The sequence features at the SOX cut site in LIMD1 and SRSF3, as well as a structural element around the site, are required for SOX cleavage.

A) Predicted structure of the 54 nt surrounding the SOX cut site in LIMD1, highlighting the A trimer (asterisk) and the cut site (arrow), as well as the ends of the 39 nt, and 33 nt insertions used in D. B) RNAfold was used to predict the structure of all 50-nt sequences surrounding SOX-specific and GFP-specific cut sites (based on a scanning window of 4 nt, a multiplicative factor of 4 and a confidence level of 99.99%). The proportion of cut site where the location of the cut was predicted to be unpaired, that had an unpaired A dimer within 5 nt of the cut or both is plotted. C-G) GFP reporters were co-expressed with SOX (“+”) or an empty vector control (“-”) in shXrn1-treated cells. The GFP mRNA was detected using Northern blotting. The empty arrowheads point to the additional cleavage fragment resulting from insertions, whereas the filled arrowheads point to the normal GFP cleavage fragment. C-D) 54–33 nt surrounding the SOX cleavage site in the LIMD1 mRNA were inserted into the GFP reporter at nt 715. In the 39 nt ΔA construct, one of the three As found at positions -7 to -5 from the cut site was deleted. A representative blot is shown (C), as well as the quantified intensity of the signal from the different RNA species (D), plotted relative to the intensity of the bands from the 54 nt insertion construct. Error bars = standard deviation, ** p p t-test). E) The A trimer preceding the SOX cut site in LIMD1 was mutated to a C, G or T trimer in the LIMD1 100 nt insertion construct. Images are representative of results from at least three experimental replicates. F) The A immediately 3’ of the SOX cut site in LIMD1 was mutated to a G in the 100 nt insertion construct. G) The A immediately 3’ of the SOX cut site in SRSF3 was mutated to a G (A → G) and the A dimer preceding the SOX cut site was mutated to a C dimer (AA → CC) in the 33 nt insertion construct.