Skip to main content
. 2021 May 1;8(13):2001701. doi: 10.1002/advs.202001701

Figure 3.

Figure 3

circARHGAP35 encodes a protein. A) Schematic representation of a putative open reading frame (ORF) in circARHGAP35. The junction is present inside the ORF. Start and stop codons are indicated in green and red, respectively. B) Schematic representation of the expression constructs. circARHGAP35 sequence was inserted into a circular RNA expression vector, which contains Alu elements to form the vector ‘p‐circARHGAP35’; Flag tag was added directly to upstream of the stop codon (TGA) to establish the construct ‘p‐circARHGAP35‐Flag’; the circARHGAP35‐Flag sequence was cloned to a linear vector to form a negative control vector ‘p‐circARHGAP35‐Flag‐NC’. The start codon and stop codon are shown in green and red, respectively. The Flag tag is shown in pale yellow. C) The indicated plasmids were transfected into HEK‐293T cells and potential proteins were detected using Western blot analysis. D) Immunoprecipitation assay was performed using ARHGAP35 antibody in SK‐Hep‐1 cells. The immunoprecipitated protein sample was subject to SDS‐PAGE and mass spectrometry analysis to identify specific sequences of the circARHGAP35 protein (red letters). E–H) Polysome profiling was performed using a linear 15% to 50% sucrose gradient. The polysomes of HCT‐116 cell cytoplasmic extracts without (HCT‐116) or with EDTA treatment (HCT‐116 + EDTA) were fractionated using sucrose density gradient centrifugation. Absorbance at 254 nm was measured. The relative levels of circARHGAP35 (E), linear ARHGAP35 mRNA (F), circPMS1 (G), and Actin mRNA (H) were analyzed by qRT‐PCR in gradient fractions in HCT‐116 cell lysates with or without EDTA treatment. circPMS1 and actin served as negative and positive controls, respectively. Relative distribution of each RNA in individual fraction represented as a percentage of total RNA. The sum of all fractions was considered as a total of one RNA. I) Methylated RNA immunoprecipitation (MeRIP) assay was performed using total RNA from SK‐Hep‐1 cells. Purified RNA was subsequently analyzed by qRT‐PCR. Nonspecific IgG was used as an isotype negative control. Statistical analysis was performed using unpaired Student's t‐test. *p < 0.05; **p < 0.01; ***p < 0.001. J) MeRIP was performed using total RNA from HEK‐293T cells ectopically expressing either vector or FTO. Purified RNA was subsequently analyzed by qRT‐PCR. Statistical analysis was performed using two‐way ANOVA and Tukey post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001. K) FTO reduces circARHGAP35 translation. Protein was analyzed by Western blot using HEK‐293T cells co‐transfected with circARHGAP35 expression vector and FTO (or vector control).