The RNA-binding protein SART3 promotes miR-34a biogenesis and G1 cell cycle arrest in lung cancer cells

Emily J Sherman,Dylan C Mitchell,Amanda L Garner

doi:10.1074/jbc.ac119.010419

Emily J Sherman, Dylan C Mitchell + Show 1 more

Open Access

https://doi.org/10.1074/jbc.ac119.010419

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Abstract

MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of most biological processes. Global miRNA biogenesis is altered in many cancers, and RNA-binding proteins play a role in miRNA biogenesis, presenting a promising avenue for targeting miRNA dysregulation in diseases. miR-34a exhibits tumor-suppressive activities by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor BCL-2, among other regulatory pathways such as Wnt, TGF-β, and Notch signaling. Many cancers exhibit down-regulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo However, the post-transcriptional mechanisms that cause miR-34a loss in cancer are not entirely understood. Here, using a proteomics-mediated approach in non-small-cell lung cancer (NSCLC) cells, we identified squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a-binding protein. SART3 is a spliceosome recycling factor and nuclear RNA-binding protein with no previously reported role in miRNA regulation. We found that SART3 binds pre-miR-34a with higher specificity than pre-let-7d (used as a negative control) and elucidated a new functional role for SART3 in NSCLC cells. SART3 overexpression increased miR-34a levels, down-regulated the miR-34a target genes CDK4/6, and caused a cell cycle arrest in the G1 phase. In vitro binding experiments revealed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Our results provide evidence for a significant role of SART3 in miR-34a biogenesis and cell cycle progression in NSCLC cells.

Highlights

MicroRNAs are small, noncoding RNAs that are implicated in the regulation of most biological processes
In further support of our findings, we noted that SART3 had been detected as a pre-miR-34a-BP via MS-based proteomics in a recent report, which identified this interaction across several cancer cell lines using a test set of 72 pre-miRs [15]
The phenotypes we observed indicate that SART3 possesses tumor-suppressive properties in non-small-cell lung cancer (NSCLC) cells, which is in contrast to reports describing SART3 as an antigen in other cancers

Summary

Results

To identify proteins in the miR-34a interactome, we first established a method for miR-binding protein discovery in mammalian cells (Fig. 1A). In further support of our findings, we noted that SART3 had been detected as a pre-miR-34a-BP via MS-based proteomics in a recent report, which identified this interaction across several cancer cell lines using a test set of 72 pre-miRs [15]. From these results, we concluded that SART3 protein interacts with pre-miR-34a with specificity relative to pre-let-7d and several other pre-miRs. Having characterized a phenotype related to SART3 expression, we sought to elucidate a potential mechanism for recognition of pre-miR-34a by SART3. Within SART3 the HAT domains have roles in pre-mRNA 3Ј-end processing, ubiquitin-specific protease (USP) recruitment, and

H7 H8 H9 H10 H11 H12 NLS

Discussion

Experimental procedures