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 cellular differentiation have shown reduced levels of mature transcript within 6-24 h, attributed to attenuation of transcription at pause sites downstream of the P2 promoter. The transcription initiation rate has been shown to be decreased in some and increased in other such studies. 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 fluorescent in-situ hybridisation, in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate. A 2,4-dinitrophenyl-labelled probe to c-myc exon 3 sequences gave a strong cytoplasmic and nucleolar hybridisation signal in undifferentiated cells, which decreased markedly after 24 h of differentiation. Nucleolar staining for c-myc RNA colocalised with that from a probe for ribosomal 28 S RNA. The signal from an exon 1 probe specific for sequences upstream of the c-myc P2 promoter was much weaker, but increased in the nuclei of differentiating cells, which possessed unusual ring-like or lamellar deposits, outside the nucleolus. These deposits appeared faintly together with nuclear staining with the exon 1 sense probe but not the exon 3 sense probe in differentiating cells. These findings demonstrate that within the first 24 h of differentiation, full-length c-myc RNA, which is compartmentalised as expected for a mature transcript, is considerably downregulated but nuclear primary RNA continues to be transcribed from exon 1. This is in a configuration similar to that reported for unspliced transcripts and is not elongated into exon 3. Antisense transcription with these RNA morphological features also occurs in exon 1 during differentiation. These results indicate significant changes in the intracellular dynamics of c-myc RNA during differentiation and support transcriptional attenuation and post-transcriptional processes, such as splicing, rather than reduced transcription initiation as the primary mechanism of c-myc downregulation in the early phases of differentiation.
Bains
M A
MA
Department of Pathology, School of Medicine, University of Southampton, Southampton General Hospital, UK.
Giles
I
I
Wright
D H
DH
D002454:Cell Differentiation / Q000235:genetics; D005091:Exons; D016259:Genes, myc / Q000235:genetics*; D018922:HL-60 Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D018613:Microscopy, Confocal; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D014158:Transcription, Genetic
D012313:RNA