Novel Non-Coding Transcript in NR4A3 Locus, LncNR4A3, Regulates RNA Processing Machinery Proteins and NR4A3 Expression

Abstract

NR4A3 is a key tumor suppressor in myeloid malignancy, mice lacking both NR4A1 and family member NR4A3 rapidly develop lethal acute myeloid leukemia (AML). We identified a long non-coding transcript in the NR4A3 locus and pursued the characterization of this anonymous transcript and the study of its role in leukemogenesis. We characterized this novel long non-coding transcript as a sense polyadenylated transcript. Bone marrow cells from AML patients expressed significantly reduced levels of lncNR4A3 compared to healthy controls (controls = 15, MDS= 20, p=0.05., AML= 21, p<0.01). Expression of NR4A3, as previously reported, was also significantly reduced in AML. Interestingly, the expression of both coding and non-coding transcripts was highly correlated (Pearson R = 0.3771, P<0.01). Transient over-expression of LncNR4A3 by nucleofection led to an increase in the RNA and protein level of NR4A3, reduction of proliferation in myeloid cell lines K-562 and KG1 (n=3 and 2 respectively, p<0.05) and reduced colony formation capacity in primary leukemic cells. A mass spectrometry-based quantitative proteomics approach was used to identify proteins dysregulated after lncNR4A3 over-expression in K-562. Enrichment analysis showed that the altered proteins are biologically connected (n=4, p<0.001) and functionally associated to RNA binding, transcription elongation, and splicing. Remarkably, we were able to validate the most significant results by WB. We showed that this novel transcript, lncNR4A3 regulates NR4A3 and we hypothesize this regulatory mechanism is mediated by the modulation of the RNA processing machinery.

Keywords: NR4A3; RNA processing; acute myeloid leukemia; long non-coding RNA; myeloid malignancy.

Figure 1

(A) Transcriptional map of the NR4A3 locus showing position of LncNR4A3 and primers used in this work. (B) EtdBr-staining bands in agarose gel electrophoresis from strand-specific PCR to identify the orientation of the transcript, lncNR4A3 is transcribed in the same orientation than NR4A3. (C, D) Quantitative RT-PCR (QRT-PCR) quantification of NR4A3 and long non-coding transcript, LncNR4A3, in normal bone marrow cells (NBM) of 15 controls, 20 myelodysplastic syndrome patients (MDS), and 21 acute myeloid leukemia patients bone marrows (AML). (E) Linear regression of the expression of LncNR4A3 plotted against NR4A3 expression and correlation analysis results (Pearson correlation P value, R coefficient). ** : <0.01, *** : <0.001.

Figure 2

(A) Protein–protein interaction network, a weighted linkage graph constructed using STRING software, inputting the differentially expressed proteins identified in the proteomics analysis. (B) Histogram plotting the Log[10] of the p-values obtained from the gene ontology enrichment analysis, cellular component, and molecular function ontology Log[10]p-values were plotted for up-regulated and down-regulated proteins obtained from proteomics analysis, threshold of 5 is denoted by the dashed line. (C) Immunoblots confirming regulation of several target proteins identified by the MS-proteomics analysis in K-562 controls and lncNR4A3 over-expressing cells, blots from three independent experiments shown in Supplementary Figure 10 , paired t-test was applied to stablish significant differences between optical density of the blots (*: <0.05, **: <0.01, ***<0.001).

Figure 3

(A) Relative quantification by quantitative RT-PCR (qRT-PCR) of NR4A3 expression in LncNR4A3 over-expressing K-562 and empty vector control cells shows significant modulation of NR4A3 at mRNA level in K-562 (mean ± SEM, n=3), similar results for KG1 cells are shown in Supplementary Figure 8 . (B) CCK-8 viability assay results showing reduced viability after lncNR4A3 over-expression in k-562, results for KG1 are shown in Supplementary Figure 8 . (C) Photographs of human hematopoietic CFUs in semi-solid culture (bar: 200µm), see additional colonies in supplementary information. Left: representative colony from control CD34+ cells after 15 day in culture. Right: representative cluster from LncNR4A3 over-expressing cells 15 days in culture. (D) Immunoblots showing the reactivation of NR4A3 protein in AML CD34+ cells, KG1 and K-562 cells. * : <0.05, *** : <0.001.