Novel requirements in peripheral structures of the extended satellite 2 hammerhead.

Abstract

A number of RNAs that infect plants self-cleave using a domain called the hammerhead. The consensus plant hammerhead has three base paired stem structures. Stems I and III flank the cleaved phosphodiester and are connected to stem II by two unpaired and highly conserved sequences. We demonstrated previously that satellite 2 transcripts from the newt, Notophthalmus viridescens, use a modified hammerhead structure for self-cleavage. Here we show that hammerheads with similar modifications occur in satellite 2 from species representing two additional families of salamanders (Ambystoma talpoideum and Amphiuma tridactylum). The distinctive features of the modified satellite 2 hammerheads include unusually short stem III regions and internally looped extensions to stem I that are required for cleavage. However, despite the strict nucleotide requirement in the internal stem I loop of the newt hammerhead, the details of the stem I extensions differ in the three salamanders. An analysis of chimeric transcripts indicated that, in order for a specific stem I extension to be functional, it must be compatible with other regions of the modified satellite 2 hammerhead. One such region is stem II, and a mutational analysis has confirmed that there are specific sequence and/or structural requirements in stem II.