Mapping the spliced and unspliced late lytic SV40 RNAs

Abstract

The sizes and map positions of the major late lytic SV40 cytoplasmic mRNAs and the abundant nuclear RNA species have been determined by the technique of Berk and Sharp (1977, 1978). From these experiments, the coding sequences (bodies) of the 16S and 19S late cytoplasmic SV40 RNAs have been located at 0.935-0.17 and 0.765-0.17 map units, respectively. The cytoplasmic 16S RNA molecules contain a leader sequence of approximately 210 nucleotides, corresponding to SV40 map positions 0.72-0.76 units, spliced to the coding sequences. In a population of the late 19S RNA molecules, there are several different leader segments, each spliced to the same coding sequences. The size of these 19S leader RNA segments was estimated to be 50-70, 100-120 and 200-210 nucleotides in length. The 5' ends of the 19S leader RNA segments were located at 0.72, 0.71, 0.695 and 0.69 map units. An analysis of the nuclear viral RNAs has provided insight into the biogenesis of the cytoplasmic messages. Poly (A)-containing nuclear RNA has a number of species in addition to those found in the cytoplasm. The 3' ends of the poly (A)-containing RNAs map at 0.17 SV40 units. The 5' ends of the more abundant nuclear molecules map approximately at 0.72, 0.70, 0.67, 0.64 and 0.59 units. Since these nuclear SV40 RNA molecules are both colinear with the viral DNA and larger than the cytoplasmic nRNAs, they may represent intermediates in a stepwise processing system. Alternatively, the variation in 5' ends of nuclear SV40 transcripts may represent a number of separate initiation sites for transcription. The presence of the intervening RNA sequences (between the leader and the coding sequences of the mature mRNAs) in these nuclear RNA molecules suggests that the synthesis of "spliced" SV40 RNA involves the direct transcription of the DNA sequences and the subsequent splicing out of the intervening seqment of RNA. Evaluation of the more abundant nonpolyadenylated nuclear RNA molecules showed that they have the same 5' ends as the poly (A)-containing nuclear RNAs. The 3' ends of the nonpolyadenylated RNA molecules map heterogeneously in a broad region extending beyond 0.28 map units. The presence of these long nuclear viral transcripts suggests that transcription of late SV40 RNA does not terminate at 0.17 map units. The location of poly (A) in mature cytoplasmic viral RNA at 0.17 map units suggest that poly (A) addition to RNA molecules may occur by a specific cleavage of the longer transcripts. Based on these analyses, we propose that the longer nonpolyadenylated viral RNA molecules in the nuclei of SV40-infected cells may represent the primary transcripts. While their 5' termini are being processed, the specific addition of poly (A) at 0.17 map units takes place. The polyadenylation of RNA is followed by splicing events to generate the cytoplasmic forms of SV40 mRNA.