We previously identified novel human ets-1 transcripts in which the normal order of exons is inverted, and demonstrated that although the order of exons is different than in the genomic DNA, splicing of these exons out of order occurs in pairs using genuine splice sites (1). Here we determine the structure of these novel transcripts, showing that they correspond to circular RNA molecules containing only exons in genomic order. These transcripts are stable molecules, localized in the cytoplasmic component of the cells. To our knowledge, this is the first case of circular transcripts being processed from nuclear pre-mRNA in eukaryotes. This new type of transcript might represent a novel aspect of gene expression and hold some interesting clues about the splicing mechanism.

The mouse insulin-like growth factor 2 (Igf2) locus is a complex genomic region that produces multiple transcripts from alternative promoters. Expression at this locus is regulated by parental imprinting. However, despite the existence of putative imprinting control elements in the Igf2 upstream region, imprinted transcriptional repression is abolished by null mutations at the linked H19 locus. To clarify the extent to which the Igf2 upstream region contains autonomous imprinting control elements we have performed functional and comparative analyses of the region in the mouse and human. Here we report the existence of multiple, overlapping imprinted (maternally repressed) sense and antisense transcripts that are associated with a tandem repeat in the mouse Igf2 upstream region. Regions f lanking the repeat exhibit tissue-specific parental allelic methylation patterns, suggesting the existence of tissue-specific control elements in the upstream region. Studies in H19 null mice indicate that both parental allelic methylation and monoallelic expression of the upstream transcripts depends on an intact H19 gene acting in cis. The homologous region in human IGF2 is structurally conserved, with the significant exception that it does not contain a tandem repeat. Our results support the proposal that tandem repeats act to target methylation to imprinted genetic loci.

Following our studies which showed that the a and [ exons of the chicken c-ets-1 gene are not conserved in the human homologue, we succeeded in identifying a novel human c-ets-1 transcript in which the normal order of exons is scrambled. By PCR and RNase protection assays, we demonstrated that while the order of exons is different from that in genomic DNA, splicing of these exons in aberrant order occurs in pairs and at the same conserved consensus splice sites used in the normally spliced transcript. The scrambled transcript is nonpolyadenylated and is expressed at much lower levels than the normal transcript. It is not the consequence of genomic rearrangement at the ets-1 locus nor is it due to the transcription of any ets-1 pseudogene. These results confirm previous observations of scrambled splicing.