Objectives: We used an unbiased approach to characterize JAKMIP1’s protein interactome. We identified an interaction with the FMRP-associated translational complex and are interested in the mechanism and purpose of the FMRP-JAKMIP1 interaction, JAKMIP1’s association and regulation of FMRP mRNA translational targets, and JAKMIP1’s role in neuronal translation.
Methods : We used Multidimensional Protein Identification Technology (MudPIT) to identify JAKMIP1’s proteomic interactome. To test JAKMIP1’s presence in polyribosomes, we conducted polyribosome fractionation, immunoprecipitation from a BacTRAP neural cell line expressing eGFP-tagged polyribosomes, and immunocytochemistry of JAKMIP1 and poly(A)-binding protein 1 (PABPC1) in neurons. To test JAKMIP1’s association with FMRP mRNA targets, we conducted quantitative RTPCR with RNA from JAKMIP1 immunoprecipitation. We tested JAKMIP1’s translational control of a subset of these targets by protein analysis at synaptosomal membranes in neural systems with reduced JAKMIP1. We used fluorescence non-canonical amino acid tagging (FUNCAT) to assess translation in neurons lacking JAKMIP1.
Results: We show that JAKMIP1 is present in the polyribosome fraction and binds FMRP protein as well as several of its mRNA targets, including Fmr1 and PSD95, both of which have increased expression in synaptosomal membranes upon Jakmip1 knockdown. Furthermore, we show that neurons from Jakmip1 knockout mice show significantly reduced nascent translation.
Conclusions : These results identify JAKMIP1 as a new protein involved in the regulation of neuronal translation and show that it interacts with the process of FMRP-related translational control. Elucidation of the precise molecular interactions occurring within the JAKMIP1 and FMRP-associated complex, its relationship to neuronal activity, as well as the interaction of JAKMIP1 with the transport machinery are exciting new directions that we are exploring.
See more of: Cell Biology
See more of: Biological Mechanisms