Novel coding, translation, and gene expression of a replicating covalently closed circular RNA of 220 nt

Abstract

The authors note, "Figs. 2, 3, and 5 were not in compliance with PNAS figure preparation policies because some gel lanes from noncontiguous experiments were spliced together. The corrected figures shown below have white spaces inserted to indicate noncontiguous experiments." 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.

Circular RNAs in cancer: an emerging key player

“…Early in 1998, Perriman et al reported that a circular mRNA containing a simple green fluorescent protein (GFP) open reading frame could direct GFP expression in Escherichia coli [16]. Subsequently, AbouHaidar et al discovered a circRNA (220 nt) of the virusoid associated with rice yellow mottle virus can code for a 16-kD protein [17]. It was shown that peptides can be translated from circRNAs in vitro [18] or in vivo [16], only when the RNAs contain internal ribosome entry site elements (IRES) [18] or prokaryotic ribosome-binding sites [16].…”

CircRNA Expression Profile in Early-Stage Lung Adenocarcinoma Patients

“…CircRNAs are a class of covalently closed circular RNAs that have been originally identified in plant viroid [6, 7], virus [8, 9], archaea [10], and metazoans [11]. Unlike linear RNAs, circRNAs exhibit the remarkable characteristic of being covalently closed continuous loops without 5’ to 3’ polarity and without a polyadenylated tail [12], which allows circRNAs to resist the degradation of major RNA enzymes.…”

Evolution of Nucleotide Punctuation Marks: From Structural to Linear Signals

“…Involving 64 nucleotide triplets called codons (Elzanowski and Ostell, 2013), the genetic code is a system coding the set of rules by which information is translated from RNA into proteins by living cells and viruses, by specifying which amino acid will be added during protein synthesis. Information encoded within genetic material also possesses superimposed cryptic messages (Popov et al, 1996), revealing highly complex semiotics (e.g., in circular virusoid RNAs, AbouHaidar et al, 2014). The rules of DNA punctuation vary among 25 recognized genetic codes, suggesting these constantly evolve.…”