Genetic test for involvement of intervening sequences in transport of nuclear RNA

Abstract

The construction of a recombinant virus in the late region of simian virus 40 is presented. The small intervening sequence of late 19S RNA (0.760 to 0.765 map unit) was cloned and inserted into the EcoRI site (1.0 map unit) in the late region of simian virus 40. This is a mutant virus that now has two intervening sequences, one at the normal position (0.760 map unit) and another out of the context of its flanking sequence and now at 1.0 map unit. The recombinant appears poisonous, as repeated attempts to plaque it as a virus with a standard helper virus were unsuccessful. The transcription of this recombinant was, therefore, studied after direct DNA transfection onto CV-1 cells. Nuclease S1 analysis of mutant RNA indicates that the major nuclear transcript was a spliced but nuclear 16S RNA species. Normally, 16S RNA is not found in the nucleus. This result was shown to be an artifact of the DNA transfection protocol. When the glycerol shock was done after infection with virus, a similar alteration in the makeup of nuclear RNA was seen. A transient stock of this double-intron mutant was finally obtained, using a nonrevertable helper virus. The transcriptional analysis of this mutant showed that unspliced 19S RNA was not transported and remained within the nucleus, whereas spliced 19S and 16S RNAs were transported. We conclude that the retention of nuclear transcripts within the nucleus is not simply due to the presence of intronic sequences, as spliced 19S and 16S RNAs which contain the second intron were efficiently transported.