Chemical probing of sub-complexes of the Pab21 sRNP.

<p>(<b>A</b> and <b>B</b>) Footprinting of proteins L7Ae, aCBF5 and aNOP10 on sRNAs Pab21 and Pab91. Reactions with lead were carried out on 5′ ³²P end-labeled Pab21 (panel <b>A</b>) or Pab91 (panel <b>B</b>). Samples were fractionated on 10% polyacrylamide denaturing gels containing 8 M urea. The protein set used for assembly of the probed protein–RNA complexes is indicated on top of each lane: L7Ae (L), aCBF5 (C), aCBF5–aNOP10 (CN), and L7Ae–aCBF5–aNOP10 (LCN). Probing was also performed on the complex obtained in presence of L7Ae–aCBF5–aNOP10 and an excess of the substrate RNA (LCN+S). Lanes OH^– and T1 correspond, respectively, to an alkaline hydrolysis ladder, and RNase T1 digestion ladder obtained under denaturing conditions. (<b>C</b> and <b>D</b>) Summary of probing data. Positions of the phosphodiester bonds cleaved upon incubation with lead of the naked sRNA and of the various sub-complexes L, C, CN, LCN, and LCN +S are indicated by stars on the secondary structure model of sRNA Pab21 (panel <b>C</b>) and Pab91 (panel <b>D</b>). The number of stars is proportional to the efficiency of the cleavages. Regions that were protected from cleavage are highlighted by blue lines and regions that displayed an increase in lead-induced digestion are highlighted by red lines. (<b>E</b>) Probing of the LCN complexes assembled with the mutant proteins of the aNOP10–L7Ae interaction, indicated with the same code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070313#pone-0070313-g001" target="_blank">Figure 1</a>. The differences in cleavage products obtained with the wild-type and mutant LCN sub-complexes are indicated with arrows along the autoradiogram.