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. 2017 Sep 15;45(20):11891–11907. doi: 10.1093/nar/gkx820

Figure 3.

Figure 3.

The native dsRBD and conserved cysteine C230 are required for RTL1 cleavage of RNA in vitro. (A) Coomassie blue staining and schematic representation of Wt His-RTL1 (R1D1), His-RTL2 (R2D2), swapped His-R1D2 and His-R2D1 or His-RTL1 with mutated cysteines C230S and C260S or truncated HisRTL1ΔdsRBD proteins. Red and yellow boxes correspond to RNase III domains while blue and light blue boxes correspond to dsRBD from RTL1 (R1 and D1) and RTL2 (R2 and D2a and b) proteins respectively. Asterisks show position of mutated Cys 230 and Cys260. (B) 32P-CTP RNA substrate-1 was incubated with 100 ng of His-RTL1(R1D1), His-R1D2, His-R2D1, His-RTL2 (R2D2), His-RTL1C230S, His-RTL1C260S or His-RTL1ΔdsRBD (lane 10) recombinant proteins or with buffer alone. Arrowhead indicates full-length RNA substrate (f0) and major cleavage fragment products (f1 and f2). DNA size markers are indicated on the left. (C) Amino acid sequence alignment of dsRBD from RTL1 (F4JK37), RTL2 (Q9LTQ0) and DCL1 (NP_171612), DCL2 (NP_566199), DCL3 (NP_189978) and DCL4 (NP_197532). Vertical arrows heads show highly conserved Cys230 and non-conserved Cys250 and Cys260 in the RTL1 sequence.