Probing substrate specificity of RNA repair protein Pnkp/Hen1 from Anabaena variabilis

Ribonucleic acid (RNA) repair;substrate specificity;ribotoxin;Pnkp/Hen1

Zhang, Can ; Huang ; Raven H.

关键词: Ribonucleic acid (RNA) repair;    substrate specificity;    ribotoxin;    Pnkp/Hen1;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/34202/Zhang_Can.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

This is an in vitro study of the substrate specificity of a RNA-repair protein Pnkp/Hen1 from bacteria Anabaena Variabilis. Following the previous discovery of a RNA repair protein—Pnkp/Hen1, which can repair ribotoxin cleaved tRNA in vitro and can add a methyl group at the cleaved site to prevent further cleavage, this study investigated the RNA secondary structure that this protein complex recognizes and repairs. First, three different truncated versions of aspartic acid and arginine tRNAs are produced by deleting the D loop (tRNA-D), TC loop (tRNA-T), or both loops (tRNA-DT) from the tRNA secondary structure. Then these three versions of RNA as well as the original tRNA are cleaved at the anticodon loops in vitro with Colicin. Then protein Pnkp/Hen1 is used to repair each pair of the cleaved RNAs, and it is found that truncated versions of RNAs could be repaired as efficiently as the original tRNA, suggesting that Pnkp/Hen1 is not tRNA specific. Next tRNAs with only stem-loop structures are produced and tested by Pnkp/Hen1, the repair result is negative in comparison to the repair of tRNA-DT as position control. Then nicked & bulged RNA secondary structures are tested and it turns out either nicked or bulged RNA structures is repaired. However the negative controls of the same experiment shows are ligated by Pnkp/Hen1. Following this discovery, pairs of single strand RNAs with similar or distinct sequences are mixed and tested, and we found that RNAs with a particular structural pattern can be ligated to the target RNA; while others can’t be ligated. The study shows that Pnkp/Hen1 repairs a relatively broader spectrum of RNA substrates than we expected but also has secondary structure specificity. Based on the experimental data, a model of preferred RNA substrates of the Pnkp/Hen1 repair system is proposed.