Satellite 2 trimethylation and the search for a Satellite 2 related small nuclear RNA

Satellite 2 (Sat2) is a 330-bp tandemly repeated DNA sequence that is evolutionarily conserved in the Caudate amphibians (newts and salamanders). Sat2 transcripts have been found in all tissues investigated, and transcription is driven by a small nuclear RNA (snRNA) type promoter. Portions of the Sat2 hammerhead core resemble conserved snRNA elements, boxes C and D. Here we show that a subset of Sat2 transcripts contain a trimethylguanosine cap structure, so far unique to the snRNAs and the small nucleolar RNAs (snoRNAs). A transcript of ∼390 nts is heavily trimethylated within the ovary germinal vesicle, and microinjection of synthetic, labeled RNA transcripts demonstrated that the region important for this trimethylation was contained within the conserved hammerhead structure near the 3′ end of the transcript. Furthermore, the second hammerhead sequence must be exposed on the 3 ′ end of the transcript to obtain efficient trimethylation. We tested Sat2 transcripts for the ability to bind to the Sm proteins (involved in snRNA trimethylation) or to the protein fibrillarin (involved in snoRNA trimethylation) and found that neither set of proteins associate with Sat2 transcripts. This suggests that Sat2 transcripts use an alternate pathway to become trimethylated, possibly involving association with a different and as yet unknown set of proteins.;These data suggest that Sat2 could be a member of the snRNA gene family, and as such Sat2 homologues would be expected to exist in other vertebrate species. A Northern blot of RNAs from various unrelated eukaryotic organisms immunoprecipitated with anti-trimethylguanosine antibodies yielded two RNAs from Xenopus laevis and four RNAs from Drosophila melanogaster. A unique protocol developed for cloning RNAs regardless of internal sequence was unsuccessful at rewarding us with the nature of the two RNAs from Xenopus. The protocol was demonstrated to work with control RNAs, but due to the potential problems with the current protocol, the RNAs from Drosophila have not been attempted. The largest RNA detected within Drosophila has shown evidence of an embryonic specific expression pattern that begins about 2 hours after fertilization and ends between 6–10 hours after fertilization. Our current model of satellite 2 represents it as a Caudate specific pseudogene family that is derived from an as yet unidentified true snRNA gene.