AN IN-VIVO AND IN-VITRO STRUCTURE-FUNCTION ANALYSIS OF THE SACCHAROMYCES-CEREVISIAE U3A SNORNP - PROTEIN-RNA CONTACTS AND BASE-PAIR INTERACTION WITH THE PRE-RIBOSOMAL-RNA

The structure and accessibility of the S. cerevisiae U3A snoRNA was st udied In semi-purified U3A snoRNPs using both chemical and enzymatic p robes and in vivo using DMS as the probe, The results obtained show th at S. cerevisiae U3A snoRNA is composed of a short 5' domain with. two stem-loop structures containing the phylogenetically conserved boxes Al and A and a large cruciform 3' domain containing boxes B, C, C' and D. A precise identification of RNA-protein contacts is provided. Prot ection by proteins in the snoRNP and ii? vivo are nearly identical and were exclusively found in the 3' domain. There are two distinct prote in anchoring sites: (i), box C' and its surrounding region, this site probably includes box D, (ii) the boxes B and C pail and the bases of stem-loop 2 and 4. Box C' is wrapped by the proteins, RNA-protein inte ractions are more loose at the level of boxes C and D and a box C and D interaction is preserved in the snoRNP. In accord with this location of the protein binding sites, an it? vivo mutational analysis showed that box Ct is important for U3A snoRNA accumulation, whereas mutation s in the 5' domain have little effect on RNA stability. Our in viva pr obing experiments strongly suggest that, in exponentially growing cell s, most of the U3A snoRNA molecules are involved in the 10-bp interact ion with the 5'-ETS region and in two of the interactions recently pro posed with 18 S rRNA sequences. Ow experimental study leads to a sligh tly revised version of the model of interaction proposed by J. Hughes. Single-stranded segments linking the heterologous helices are highly sensitive to DMS in vivo and their functional importance was tested by a mutational analysis. (C) 1997 Academic Press Limited.