Presentation/Session Information

Session Information

Session Title: Plenary Session 2 Session Type: Plenary
Session Location: Royce Hall Session Time: Thu, Jun 25 3:00PM - 6:00PM

Presentation Information

Program Number: 72 Presentation Time: 5:48PM

Presentation Content

The P-granule assembly protein, PGL-1, is a base-specific RNA nuclease.Scott Takeo Aoki 1, Aaron M. Kershner 1,2, Marvin Wickens 1, Craig Bingman 1, Judith Kimble 1,2. 1)Department of Biochemistry, University of Wisconsin-Madison, Madison, WI; 2)Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI

P-granules are RNA-protein assemblies required for germ cell development. Loss of P-granules leads to adult germline deterioration (reviewed in 1-2) and differentiation of germ cells into somatic cell types (3). Granule formation requires PGL-1 and PGL-3 (PGLs), protein paralogs capable of granule self-assembly (4-5). We set out to structurally characterize the PGLs to better understand their molecular function in P-granules. Our biochemical analyses of recombinant PGL-1 identified a dimerization domain in the central region of the protein, referred to as “PGL-1 DD.” We then determined the 1.6 Å and 3.6 Å crystal structures of C. remanei and C. elegans PGL-1 DD, respectively. PGL-1 DD has a novel, alpha helical fold. The proposed dimer forms a positively charged channel with the appropriate dimensions to fit single stranded RNA. Unexpectedly, incubation of PGL-1 DD with oligonucleotides resulted in RNA cleavage. Additional in vitro results demonstrate PGL-1 DD to be a guanosine-specific, single-stranded RNase. The crystal structure allowed us to identify a site in the dimer channel that brought together conserved glutamine and lysine side chains. We postulated these side chains might contribute to PGL’s RNase activity. Mutation of this glutamine to alanine abrogated RNA cleavage without affecting either dimerization or RNA binding. We have now created the glutamine to alanine RNase mutants in the endogenous pgl-1 and pgl-3 genes using CRISPR-Cas methods, and we are assessing the role of PGL's enzymatic function in germ cell development. Our results support the notion that PGL-1 may have a dual function in P-granules as an assembly protein and as an RNase. Our results also raise the question of whether other germ granule scaffold proteins, like Drosophila Oskar and zebrafish Bucky Ball, may also have enzymatic activities affecting RNA regulation.

1. Updike, D., Strome, S. J Androl 2010; 31(1):53-60.

2. Voronina E, Seydoux G, Sassone-Corsi P, Nagamori I. Cold Spring Harb Perspect Biol. 2011 Dec 1;3(12):a002774.

3. Updike DL, Knutson AK, Egelhofer TA, Campbell AC, Strome S. Curr Biol. 2014 May 5;24(9):970-5.

4. Updike DL, Hachey SJ, Kreher J, Strome S. J Cell Biol. 2011 Mar 21;192(6):939-48.

5. Hanazawa M, Yonetani M, Sugimoto A. J Cell Biol. 2011 Mar 21;192(6):929-37.

Please note: Abstract shown here should NOT be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

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