P3.05. MUSCLEBLIND-LIKE RNA BINDING PROTEIN TRAFFICKING DYNAMICS ARE REGULATED BY MOLECULAR INTERACTIONS WITH EARLY AND RECYCLING ENDOSOMES
Aleksandra Janusz-Kamińska1, Annie Shen1, Paul Fernandez2, Luke Knudson1, Eric Zhou1, Ryan Hildebrandt2, Eric T. Wang2, Gary J. Bassell1
1 Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA
2 Center for Neurogenetics, Dept. of Molecular Genetics, University of Florida, Gainesville, FL, USA
INTRODUCTION: Transport of mRNAs and localized translation are crucial for neurodevelopment and function. Recent findings revealed a role of endosomes in RNA transport and localized translation in conjunction with RNA binding proteins (RBPs). Muscleblind-like (MBNL) proteins are RBPs that regulate splicing and mRNA localization. MBNLs are depleted from the cytoplasm in myotonic dystrophy type 1 (DM1) by sequestration on the expanded CTG repeats in the 3' UTR of dystrophia myotonica protein kinase (DMPK) transcripts. We have shown a role for MBNL-kinesin interactions in mRNA localization although the role of membrane association is not understood.
AIM(S): We hypothesized that the unstructured carboxy terminus (C-term) of MBNL1 regulates endomembrane attachment necessary for mRNA localization.
METHOD(S): Fixed and live cell imaging in cell lines and mouse neurons. Overexpression of EGFP-MBNL1 with or without C-term, followed with immunofluorescence (IF) for endo- and lysosome markers (Rab5, Rab7, Rab11, Lamp1) in n2a cells. Widefield live imaging of EGFP-MBNL1 with endo- and lysosomes (Rab5, Rab11, Rab7 and Lamp1), then MBNL1 and Rab5/Rab11 endosomes with the expression of mutants (dominant negative Rab5 S43N, MBNL1 with C-term deletion). Superresolution (STED) live imaging with MBNL1 RNA binding deficient mutant fused to MCP-Halo, 24xMS2 construct and Rab5 or its S43N mutant. Single-molecule in-situ hybridization and IF for SNAP25 mRNA, an MBNL1 target, Rab5 and EGFP-MBNL1 WT or its C-term deletion mutant.
RESULTS: MBNL1 mRNA granules are located on early endosomes and the C-term of MBNL1 bi-directionally regulates its mobility with endosomes. Work in progress is to evaluate the role of the C-term of MBNL1 in endosome coupled local translation.
CONCLUSIONS: Our findings suggest that one of the mechanisms for MBNL1 to regulate mRNA cargo distribution and localized translation in neurons is endomembrane clustering.
FINANCIAL SUPPORT: NIH R01 NS114253 grant awarded to G.J.B and E.T.W.