Extended Data Fig. 1: Confirmation of the firefly luciferase reporter cassettes.
From: N6-adenosine methylation enhances nuclear mRNA export through METTL3 and NUP93
a, Validation of site-specific m6A modification on firefly luciferase reporter cassettes (Fluc-no m6A, Fluc−2x m6A, and Fluc−10x m6A) using SELECT-qPCR. b, Schematic illustration of the dCas13b-ALKBH5-based m6A-editing system with sgRNA targeting m6A sites on firefly luciferase mRNA. c, Western blot analysis of indicated proteins in HeLa cells expressing GFP-POM121, which were transiently transfected with the dCas13b-ALKBH5-based m6A-editing system including control sgRNA (sgCtrl) or three independent target sgRNAs (sgFluc-A, -B, and -C). Blots are representative of two independent experiments with similar results. d,e, m6A-MeRIP-qPCR analysis of Fluc-mRNAs following transient transfection with wild-type dCas13b-ALKBH5 (d) or the catalytically inactive mutant dCas13b-ALKBH5 H204A (e), together with sgCtrl or sgFluc. The percentages of m6A levels on Fluc-mRNAs were obtained by calculating MeRIP signals over input RNA signals. f, Validation of site-specific m6A modification on firefly luciferase reporter cassettes (Fluc-no m6A, Fluc−2x m6A, and Fluc−10x m6A) using SELECT-qPCR in GFP-POM121-expressing HeLa cells treated with DMSO or 10 μM STM2457 for 24 hrs. g, Typical types of nuclear export and trajectories of mRNPs in SPEED microscopy. Classification of Successful and Abortive Export. The centroid of the membrane was determined (red dashed line) via brightfield and used to determine whether a single-molecule trace was successfully exported or aborted. Successful export (left) is defined as having an origin point in the nucleus and an exit point in the cytoplasm. Abortive export (right) is defined as having an origin point in the nucleus, a minimum of one point on the cytoplasmic side of the centroid, and a termination point within the nucleus. Data in (a, d-f) are present as mean ± s.d. from three independent experiments. P values were calculated by two-sided student’s t-test. Source numerical data, unprocessed gels and blots are provided in the Source Data.
