The position of yeast snoRNA-coding regions within host introns is essential for their biosynthesis and for efficient splicing of the host pre-mRNA.

Genomic location of sequences encoding small nucleolar RNAs (snoRNAs) is peculiar in all eukaryotes from yeast to mammals: most of them are encoded within the introns of host genes. In Saccharomyces cerevisiae, seven snoRNAs show this location. In this work we demonstrate that the position of snoRNA-coding regions with respect to splicing consensus sequences is critical: yeast strains expressing mutant constructs containing shorter or longer spacers (the regions between snoRNA ends and intron splice sites) show a drop in accumulation of U24 and U18 snoRNAs. Further mutational analysis demonstrates that altering the distance between the 3' end of the snoRNA and the branch point is the most important constraint for snoRNA biosynthesis, and that stable external stems, which are sometimes present in introns containing snoRNAs, can overcome the positional effect. Surprisingly enough, splicing of the host introns is clearly affected in most of these constructs indicating that, at least in S. cerevisiae, an incorrect location of snoRNA-coding sequences within the host intron is detrimental to the splicing process. This is different with respect to what was demonstrated in mammals, where the activity of the splicing machinery seems to be dominant with respect to the assembly of snoRNPs, and it is not affected by the location of snoRNA sequences. We also show that intronic box C/D snoRNA recognition and assembly of snoRNPs occur during transcription when splicing sequences are recognized.