The intervening sequence of the ribosomal RNA precursor is converted to a circular RNA in isolated nuclei of Tetrahymena

Abstract

The Tetrahymena thermophila ribosomal RNA gene contains an intervening sequence (IVS), which is transcribed as part of the precursor RNA and subsequently removed by splicing. We have found previously that the IVS is excised as a 0.4 kb RNA in isolated nuclei. We now report the finding of a novel RNA molecule, which is an electrophoretic variant (EV) of this 0.4 kb IVS RNA. The EV was identified as a form of the IVS RNA by Southern hybridization, RNA fingerprinting and R-loop mapping. A pulse-chase experiment established that in vitro the excised IVS RNA is converted to the EV by a post-splicing event. This conversion is enhanced at 39 degrees C compared to 30 degrees C and is irreversible under our experimental conditions. The EV of the IVS is a circular RNA. This structure was first suggested by its anomalous electrophoretic mobility on denaturing compared to nondenaturing gels. When the EV was prepared for electron microscopy under totally denaturing conditions, 0.4 kb circular molecules were observed. Furthermore, we have converted the circular form to a linear form by limited T1 RNAase digestion. The circular RNA survived treatment with DNAase, protease, glyoxal and various denaturants, which suggests that it is a covalently closed RNA circle.