The Fusion of CLEC12A and MIR223HG Arises from a trans-Splicing Event in Normal and Transformed Human Cells

Abstract

Chimeric RNAs are often associated with chromosomal rearrangements in cancer. In addition, they are also widely detected in normal tissues, contributing to transcriptomic complexity. Despite their prevalence, little is known about the characteristics and functions of chimeric RNAs. Here, we examine the genetic structure and biological roles of CLEC12A-MIR223HG, a novel chimeric transcript produced by the fusion of the cell surface receptor CLEC12A and the miRNA-223 host gene (MIR223HG), first identified in chronic myeloid leukemia (CML) patients. Surprisingly, we observed that CLEC12A-MIR223HG is not just expressed in CML, but also in a variety of normal tissues and cell lines. CLEC12A-MIR223HG expression is elevated in pro-monocytic cells resistant to chemotherapy and during monocyte-to-macrophage differentiation. We observed that CLEC12A-MIR223HG is a product of trans-splicing rather than a chromosomal rearrangement and that transcriptional activation of CLEC12A with the CRISPR/Cas9 Synergistic Activation Mediator (SAM) system increases CLEC12A-MIR223HG expression. CLEC12A-MIR223HG translates into a chimeric protein, which largely resembles CLEC12A but harbours an altered C-type lectin domain altering key disulphide bonds. These alterations result in differences in post-translational modifications, cellular localization, and protein-protein interactions. Taken together, our observations support a possible involvement of CLEC12A-MIR223HG in the regulation of CLEC12A function. Our workflow also serves as a template to study other uncharacterized chimeric RNAs.

Keywords: C-type lectin; CCL1; Fusion RNAs encoding protein; alternative splicing; chimeric RNAs; chronic myeloid leukemia; fusion transcript; linc-223; miR-223 host gene; myeloid cell differentiation; trans-splicing.

Figure 1

CLEC12A-MIR223HG detection in CML patients, controls, normal tissues, and cell lines. (A) Reads mapping across the CLEC12A/MIR223HG breakpoint. (B) Read counts that span across the fusion breakpoint in representative CML diagnostic and control samples. (C) RT-PCR-based detection of CLEC12A-MIR223HG in CML diagnostic and control samples with primers flanking the breakpoint. Amplicon sizes expected: B2M: 96 bp, CLEC12A-MIR223HG: 207 bp. (D) RT-PCR-based detection of CLEC12A-MIR223HG in diverse normal tissues and RNA sequencing-based quantification of CLEC12A and MIR223HG transcript levels. (E) Contour plot illustrating correlation between CLEC12A and MIR223HG across different tissues. Correlation coefficient (r) was determined using Pearson correlation and the red line indicates the line of best fit. (F) RT-PCR-based detection of CLEC12A-MIR223HG in leukemic cell lines. TPM (transcripts per million) values for tissues were obtained from the Genotype-Tissue Expression (GTEx) Portal V8 (https://gtexportal.org) (accessed on 25 October 2021). TPM values for cell lines were obtained from the Cancer Cell Line Encyclopedia project portal (https://depmap.org/portal/ccle/) (accessed on 25 October 2021).

Figure 2

Transcript structure of CLEC12A-MIR223HG. (A) Illustration of the breakpoint location and the resulting fusion transcript. (B) RT-PCR on U-937 mRNA to identify the full length CLEC12A-MIR223HG transcript. Expected amplicon sizes are as follows: A/B: 207 bp, A/C: 361 bp, A/D: 371 bp, A/E: 564 bp, A/F: 579 bp, H/B: 829 bp, G/B: 962 bp. The chimeric transcript is produced by the substitution of the final exon of CLEC12A-201 isoform with a part of MIR223HG including an in-frame stop codon followed by a poly(A) sequence. (C) Illustration of the CRISPR/SAM-based transcriptional activation system used to increase the endogenous expression of CLEC12A. (D) Three independent replicates of qRT-PCR accessing expression levels of CLEC12A and CLEC12A-MIR223HG after CRISPR/SAM-mediated transcriptional activation of CLEC12A in HEK293 cells. Expected amplicon sizes are as follows: B2M: 91 bp, CLEC12A: 105 bp, CLEC12A-MIR223HG: 206 bp. B2M was used to determine the dCt values.

Figure 3

CLEC12A-MIR223HG encodes a chimeric protein distinct from CLEC12A. (A) Schematic of CLEC12A and CLEC12A-MIR223HG protein architectures. (B) Western blot image of cell lysates from HEK293 cells transduced with lentiviral vectors expressing either CLEC12A or CLEC12A-MIR223HG. (C) Western blot image of PNGase F-treated and untreated U-937 cell lysates overexpressing CLEC12A or CLEC12A-MIR223HG. (D) Immunoprecipitation with anti-FLAG antibody coupled with mass spectrometry of U-937 cells expressing either CLEC12A or CLEC12A-MIR223HG. Total peptide intensity divided by the number of observable peptides for a particular protein (iBAQ) is depicted. Compared to CLEC12A, the chimeric protein has gained interacting partners and lost at least one. Three separate replicates were performed. (E) Representative images of immunofluorescence with anti-FLAG antibody (green) and DAPI (blue) of U-937 cells expressing either CLEC12A or CLEC12A-MIR223HG. Image analysis was performed using Biplane Imaris software. Three different angels of the same cell are depicted.

Figure 4

Biological roles of CLEC12A-MIR223HG. (A) Fold change in the expression of CLEC12A and CLEC12A-MIR223HG in U-937 cells treated with two different doses of cytarabine (AraC) measured by qRT-PCR. (B) Percentage of apoptotic cells (Annexin V-positive cells) after the treatment of U-937 cells overexpressing CLEC12A or CLEC12A-MIR223HG with cytarabine. (C) Representative flow cytometry plots depicting percentage of apoptotic U-937 cells overexpressing either CLEC12A or CLEC12A-MIR223HG after AraC treatment. X-axis: Annexin V, Y-axis: mCherry (depicting transduction efficiency). (D) Fold change in the expression of CLEC12A and CLEC12A-MIR223HG during the differentiation of U-937 monocytes induced by phorbol 12-myristate 13-acetate (PMA) treatment (measured by qRT-PCR). (E) Percentage of either CD45+ or CD44+ cells after the treatment of U-937 cells overexpressing CLEC12A or CLEC12A-MIR223HG with PMA. (F) Representative flow cytometry plots depicting the percentage of either or CD44 (x-axis) or CD45 (y-axis) in PMA-treated U-937 monocytes overexpressing CLEC12A or CLEC12A-MIR223HG (n > 3, * p < 0.05, ** p < 0.01).