Correction MICROBIOLOGY Correction for “Novel coding, translation, and gene expression of The authors note, “Figs. 2, 3, and 5 were not in compliance a replicating covalently closed circular RNA of 220 nt,” by Mounir with PNAS figure preparation policies because some gel lanes Georges AbouHaidar, Srividhya Venkataraman, Ashkan Golshani, from noncontiguous experiments were spliced together. The cor- Bolin Liu, and Tauqeer Ahmad, which appeared in issue 40, Oc- rected figures shown below have white spaces inserted to indicate tober 7, 2014, of Proc Natl Acad Sci USA (111:14542–14547; first noncontiguous experiments.” published September 24, 2014; 10.1073/pnas.1402814111). Fig. 2. In vitro transcription/translation and immunoprecipitation of translated products using antiserum raised against the scRYMV ORF-encoded protein. (A) Lane 2 shows 19-kDa and 16-kDa proteins from reaction using pET52 DNA, whereas lane 3 shows only the 19 kDa from reaction using the truncated pETdimer DNA. Lane 1: Negative control reaction using pET29 (empty vector). Arrows on left indicate molecular size of proteins, from top to bottom: 36 kDa, 19 kDa, and 16 kDa. (B) In vitro translation reaction depicting the 16-kDa protein from the scRYMV circular RNA (purified from denaturing polyacrylamide gels) is shown in lane 3, whereas that of the reaction from total RNA of RYMV-infected rice is shown in lane 2. Lane 5 demonstrates the 16-kDa product from reaction using total viral RNA extracted from RYMV virus particles, and lane 4 shows the enhanced 16-kDa signal from reaction using RYMV total viral RNA but supplemented with the same amount of gel-purified circular RNA as that used in the reaction of lane 3. Lanes 1 and 6 represent negative control reactions using healthy rice and the endogenous empty lysate, respectively. Arrows on right depict the position of molecular weight markers 25, 16, and 14 kDa from top to bottom, respectively. (C) Northern analysis to detect the nature of scRYMV RNA species. Denaturing 4–20% PAGE in presence of 8 M urea was carried out. Lanes 2 and 3 demonstrate presence of linear (marked as “L”), circular (“C”), dimer (“D”), and trimer (“T”) forms of the scRYMV RNA in total RNA preparations from RYMV-infected rice and RYMV particles, respectively. Lane 1 shows the negative control of total RNA from healthy rice. Lane 4: 7 M urea- PAGE stained with ethidium bromide and showing the purified circular RNA extracted from the band corresponding to circular RNA (shown in lane 3). This purified circular RNA is used for in vitro translation. Arrows indicate the positions of RNA size markers: 220 (marked as “L” or “C”), 440 (“D”), and 660 (“T”)nt. E5252–E5253 | PNAS | August 30, 2016 | vol. 113 | no. 35 www.pnas.org Downloaded by guest on September 27, 2021 Fig. 3. SDS/PAGE, Western detection, and time course of expression of the scRYMV-encoded proteins. (A) Western analysis using antibodies specific to the scRYMV ORF-encoded protein to detect the 16-kDa proteins among total plant proteins from RYMV infected (lane 1), healthy uninfected plant (lane 3), and purified RYMV virus (lane 2). Arrows on right indicate the positions of the 39-, 32-, and 16-kDa proteins (from top to bottom). (B) Western analysis time course of the appearance of the scRYMV 16 kDa during infection in rice plants. Lane 1 contains total proteins extracted from noninfected rice plants (negative control). Lane 2 contains total proteins from RYMV-infected plants at 4 d postinoculation. Lanes 4–7 contain total rice proteins extracted from RYMV-infected plants at 10, 14, 21, and 28 d post- inoculation, respectively. Lane 3 and arrow (16 kDa) depict molecular weight size markers 116, 66, 45, 35, 25, and 18 kDa, from top to bottom, respectively. (C) North- Western analysis showing the RNA-binding activity of scRYMV-encoded proteins. Lane 1 shows the presence of the 16-kDa RNA-binding protein in infected rice, whereas lanes 2 and 9 demonstrate the detection of the 16-kDa, 18-kDa, and 19-kDa RNA-binding proteins in protein extracts from E. coli carrying pET52. Lanes 4 and 5 are the negative controls from healthy rice and pET29 protein extracts, respectively. Lanes 6 and 7 are negative control from E. coli alone and E. coli carrying pET29, respectively. Lanes 3 and 8 contain the molecular weight markers including the 14-kDa positively charged lysozyme. The 35S-labeled scRYMV and PVX probes were used in lanes 1–5and6–9, respectively. Arrows on left indicate the positions of molecular size markers (from top to bottom, 19, 18, 16, and 14 kDa, respectively). Fig. 5. Mutational analysis of scRYMV. (A) In vitro transcription/translation and immunoprecipitation profile of scRYMV AUG to AUU (in the sequence UGAUGA) mutant shows abolition of 16-kDa translation. Lane 1: radiolabeled dots indicating the positions of protein molecular weight size markers (top to bottom, 35, 25, 20, 17, 11, and 5 kDa). Lane 2: in vitro reaction (with scRYMV 19-kDa antibody) with purified RYMV genomic RNA alone (without scRYMV) used as template shows no 16-kDa protein and was used as infectious transcript in plants. Lane 3: same reaction as in lane 2 except for addition of mutated scRYMV construct (AUG to AAU mutant pBS-trimer). Lane 4: empty lane. Lane 5: same reaction as in lane 2 except for the addition of pBS-trimer (intact construct) demonstrating the synthesis of a 16-kDa protein. Lane 6: reaction containing total RYMV viral RNA obtained from purified virus containing scRYMV. Lane 7: reaction containing purified WT scRYMV RNA alone (obtained from virus particles). (B) Replication of AUG to AAU scRYMV mutant is abolished in plants. Lane 1: RYMV viral RNA profile from purified virus particles of plants infected with infectious RYMV RNA along with T7 polymerase- generated in vitro transcripts from WT pBS-trimer scRYMV clone. Lane 2: RYMV infectious RNA plus in vitro transcripts from AUG to AAU mutant pBS-trimer clone showing complete absence of the scRYMV RNA (no replication). Upper arrow indicates the position of genomic RYMV RNA (4450 nt) and the bottom arrow indicates the position of circular scRYMV RNA (220 nt). (C) UGAUGA codons influence translation termination: Western blotting analysis of total E. coli proteins using pET dimer and anti–His-tag antibody for intact and a UGAUGA-to-CUCGAG mutant construct. Lane 1: protein molecular weight ladder (top to bottom, 135, 100, 75, 63, 48, 35, 25, 20, 17, and 11 kDa). Lane 2: E. coli containing empty pET29 plasmid. Lane 3: E. coli containing intact construct with UGAUGA terminator upstream of a His-tag. No protein product is detected. Lane 4: E. coli containing UGAUGA to CUCGAG mutant construct. A 20-kDa protein is detected by using anti–His-tag antibody. This is in accord with a translation read-through over the mutated UGAUGA-to-CUCGAG region producing a His-tagged fusion protein. www.pnas.org/cgi/doi/10.1073/pnas.1611407113 CORRECTION PNAS | August 30, 2016 | vol. 113 | no. 35 | E5253 Downloaded by guest on September 27, 2021 Novel coding, translation, and gene expression of a replicating covalently closed circular RNA of 220 nt Mounir Georges AbouHaidara,1, Srividhya Venkataramana, Ashkan Golshanib, Bolin Liua, and Tauqeer Ahmada aDepartment of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3B2; and bBiology Department, Carleton University, Ottawa, ON, Canada K1S 5B6 Edited by David C. Baulcombe, University of Cambridge, Cambridge, United Kingdom, and approved August 28, 2014 (received for review February 14, 2014) The highly structured (64% GC) covalently closed circular (CCC) N- and C-terminal halves of the 16-kDa protein translated RNA (220 nt) of the virusoid associated with rice yellow mottle from the same 220-nt circular sequence and the generation of virus codes for a 16-kDa highly basic protein using novel modali- read-through proteins were examined. We discuss the evolu- ties for coding, translation, and gene expression. This CCC RNA is tionary implications of the genetic information and biological the smallest among all known viroids and virusoids and the only functions densely packed into a 220-nt nanogenome. one that codes proteins. Its sequence possesses an internal ribo- Results some entry site and is directly translated through two (or three) completely overlapping ORFs (shifting to a new reading frame at Expression in Escherichia coli of a Head-to-Tail Trimer Copy of a the end of each round). The initiation and termination codons scRYMV cDNA Clone. The original isolation, cloning, and several overlap UGAUGA (underline highlights the initiation codon AUG other properties of scRYMV RNA are described elsewhere within the combined initiation-termination sequence). Termina- (9, 11). The putative ORF generated from a linear trimeric head- tion codons can be ignored to obtain larger read-through proteins. to-tail cDNA copy of the 220 nt of scRYMV RNA (representing This circular RNA with no noncoding sequences is a unique natural a three-frame overlapped ORF present in the monomeric form supercompact “nanogenome.” of circular RNA) was constructed as a fusion protein with S-Tag/ thrombin under the T7 transcriptional and translational controls hammerhead ribozyme | sobemovirus | circular RNA translation | in plasmid expression tag (pET)29(c) vector. The resulting clone leaky termination codons was designated pET52 (Fig. 1). Thirteen amino acids at the N terminus of the putative scRYMV protein were replaced by 39 aa from thrombin to generate a fusion protein. Upon induction of iroids and virusoids (viroid-like satellite RNAs) are typically pET52 in E. coli cells, a major protein species of 19 kDa was de- small (220–450 nt) covalently closed circular (CCC) RNAs V tectable by SDS/PAGE and Coomassie brilliant blue R-250 staining with no coding capacity (i.e., no genetic information) (1–3) and (Fig.