Figure 3
From: A universal approach to investigate circRNA protein coding function
Translation of circRtn4. (A) Schematic illustration for the insertion of a FLAG-tag (magenta) with and without stop codon (red) into exon 2 (blue) of the circRtn4 open reading frame. Exons 2 (blue) and 3 (yellow) constitute the circRNA open reading frame; the green arrow, denotes the presumed AUG start codon, magenta arrow denotes the position of the FLAG-tag in pCircRNA-DMo-Rtn4-FLAG and derivatives; the red rectangle denotes the stop codon (UGA) in the pCircRNA-DMo-Rtn4-Stop construct. (B) Representation of the “infinite” circRtn4 open reading frame (ORF). The inner circle denotes the circular RNA with exon 2 and exon 3 in blue and yellow. The presumed AUG translation start codon is indicated by a green arrow on the outer circle showing the presumed circRtn4 translation products(s). (C) Western blot of circRtn4 translated polypeptides in HEK293 cells detected with antibodies targeting Nogo-A (α-Nogo-A). Control, i.e., the empty vector; BE-Rtn4, pCircRNA-BE-Rtn4; DMo-Rtn4, pCircRNA-DMo-Rtn4; Rtn4-Exon2-Exon3, pCMV-Rtn4-Exon2-Exon3. On the right, polypeptides of higher molecular weights are presumed products of more than one round of circRtn4 circular translation; RTN4-fl indicates the endogenous RTN4 full length protein (Reticulon 4 or Nogo-A); the “monomer” presumably represents a single round of circRtn4 circular translation. The calculated MW of the theoretical “monomer” is 88.2 kDa. Possibly due to its highly acidic pI (4.3) and/or high proline content, it migrates significantly slower in the gel, thus corresponding to 150 kDa39,40. For calculating the relative protein level, protein from pCMV-Rtn4-Exon2-Exon3 was set as 1; pCircRNA-DMo-Rtn4 expressed less “monomer” protein by a factor of 0.25. (D) The putative open reading frame of Rtn4-FLAG circRNA. Annotation as in B: In addition, the aspartic acid (blue D) is resulting from the junction site of backsplicing; the FLAG-tag peptide is in magenta; the glutamic acid residue (E, highlighted in blue) is resulting from the junction site of exon2-exon3; the green methionine (M) is the presumed start codon and the isoleucine (I, orange) is directly N-terminal to M and supports a further round of translation (see below). (E) Western blot hybridisation of pCircRNA-DMo-Rtn4-FLAG expression in N2a cells with the anti-FLAG antibody (α-FLAG). Control, the empty vector; DMo-Rtn4-FLAG, pCircRNA-DMo-Rtn4-FLAG; of note, compared to C, the intensity of repeating peptides is higher than “monomer”; the reason is unknown. (F) The putative open reading frame of Rtn4-stop circRNA is delineated. Annotation as in B: In addition, the red bar indicates the in-frame stop codon (UGA). (G) Western blot showing expression of pCircRNA-DMo-Rtn4-Stop transfected in HEK293 cells with an anti-Nogo A antibody (α-Nogo-A). Control, the empty vector; DMo-Rtn4-Stop, pCircRNA-DMo-Rtn4-Stop; DMo-Rtn4, pCircRNA-DMo-Rtn4. (H) Nucleotide sequence and predicted translation products surrounding the inserted sequences encoding the FLAG peptide (magenta) as part of exon 2, the region where exons 2 and 3 are joined, and the site of circularization for construct circRtn4-FLAG. Exon 2 nucleotide sequence is highlighted in blue, and black upper-case letters indicate Rtn4 exon 3. The nucleotides encoding the FLAG-tag are displayed in black lower-case letters. The last G residue (position 2448) is joined to the first nucleotide by circularization yielding a GAU triplet (highlighted in yellow), encoding aspartic acid (D, bold, blue letter, in brackets). Nucleotides are arranged in blocks of 12. The predicted amino acid sequence is given in the IUPAC one-letter amino acid code. Tryptic peptide 46 (Supplementary Fig. 6) beginning with TSDETL, bridging the circularization site and containing the FLAG peptide (bold and magenta lettering) is underlined. The glutamic acid residue bridging exons 2 and 3 is highlighted as bold blue letter. Unless additional start codon(s) are used, tryptic peptide 52 (Supplementary Fig. 6) starting with IMDLKEQPG (broken line) must be derived from translation into the second circular round across the presumed AUG start codon (marked by a horizontal green arrow). Amino acids are highlighted as above. (I) Mass spectrometry of peptide 46, providing direct evidence of circular translation of the junction site in circRtn4-FLAG. Amino acids are highlighted as above. (J) Mass spectrometry of peptide 52, providing evidence of circular translation into a second round of circRtn4-FLAG. Amino acids are highlighted as above. (K) Open reading frame (ORF) of circRtn4-FLAG-ac. Annotation as in B: In addition, the AC insertion is marked in red and the junction amino acid glutamine (Q, red) is marked. The different ORF beyond the first round of translation is marked in red and the extension of the first round in red and “69 aa”. The substitution to generate the stop codon (UAA) some 207 nucleotides further downstream is not shown in this C-terminal segment (Supplementary Fig. 2B). (L) Western blot analysis of pCircRNA-DMo-Rtn4-ac transfected in N2a cells using an anti-Nogo A antibody (α-Nogo-A). DMo-Rtn4-FLAG, pCircRNA-DMo-Rtn4-FLAG; DMo-Rtn4-FLAG-ac, pCircRNA-DMo-Rtn4-FLAG-ac.
