Aimp2 Gene

[Endike Baur]

TheJTV1 gene is located on chromosome 7p22 flanked by two genes, HRI and PMS2. JTV1 and HRI overlap slightly and are arranged in a tail-to-tail fashion. JTV1 and PMS2 are separated by approximately 200 base pairs and are arranged head-to-head. JTV1 is transcribed in the opposite direction compared to HRI and PMS2. The function of the JTV1 gene product is unknown.[7]

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Aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2) is a non-enzymatic component required for the multi-tRNA synthetase complex. While exon 2 skipping alternatively spliced variant of AIMP2 (AIMP2-DX2) compromises AIMP2 activity and is associated with carcinogenesis, its clinical potential awaits further validation. Here, we found that AIMP2-DX2/AIMP2 expression ratio is strongly correlated with major cancer signaling pathways and poor prognosis, particularly in acute myeloid leukemia (AML). Analysis of a clinical patient cohort revealed that AIMP2-DX2 positive AML patients show decreased overall survival and progression-free survival. We also developed targeted RNA-sequencing and single-molecule RNA-FISH tools to quantitatively analyze AIMP2-DX2/AIMP2 ratios at the single-cell level. By subclassifying hematologic cancer cells based on their AIMP2-DX2/AIMP2 ratios, we found that downregulating AIMP2-DX2 sensitizes cells to anticancer drugs only for a subgroup of cells while it has adverse effects on others. Collectively, our study establishes AIMP2-DX2 as a potential biomarker and a therapeutic target for hematologic cancer.

Aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2) is a component of a macromolecular protein complex consisting of several different aminoacyl-tRNA synthetases (MSC). It is a nonenzymatic auxiliary component required for the integration and stability of this translational complex1,2. In addition, AIMP2 can also act as a potent tumor suppressor3. In response to DNA damage, a fraction of AIMP2 dissociates from the MSC, and induces apoptosis by binding to p53 and protecting p53 from degradation through ubiquitination by murine double minute 2 (MDM2)4. In addition, AIMP2 augments tumor necrosis factor-α-induced apoptotic signaling and exerts antiproliferative activities in TGF-β and Wnt pathways via distinct working mechanisms3,5,6. Given that these pathways are critically implicated in the control of tumorigenesis, AIMP2 is expected to act as a potent tumor suppressor with broad coverage against various types of cancer. In fact, AIMP2 haploid mice showed increased tumor susceptibility compared to the wild-type littermates to carcinogenic treatment, confirming its tumor-suppressive activity in vivo3.

The full-length AIMP2 transcript consists of four exons, but a small fraction of the pre-mRNA undergoes alternative splicing to produce a variant lacking the second exon (AIMP2-DX2). AIMP2-DX2 protein compromises the tumor-suppressive activity of AIMP2 via competitive binding to p53, but fails to protect p53 from MDM2-mediated ubiquitination7. In contrast to AIMP2, which is mainly bound to the MSC, AIMP2-DX2 cannot work as a scaffold for MSC assembly, and thus works as a potent competitor for the tumor-suppressive activities of AIMP27.

AIMP2-DX2 is receiving increasing attention as an attractive biomarker for diagnosis and prognosis7,8. Moreover, AIMP2-DX2 showed potential as a therapeutic target, since the downregulation of AIMP2-DX2 suppressed the growth of cancer cells and tumors in vivo7,8. Therefore, quantifying AIMP2-DX2 expression would allow subclassification of cancer patients and identify those who may undergo AIMP2-DX2 targeting treatment. Despite the mounting pieces of evidence, the expression of AIMP2-DX2 and its clinical implications in various types of cancer have not yet been clearly demonstrated.

The clinical application of AIMP2-DX2 has been limited due to the lack of a detection technique that allows a quantitative assessment of the AIMP2-DX2/AIMP2 expression ratio. Currently, the primary experimental approach relies on PCR amplification and examining the size difference between the two splicing variants through electrophoresis, which cannot be applied to analyze patient samples. Molecular beacon-based detection technique has been developed9, but its clinical applicability is questionable. Moreover, molecular beacon fails to examine both AIMP2 and AIMP2-DX2 mRNAs simultaneously in the same group of cells. Considering the competitive situation of AIMP2-DX2 and AIMP2 in carcinogenesis, simultaneous quantitation of the two variants is expected to provide a more relevant marker for accurate assessment of patient samples.

In situ hybridization (ISH) uses nucleic acid probes that are complementary to the target DNA/RNA sequences to detect and visualize the target. Clinically, DNA-ISH has been widely used to visualize DNA pathogenic variants or chromosomal structures10. However, as DNA does not provide information on gene expression, in particular those of alternatively spliced RNA variants, RNA-ISH is an alternative approach to investigate mRNA expressions. In addition, RNA-ISH allows analysis at a single-cell level with minimal sample disruption, which makes it an attractive clinical tool. Moreover, using multiplex single-molecule fluorescence ISH (smFISH), expression levels of both AIMP2 and its splicing variant AIMP2-DX2 mRNAs can be quantified and compared together in the same cells.

In the present study, we investigated the significance and clinical implications of AIMP2-DX2 by analyzing samples from the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) database, and validated results with a clinical patient cohort of acute myeloid leukemia (AML), which was found to have the most significant association with AIMP2-DX2/AIMP2 expression ratio in terms of cancer signaling pathways. The potential of AIMP2-DX2 as a key regulator of major cancer signaling pathways in AML was further analyzed using AML cell line, which further supported our analysis of public databases. Moreover, to satisfy the unmet clinical need for a quantitative assessment of the AIMP2-DX2 expression ratio at the single-cell level, we developed an RNA-smFISH-based image analysis tool to measure AIMP2-DX2/AIMP2 expression ratio. Our image analysis showed good concordance with targeted RNA-sequencing, an alternative method to quantitate the AIMP2-DX2/AIMP2 expression ratio. Applying our image-based quantification tool, we subclassified seven hematologic cancers based on their AIMP2-DX2/AIMP2 expression ratios. More importantly, we showed that targeting AIMP2-DX2 expression in those cells with a high AIMP2-DX2/AIMP2 expression ratio could sensitize cells to anticancer drugs. To our surprise, we also found that targeting AIMP2-DX2 in cells with a low AIMP2-DX2/AIMP2 expression ratio had adverse effects and made cells more resistant to anticancer drugs. Collectively, our work provides the development of tools for quantitative assessment of alternatively spliced variants and clinical implications of AIMP2-DX2, as a potential biomarker and therapeutic target in hematologic cancer.

To examine the pathophysiological implications of AIMP2 and AIMP2-DX2 in carcinogenesis, we assessed their basal mRNA expression levels using the multiplex RNAscope smFISH technique11. We first designed smFISH ZZ probe pairs that targeted only the exon 2 and tagged them as channel 1 (C1). We designed another set of ZZ probe pairs that recognized exons 1, 3, and 4, and tagged them as channel 2 (C2). By simultaneously hybridizing and amplifying these two sets of probes, we could visualize AIMP2 mRNAs in one color (green; C1), while another color can be designated to visualize both AIMP2 and AIMP2-DX2 mRNAs (red; C2; Fig. 1a). We tested our design using HeLa cells, and found that both C1 and C2 probes yielded foci-like signal patterns (Fig. 1b). As a control, we performed RNA-smFISH without hybridization of C1 and C2 probes, which resulted in no fluorescent signal (Fig. 1b). By quantifying and determining the red-to-green signal ratios, we can infer the relative expression ratio of AIMP2-DX2/AIMP2 mRNA variants (Fig. 1b).

To examine whether our RNA-smFISH approach can be applied in cells other than HeLa, particularly for lung carcinomas where AIMP2-DX2 was reported for the first time7, we quantified the AIMP2-DX2/AIMP2 expression ratio in A549 lung adenocarcinoma cells (Fig. 1g, h). We again confirmed the specificity of our quantification using siRNAs (Fig. 1h). We then compared the AIMP2-DX2 expression ratios between HeLa and A549 cells. A549 cells showed a red-to-green ratio that was significantly higher than that of HeLa cells (Fig. 1i), which is consistent with the previous reports that the AIMP2-DX2 variant is frequently observed in lung carcinomas7. Of note, due to differences in the number of ZZ pairs between the two channels, we found that the efficiencies of the probes in capturing their target mRNAs were different. This resulted in only a few overlapping foci because there are always more red foci than the green. Consequently, we could not quantitate the absolute expression ratio of AIMP2-DX2/AIMP2 mRNAs. Nevertheless, the quantified fluorescence ratios consistently reflected changes in AIMP2 and AIMP2-DX2 mRNAs in siRNA-transfected samples. Therefore, our approach semiquantitatively reflects changes in the ratios of the two variants upon siRNA transfection and in different cellular contexts.

In a differentially expressed gene (DEG) set analysis, 10 out of 13 predefined major cancer pathways were shown to correlate with the AIMP2-DX2/AIMP2 expression ratio to different degrees and directions among 14 cancer types (Fig. 2c), while the other 9 cancer types (ovarian cancer, breast invasive carcinoma, colon adenocarcinoma, melanoma, rectum adenocarcinoma, kidney renal clear cell carcinoma, prostate adenocarcinoma, head and neck squamous cell carcinoma, and uterine corpus endometrial carcinoma) did not show clear correlations. Interestingly, most of the major cancer pathways in AML were highly upregulated in patients with high AIMP2-DX2/AIMP2 expression ratios (Fig. 2c). Whole differentially expressed pathways by the AIMP2-DX2/AIMP2 expression ratio are available in Supplementary Fig. 2.

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