RNA comprising the 28S/R2 5' junction from many arthropods can function as a self-cleaving ribozyme.

<p>(A) Diagram of a generic 28S gene (blue box)/ R2 5' end (white box) junction. Arrows labeled <i>a</i> through <i>y</i> represent the <i>in</i> <i>vitro</i> generated RNAs tested for self-cleavage from the eight species listed on the left. <i>Ds</i>, <i>D</i><i>. simulans</i>. RNAs (except <i>y</i>) begin at position -95 relative to the R2 insertion site and end at the noted nucleotide within the R2 element. RNA <i>k</i> has a 'G' to 'A' change in the active site of the ribozyme; RNA <i>y</i> has a generated 'G' to 'C' change to improve the P1 stem (see text). The short vertical lines demarcate the extent of the predicted ribozymes. To the far right of each arrow is the average fraction of RNA cleaved in at least two independent co-transcription/cleavage assays. (B) A 5% denaturing acrylamide gel showing the cleavage products for select RNAs in the co-transcription/self-cleavage assays. A longer exposure of the lower portion of the gel is shown to better visualize the short upstream cleavage product. The uncleaved RNA (solid red circles) and cleavage products (open red circles) are indicated. Unmarked bands are alternative, stable RNA structures. Lanes are labeled with the corresponding letter from panel (A). Lane M, RNA length markers with sizes indicated. (C) An 8% denaturing acrylamide gel showing the upstream cleavage products at single nucleotide resolution. RNAs are the same as in (B) except RNA from the silkmoth was from <i>S</i><i>. cynthia</i>. DNA ladder corresponds to combined G, A, T sequencing reactions with nucleotide position from the -40 primer in the m13 vector shown to the right.