Experimental confirmation of snoRNA methylation guide function

Loss of specific rRNA 2'-O-ribose methyl site(s) for snoRNA disruption mutants

• The polyacrylamide gel to the right displays AMV reverse transcriptase (RT) primer extensions on rRNA from wildtype (wt) and snoRNA disruption mutant yeast strains (snRx)
• Primer extensions either contain high dNTP concentration (1.0 mM; left lane of pairs) or low dNTP concentration (0.004 mM; right lane of pairs)

• Bands due to known 2'-O-methyl modifications are labelled by arrows. These modifications appear as strong primer extension stops in low but not high dNTP concentration lanes

• Specific loss of 2'-O-methyl band for snoRNA disruption mutant relative to the wildtype strain confirms snoRNA guide function

• rRNA sequencing ladder lanes are labelled by U, G, C, A
  Note: 2'-O-methyl modifications appear as a band one nucleotide 3' (below) the actual modfied nucleotide
• Primer extensions use a ³²P end-labelled, gel-purified primer (primer LSU-R2305 for this experiment [primer sequence])
• The dNTP concentration-dependent primer extension assay used in these experiments (available here) is a slightly modified version of protocols previously described by Maden et al. (1995) Biochemie 77, 22-29; and Kiss-Laszlo et al. (1996) Cell 85, 1077-1088.