An additional long-range interaction in human U1 snRNA.

We present evidence for the existence of an additional long-range interaction in vertebrate U1 snRNAs. By submitting human U1 snRNP, HeLa nuclear extracts, authentic human or X. laevis in vitro transcribed U1 snRNAs to RNase V1, a nuclease specific for double-stranded regions, cleavages occurred in the sequence psi psi ACC (positions 5-9) residing in the 5' terminal region of the RNA. The RNase V1 sensitive region is insensitive to single-stranded probes, something unexpected knowing that it was considered single-stranded in order to base-pair to pre-mRNA 5' splice site. We have identified the sequence GGUAG (positions 132-136) as the only possible 3' partner. Mutants, either abolishing or restoring the interaction between the partners, coupled to an RNase V1 assay, served to substantiate this base-pairing model. The presence of this additional helix, even detected in nuclear extracts under in vitro splicing conditions, implies that a conformational change must occur to release a free U1 snRNA 5' end.