PMC:5985359 / 25513-27410 JSONTXT

{"project":"2_test","denotations":[{"id":"29526280-25593321-2044789","span":{"begin":760,"end":762},"obj":"25593321"},{"id":"29526280-10970838-2044790","span":{"begin":1290,"end":1292},"obj":"10970838"},{"id":"29526280-19095965-2044791","span":{"begin":1294,"end":1296},"obj":"19095965"},{"id":"29526280-28442996-2044792","span":{"begin":1298,"end":1300},"obj":"28442996"},{"id":"29526280-22884328-2044793","span":{"begin":1302,"end":1304},"obj":"22884328"},{"id":"29526280-20098426-2044794","span":{"begin":1306,"end":1308},"obj":"20098426"},{"id":"29526280-28949831-2044795","span":{"begin":1310,"end":1312},"obj":"28949831"}],"text":"RNA Splicing Factors MBNL1 and MBNL2 Are Sequestered by Nuclear RNA Foci\nTo determine whether RNA binding proteins were being sequestered by the RNA foci, in the cultured FECD CECs, we employed a dual FISH and ICC approach. The nuclear distribution of MBNL1 and MBNL2 was investigated in multiple CEC lines derived from individuals with and without expanded copies of the repeat (Table S5). MBNL1 displayed a diffuse nuclear localization in three non-expanded (NE) CEC lines and no foci were detected, as anticipated (Figure 3A). Striking co-localization was observed between MBNL1 and the CUG-specific RNA foci in all three independent CTG18.1 expansion-positive lines examined (Figure 3A), concordant with a previous observation made in FECD-diseased tissue.15 Similarly, the recruitment of MBNL2 to CUG-specific nuclear RNA foci was also detected in expansion-positive CECs (n = 3) and MBNL2 displayed diffuse nuclear localization in expansion-negative and control CEC lines (n = 4) (Figure 3B). These data demonstrate that RNA splicing factors are recruited to CUG RNA foci and suggest that this recruitment may induce a functional deficiency of proteins that would be predicted to alter alternative splicing events, in a similar downstream pathway to what has been established for DM1.18, 19, 20, 21, 22, 36\nFigure 3 MBNL1 and MBNL2 Are Sequestered to RNA Foci in Corneal Endothelial Cells (CECs) Derived from FECD-Affected Subjects with CTG18.1 Expansions\nRepresentative images of MBNL1 (A) and MBNL2 (B) protein nuclear localization in cell lines derived from expansion-positive FECD-affected subjects, expansion-negative FECD-affected subjects, and CECs derived from healthy individuals. RNA foci are labeled with a Cy3-(CAG)7 FISH probe and DAPI is used to stain nuclei. Co-localization of the MBNL proteins and RNA foci is represented in the bottom row of both panels. Scale bars, 10 μm."}