Purification and cloning of the Xenopus laevis double-stranded RNA adenosine deaminase

This dissertation describes both the purification of a single Xenopus laevis protein with dsRNA adenosine deaminase (dsRAD) activity, and the cloning of two distinct Xenopus dsRAD cDNAs. Purified Xenopus dsRAD is a 120 kDa protein that produces inosine from adenosines within dsRNA by hydrolytic deamination. This reaction does not require additional cofactors, nor an energy source to gain access to adenosines buried within the major groove of an A-form RNA helix. A full length Xenopus cDNA encodes a 1270 amino acid dsRAD-1 protein. A second, nearly complete Xenopus dsRAD-2 cDNA, encodes a protein that shares ~ 85%, amino acid identity with dsRAD-1. At least four mRNAs are expressed in Xenopus oocytes that correspond to long and short forms of each cDNA type. Isolation of C. elegans cDNAs (T20H4.4) related to dsRAD is also described. These cDNAs contain 5' sequences corresponding to a novel class of trans-spliced leaders (SLs), observed to correlate with downstream genes in C. elegans operons. A consensus derived from an amino acid sequence alignment of the highly conserved carboxyl termini of five known, and two putative dsRNA adenosine deaminases, including C. elegans T20H4.4, revealed motifs remarkably similar to the signature motifs of the DNA-(adenine-N6 alpha)-aminomethyltransferases and the DNA-(cytosine-5)-methyltransferases.