The slj Mutation Targets Interaction of Pol III Subunits Rpc2/Polr3b and Rpc11/Polr3k in S. pombe and Zebrafish (A) Deletion of the region between aa267 and aa308 of S. pombe Rpc2 results in a deficiency of Rpc11p in the purified Pol III complex. Wild-type Pol III and mutant Pol III containing rpc2-Δ, in which the region between aa267 and aa308 of S. pombe Rpc2 was deleted, were purified from extracts prepared from FLAG-His6-Rpc53p strains expressing Rpc2p (“FH-Rpc53/rpc2”), Rpc2-Δp (“FH-Rpc53/rpc2-Δ”), or empty vector (“FH-Rpc53/3X”) as described in Materials and Methods. Mock purification was also performed with control extracts prepared from a wild-type untagged strain (“Rpc53/rpc2”). The input extracts (lanes 1–4) and the purified Pol III (lanes 5–8) were fractionated by 4%–20% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and analyzed by immunoblotting using antisera indicated on the left; the detected proteins are indicated on the right. The amounts of input loaded relative to that used for purification are 0.4% lane 1; 0.13% lane 2; 0.1% lane 3, and 0.1% lane 4. A total of 5% of the purified material was loaded in lanes 5–8. (B) Lateral view of the pancreas from a 5-dpf wild-type (wt) larva, a 5-dpf slj larva (slj), and two 5-dpf slj larvae that were microinjected with a hsp70/polr3k expression construct (slj-polr3k), following heat shock (as described in Materials and Methods), and carboxypeptidase A (green) and insulin (red) immunohistochemistry. Expression of the polr3k cDNA from the hsp70 heat shock promoter induces partial rescue of the slj exocrine pancreas phenotype. (C) Lateral views of a 5-dpf control wild-type (wt) and polr3k Morpholino-injected larvae (polr3k-MO). The polr3k knockdown produces a phenotype that is similar to that caused by the slj mutation (Figure 1B). The intestine (green arrow), cranial morphology (green arrowhead), and terminal branchial arches (red arrowhead) are reduced with polr3k knockdown.
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