Position within the host intron is critical for efficient processing of box C/D snoRNAs in mammalian cells

In mammalian cells, all small nucleolar RNAs (snoRNAs) that guide rRNA modification are encoded within the introns of host genes. A database analysis of human box C/D snoRNAs revealed conservation of their intronic location, with a preference for 70-80 nt upstream of the 3' splice site. Transfection experiments showed that synthesis of gas5-encoded U75 and U76 snoRNAs dropped significantly for mutant constructs possessing longer or shorter spacers between the snoRNA and the 3' splice site. However, the position of the snoRNA did not affect splicing of the host intron. Substitution mutations within the spacer indicated that the length, but not the specific sequence, is important. A in vitro system that couples pre-mRNA splicing and processing of U75 has been developed. U75 synthesis in vitro depends on its box C and D sequences and requires an appropriate spacer length. Further mutational analyses both in vivo and in vitro, with subsequent mapping of the branch points, revealed that the critical distance is from the snoRNA coding region to the branch point, suggesting synergy between splicing and snoRNA release.