FIG 7.

The G₃(A/U)₄ motif is necessary and sufficient to initiate the synthesis of ttsgR and similar TCV lncRNAs. (a) wt TCV sequences at nucleotide positions 3401 to 3410 and 3771 to 3781 and sequences of various mutants that altered one or both of these regions. The sequences are separated by spaces into codons in CP ORF. The wild-type GGGUAAA motif responsible for ttsgR initiation is highlighted blue. All mutated nucleotides are highlighted in red. (b, c, d, and e) Northern blots showing the impact of various mutations on the levels of ttsgR and related lncRNAs initiated from replicons. (b) TCV_sg2R-m1 and wt TCV-m1, both containing the same three single-nucleotide mutations within the GGGUAAA motif, suffered a near-complete loss of ttsgR, with or without p88. The two arrowheads denote an additional lncRNA present in wt TCV lanes, which could have initiated from a G₃(A/U)₄ motif within the TCV CP region that was by mCherry (nucleotide positions 3290 to 3296, GGGUUUA) in TCV_sg2R. (c) The impact of several single-nucleotide mutations (m1a, m1b, m1c, m1c-i, and m1c-ii) on the level of ttsgR in the TCV_sg2R background. (d) The impact of m1a, m1b, and m1c mutations on the level of ttsgR in the wt TCV background. Note the arrowhead denoting the position of the additional lncRNA unique to wt TCV. (e) Creation of a new G₃(A/U)₄ motif (m2, GGGUUAA at nucleotide position 3401 to 3407) was sufficient to program the production of a new lncRNA, in both TCV_sg2R and wt TCV backgrounds.