drawing summarizes the data obtained from the pull-downs (right). d Total RNA from A673 stable clones constitutively expressing sh1 or sh4 shRNA sequences was analyzed by RT-qPCR to assess HMGA2 and RPSAP52 transcripts levels (lower graph) or let-7 miRNAs levels (upper graph). Graphs represent the mean ±SD of three independent replicates. e Western blot on A673 clones to analyze protein levels upon stable knockdown of RPSAP52 transcripts. f Growth-inhibitory effect of RPSAP52 knockdown in A673 mice tumor xenografts. Upper graph: tumor volume (n = 10) was monitored over time. Mean values are shown ±SEM. Lower graph: tumors were excised and weighed at 25 days. The photograph shows the relative size of all tumors extracted. Scale bar = 10 mm. Source data are provided as a Source Data file
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