16; 2, orb2ΔQ, LI = 2.15) ( Figure 3; Table S4), suggesting that the residual memory of the orb2ΔA mutants might RO4929097 cell line be mediated by the Q domain
of Orb2B. Since the orb2ΔQΔB mutation was lethal when homozygous, we tested this allele in combination with the viable orb2ΔA allele. These flies, which lack the Q domain specifically in Orb2A, had a normal short-term memory ( Table S5D) but no long-term memory (5, orb2ΔQΔB/orb2ΔA, LI = 2.86) ( Figure 3; Table S4). This lack of memory shows that the Q domain in Orb2A is essential, and that of Orb2B insufficient, for long-term memory. To test for the sufficiency of the Q domain in Orb2A, we tested the memory of the transheterozygotes in which the Q domain is present only in Orb2A. The learning index of these mutants was indistinguishable from control flies in which both isoforms are intact (6, orb2ΔB/orb2ΔQΔA, LI = 16.97; 7, orb2ΔB/orb2ΔA LI = 20.83) ( Figure 3; Table S4). These results indicate that Orb2A has a specific role in long-term memory that requires the Q domain, which in Orb2B is both dispensable and insufficient. Epacadostat solubility dmso To assess the role of the RBD in long-term memory, as a first step we chose to replace the Orb2 RBD with the RBDs of other CPEBs,
reasoning that such chimeric proteins might retain activity toward conserved and common RNA targets but not Orb2-specific targets involved in long-term memory formation. A swap of the Orb2 RBD with the RBD of Orb1 (3, orb2orb1RBD) did not rescue viability, whereas the swap with the RBD of the mCPEB2 (4, orb2mCPEB2RBD) rendered flies viable and healthy ( Figure 4A; Table S5A). This indicated that RNA binding properties of this domain are required during the development, and moreover
suggested a potential conservation in RNA targets between the CPEB II family members at least in development. The conservation tuclazepam of RNA targets is consistent with the high homology in this region, ∼90% ( Theis et al., 2003). Interestingly, orb2mCPEB2RBD mutants showed strong long-term memory impairment in comparison to the control flies (4, orb2mCPEB2RBD LI = 6.16; 1, orb2+, LI = 32.39) ( Figure 4A; Table S5A). In contrast, short-term memory was normal (4, orb2mCPEB2RBD, LI = 40.0; 1, orb2+, LI = 42.34) ( Figure 4A; Table S5A′), indicating that the long-term memory impairment is unlikely the result of developmental defects caused by the RBD swap. Most importantly, this allele provided us with the unique opportunity to assess the role of the RBD in Orb2B in long-term memory, independently of its role in development. In the orb2mCPEB2RBD background, expression of the wild-type Orb2B, but not Orb2A, fully rescued memory (3, orb2ΔA/orb2mCPEB2RBD, LI = 28.5; 4, orb2ΔB/orb2mCPEB2RBD, LI = 1.68) ( Figure 4B; Table S5B), and this rescue was dependent on its RBD (5, orb2RBD∗ΔA/orb2mCPEB2RBD, LI = 1.04, see the paragraph below on the mutated RBD∗).