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. 2016 Feb;22(2):216–224. doi: 10.1261/rna.039842.113

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© 2016 Sheppard et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

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FIGURE 4. — Endonucleolytic cleavage of ssRNA by Csx1. (A) Radiolabeled linear (L) and circular (C) ssRNAs (67mer) were incubated with no protein (−), Terminator 5′-phosphate-dependent exonuclease (TEX), or Csx1 for the indicated time, then resolved by denaturing gel electrophoresis. The full-length linear and circular RNA are indicated by arrows, while the Csx1 cleavage products are indicated by the bracket. (B) 5′-Radiolabeled RNA (45mer A) was treated with no protein (−), Csx1, poly(A) polymerase (PAP), or Csx1 followed by PAP, and resolved by denaturing gel electrophoresis. The arrow indicates RNA elongated by PAP, while the bracket indicates Csx1 cleavage products. (C) 3′-Radiolabeled RNA (45mer A) was treated with no protein (−), with Csx1, with TEX, or with Csx1 followed by Tex, while 5′-radiolabeled RNA was treated with or without TEX. The samples were resolved by denaturing gel electrophoresis. The arrow indicates the expected TEX cleavage product, while the bracket indicates Csx1 cleavage products. (D) A diagram depicting the cleavage method of RNA by Csx1 as suggested by the resistance of the cleavage products to TEX activity and protection from elongation by PAP.