Thursday, November 21, 2024
11/21/2024
Project Summary/Abstract Mirtrons are a new class of small RNAs. Previous findings show mirtrons are generated by splicing and are less stable than canonical miRNAs. Thus, the expression of mirtrons is often negligible. Additionally, most mirtrons exhibit distinct evolutionary properties relative to solo and mammals have way more mirtrons than lower organisms suggesting some unknown importance of mirtrons in mammals. Recently, elevation of mirtrons has been linked to human cancers and immune disorders. However, the biological significance of mirtrons in humans is not well understood due to very limited efforts of mirtron research in humans. It is therefore important to investigate how biogenesis of mirtrons is stabilized during disease development and how increased mirtron expression promotes tumor/disease progression. In ongoing experiments, we show most mirtrons elevated in human cancers contain guanine rich sequence that forms G-quadruplex, a secondary structure that has been implicated as a strategy adopted by some RNA viruses to evade the host exoribonucleases, XRN1/2 mediated digestion. We also show XRN/1/2 are crucial enzymes for mirtron stability that mirtrons in cancer cells significantly increase when XRN1/2 are knocked down. Additionally, all cancer mirtrons are featured with many (long) terminal nucleotide additions which result in abundance of mirtrons present in the cancer derived exosomes. Furthermore, we also show mirtrons from the cancer derived exosomes bind to the endosomal Toll-like receptor 7 (TLR7) of macrophages suggesting mirtrons are novel ligands of TLR7 and potentially modulate immune responses. Given these findings, we hypothesize that cancer cells selectively express mirtrons exhibiting high guanine contents that could form G-quadruplex structure to bypass XRN1/2 mediated degradation. The nucleotide addition to the 3’ tail provides preferential sorting of mirtrons into exosomes. Cancer exosomes can be taken by immune cells through endocytosis providing the opportunity for mirtrons to bind to TLR7. The binding of mirtron to TLR7 serves as an allosteric inhibitor to block TLR7 signaling and the associated downstream immunological responses. These hypotheses will be addressed in the experiments of the following Specific Aims: (1) to determine the impact of G-quadruplex structure of mirtrons on the resistance to exoribonucleases, XRN1/2; (2) to determine the consequence of altered mirtron precursor tailing on exosomal sorting; and (3) to evaluate the immune modulatory role of cancer mirtrons through binding to TLR7. Should this exploratory study reveal novel mechanisms of cancer mirtron stabilization and sorting into exosomes, and should these cancer mirtrons negatively regulate the TLR7 signaling and the related immune functions, novel therapeutics aimed to destabilize mirtron, to prevent their sorting into exosomes, and/or to compete mirtron binding to TLR7 may provide benefits to cancer treatment in addition to advance current knowledge of mirtrons in humans.
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