Oncogenic Role of THOR, a Conserved Cancer/Testis Long Non-coding RNA

Abstract

Summary Large scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality

“…We confirmed that an MS2-MCP tagged mRNA bearing the firefly luciferase (FL) coding sequence (CDS) and an artificial 3`untranslated region (3`UTR) bearing six tandem miRNA response elements (MREs) for let-7 (l7-6x) was translated and regulated much like its untagged counterpart ( Figure S1I). We additionally showed that the tagged lncRNA THOR, which binds PB-enriched IGF2BP1 protein (Hubstenberger et al, 2017), mediated an oncogenic phenotype by promoting cell growth and stimulating oncogene expression (Figure S1J-L) as expected (Hosono et al, 2017). We then adapted a super-registration fluorescence microscopy-based tool (Grunwald and Singer, 2010) that measures intermolecular distances of spectrally distinct fluorescent molecules with nanometer (nm) precision, thereby enabling the capture of transient RNA-PB interactions in living cells and the precise quantification of RNA localization patterns within PBs in fixed cells (Methods).…”

“…Mature regulatory miRNAs, whose size (~22 nt per strand) precludes endogenous labeling strategies (Pitchiaya et al, 2014), were chemically synthesized with a fluorescent Cy5 dye at the 3`end of one of their two complementary strands, typically the guide strand. Since transfection results in the sequestration of RNA within subcellular vesicles (Cardarelli et al, 2016), we chose to deliver these miRNAs via microinjection ( Figure 1A-C), which defines a clear starting point for our assays by instantaneously exposing RNAs to the cellular milieu (Pitchiaya et al, 2012;Pitchiaya et al, 2017;Pitchiaya et al, 2013). We confirmed that fluorophore labeling and microinjection did not affect the gene-repressive function ( Figure S1E-H) of a tumor suppressive let-7 (l7/l7* and l7-Cy5/l7*) miRNA (Pitchiaya et al, 2012).…”

“…We then adapted a super-registration fluorescence microscopy-based tool (Grunwald and Singer, 2010) that measures intermolecular distances of spectrally distinct fluorescent molecules with nanometer (nm) precision, thereby enabling the capture of transient RNA-PB interactions in living cells and the precise quantification of RNA localization patterns within PBs in fixed cells (Methods). By combining this tool with intracellular single molecule, high-resolution localization and counting (iSHiRLoC) (Pitchiaya et al, 2012;Pitchiaya et al, 2017;Pitchiaya et al, 2013) and single mRNA imaging, we were able to probe the PB-associated dynamics, stoichiometry and localization of either microinjected miRNAs ( Figure 1A-C, Supplementary movie 1) or ectopically expressed MS2-MCP tagged m/lncRNAs ( Figure 1D-F) at a spatial accuracy of 30 nm and temporal resolution of 50 ms. We found that RNAs diffused ~100-1,000fold slower while at PBs compared to the cytosol ( Figure 1G), supporting the notion that they physically dock to form higher order complexes at PBs. Concordantly, cytoplasmic RNAs were predominantly monomeric, whereas a significant fraction of RNAs in PBs appeared multimeric ( Figure 1H-J).…”

“…Next, we probed whether the specific RNA sequence within an RNA class influences its RNA-PB interactions. To this end, we compared the PB-localization of double-stranded l7-Cy5/l7* miRNA, whose regulatory functionality is clearly defined ( Figure S1A), to that of two small RNA constructs and a small DNA construct of distinct intracellular stability (Pitchiaya et al, 2017) and regulatory potential: 1) l7/l7*-Cy5, i.e., let-7 miRNA Cy5labeled on the passenger instead of the guide strand, where the passenger strand has very few endogenous targets and is at least 8-fold less stable than the guide strand; 2) ml7-Cy5/ml7*, a seed-sequence mutated let-7 miRNA variant that cannot bind endogenous let-7 targets and is at least 4-fold less stable than let-7 miRNA; and 3) dl7-Cy5/dl7*, a control DNA oligonucleotide of the same sequence as let-7 miRNA. As expected, the control DNA neither localized to, nor was enriched at PBs ( Figure 4A By contrast, the fractional extents of PB localization and enrichment were significant and similar for all three miRNAs ( Figure 4A-B), suggesting that miRNA functionality is not necessary for PB localization.…”

Meiosis related cancer-testis genes correlate with tumor aneuploidy and immune infiltration in 21 cancer types

“…Several CT genes, such as MAGEA3, FATE1, and LIN28B have been proved to contribute to the infinite proliferation of cancer cells (3)(4)(5). Besides protein coding CT genes, our previous work found that a number of non-coding genes also presented the cancer-testis expression patterns and the function of CT-lncRNA THOR and LIN28B-AS1 in cancers were also deciphered recently (2,3,6). The results mentioned above suggested that protein-coding and non-coding CT genes could be the molecular basis of proliferation similarity between cancer cells and germ cells.…”