RNAi of Ubiquitin Conjugating Enzymes in C. elegans. The Q82:GFP strain of C. elegans expresses polyglutamine aggregates in body wall muscle cells. Synchronized worms from the Q82:GFP strain were treated with RNAi for 48 hours until they reached approximately the L4 larval stage. RNAi treatments included the knockdown of ubiquitin (ubq-1), a subunit of the proteasome (rpt-1) and 11 different ubiquitin conjugating enzymes. Worms continue to express aggregates after these RNAi treatments, however, significant alterations in numbers and sizes of aggregates occur upon knockdown of specific Ubc's. A) Fluorescent images of control and RNAi treated worms showing the Q82:GFP aggregates. Scale bar is 100 μm. B) The average number of aggregates was counted manually under 40× magnification. RNAi knockdown of ubiquitin (ubq-1), proteasome (rpt-1) or ubc-1 resulted in an increase in the number of aggregates whereas RNAi of either ubc-2 or ubc-22 showed a significant decrease in the number of aggregates. (* indicates statistical significance at p < .05). C) The average size of aggregates was determined following knockdown of each Ubc after 48 hours of RNAi treatment. The control was found to have an average aggregate area of 2.23 μm2. Knockdown of ubq-1, ubc-1, or uev-1 resulted in a significant decrease in aggregate sizes. Knockdown of rpt-1, ubc-2 or ubc-22 resulted in a significant increase in aggregate sizes. (* indicates statistical significance at p < .05). D) Same procedure as in A except that bacterial feeding cultures were combined to achieve knockdown of more than one Ubc. The ubc-1 phenotype is epistatic to both ubc-2 and ubc-22. There is no additive effect between ubc-2 and ubc-22. (* indicates statistical significance at p < .05). E) RT-PCR of RNAi treated worms confirms RNAi knockdown. Primers for ubc-1, ubc-2, and act-1 were used to test RNA levels after RNAi treatments. For each set of primers, PCR template for lane C is cDNA from control (pL4440) worms, lane 1 ubc-1(RNAi), lane 2 ubc-2(RNAi), and lane 1+2 ubc-1 + ubc-2(RNAi).
Howard et al. BMC Cell Biology 2007 8:32 doi:10.1186/1471-2121-8-32