Identification of six new box C/D snoRNA gene clusters from rice.

Here we have identified 18 different box C/D snoRNAs encoded in six new gene clusters by the screening of Oryza sativa (rice) genome sequences using a computer program that we have designed to search for non-canonical box C and D sequences. Among this snoRNA collection, five are homologues of snoRNAs previously reported in other plants. One snoRNA is implicated to guide methylation at a corresponding site also modified in yeast rRNA but not in any other plants species yet examined. The other 12 snoRNAs are implicated in guiding 12 completely novel rRNA 2'-O-ribose methylation sites. We have verified the methylation of these rRNA sites by dNTP-concentration dependent primer extension assay. The expression of all snoRNAs reported here was experimentally confirmed by primer extension assay. Polycistronic precursor transcripts from the gene clusters have been demonstrated by RT-PCR. Thus it is highly likely that each of the rice snoRNA gene clusters identified here is transcribed in a polycistronic way, regardless of whether or not they are encoded within introns. This indicates that the mode of polycistronic expression of snoRNA clusters is widespread in rice, as is the case in Arabidopsis thaliana.