DISTRIBUTION AND CONFIGURATION OF C-MYC RNA DURING TRANSCRIPTIONAL ATTENUATION IN DIFFERENTIATING CELLS IN-SITU

Previous northern studies of c-myc RNA downregulation during early cel lular differentiation have shown reduced levels of mature transcript w ithin 6-24 h, attributed to attenuation of transcription at pause site s downstream of the P2 promoter. The transcription initiation rate has been shown to be decreased in some and increased in other such studie s. We assessed the contribution of RNA trafficking to c-myc reduction during differentiation by examining the localisation and configuration of exon-specific transcripts, using oligonucleotide probes and fluore scent in-situ hybridisation, in HL60 cells induced to differentiate wi th 12-O-tetradecanoylphorbol-13-acetate. A 2,4-dinitrophenyl-labelled probe to c-myc exon 3 sequences gave a strong cytoplasmic and nucleola r hybridisation signal in undifferentiated cells, which decreased mark edly after 24 h of differentiation. Nucleolar staining for c-myc RNA c olocalised with that from a probe for ribosomal 28 S RNA. The signal f rom an exon 1 probe specific for sequences upstream of the c-myc P2 pr omoter was much weaker, but increased in the nuclei of differentiating cells, which possessed unusual ring like or lamellar deposits, outsid e the nucleolus. These deposits appeared faintly together with nuclear staining with the exon 1 sense probe but not the exon 3 sense probe i n differentiating cells. These findings demonstrate that within the fi rst 24 h of differentiation, full-length c-myc RNA, which is compartme ntalised as expected for a mature transcript, is considerably downregu lated but nuclear primary RNA continues to be transcribed from exon 1. This is in a configuration similar to that reported for unspliced tra nscripts and is not elongated into exon 3. Antisense transcription wit h these RNA morphological features also occurs in exon 1 during differ entiation. These results indicate significant changes in the intracell ular dynamics of c-myc RNA during differentiation and support transcri ptional attenuation and post-transcriptional processes, such as splici ng, rather than reduced transcription initiation as the primary mechan ism of c-myc downregulation in the early phases of differentiation.