PgmNr W399: Dynein subunit DLC-1 promotes localization and function of stem cell regulator FBF-2 in C. elegans
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Authors:
X. Wang 1 ; M. Ellenbecker 1 ; J. Olson 1 ; J. Bailey 1 ; D. Rasoloson 2 ; E. Voronina 1


Institutes
1) University of Montana, Missoula, MT; 2) Johns Hopkins University School of Medicine/HHMI, Baltimore, MD.


Keyword: Germ line stem

Abstract:

PUF family RNA-binding proteins are conserved eukaryotic mRNA regulators. FBF-1 and FBF-2, two PUF family proteins in C. elegans, are translational repressors required for maintenance of germline stem cells (Crittenden et al., 2002).  FBF-1 and FBF-2 are very similar to each other but employ different mechanisms to silence target mRNAs. FBF-2 function depends on its localization to P granules, RNA granules of germ cells (Voronina et al., 2012). In this study, we found that dynein light chain DLC-1 is required for FBF-2 recruitment to P granules. To understand the role of cofactors in FBF-2 function, we characterized FBF-2 interactome by mass-spectroscopy. By genetic interaction assay, dlc-1 was identified as a component of FBF-2 RNP specifically contributing to FBF-2-mediated regulation. One known role of DLC-1 is aiding assembly of the dynein motor complex involved in trafficking of organelles, proteins and mRNAs. Knock-down of dlc-1 by RNAi leads to dispersion of FBF-2 from P granules and decreased FBF-2 regulatory activity. However, FBF-2 localization and activity are not affected by depletion of dynein motor subunit, suggesting DLC-1 cooperates with FBF-2 in dynein-independent manner. In the in vitro pulldown assay, DLC-1 interacts with FBF-2, but not FBF-1. Typically, LC8 associates with its partner proteins through the KXTQT or GIQVD recognition sequence. However, FBF-2 does not contain canonical LC8 recognition sites. To investigate how FBF-2 interacts with DLC-1, we are determining the structure of DLC-1 bound to one of the DLC-1-binding regions of FBF-2. Preliminary data suggests that FBF-2 peptide binds the same site on DLC-1 as DLC-1’s conventional partners. To test whether FBF-2 localization depends on direct interaction with DLC-1 in vivo, we generated FBF-2 mutant transgene with mutations of DLC-1 binding sites. The FBF-2 mutant dispersed away from P granules, suggesting that direct interaction with DLC-1 is required for FBF-2 perinuclear localization in vivo.  In vitro RNA binding assay suggested that FBF-2 mutant binds target RNA with same affinity as wildtype FBF-2.

LC8 light chains are now recognized as regulatory hub proteins essential for diverse protein networks. Our finding of cooperation between FBF-2 and DLC-1 introduces the idea that DLC-1 homologs directly impact regulatory networks affecting germline stem cell maintenance. This insight will advance understanding of regulatory mechanisms of germline stem cell maintenance in other organisms, including humans.