C. elegans RNA-binding proteins PUF-8 and MEX-3 function redundantly to promote germline stem cell mitosis

Abstract

Maintenance of mitotically cycling germline stem cells (GSCs) is vital for continuous production of gametes. In worms and insects, signaling from surrounding somatic cells play an essential role in the maintenance of GSCs by preventing premature differentiation. In addition, germ cell proteins such as the Drosophila Pumilio and Caenorhabditis elegans FBF, both members of the PUF family translational regulators, contribute to GSC maintenance. FBF functions by suppressing GLD-1, which promotes meiotic entry. However, factors that directly promote GSC proliferation, rather than prevent differentiation, are not known. Here we show that PUF-8, another C. elegans member of the PUF family and MEX-3, a KH domain translational regulator, function redundantly to promote GSC mitosis. We find that PUF-8 protein is highly enriched in mitotic germ cells, which is similar to the expression pattern of MEX-3 described earlier. The puf-8(−) mex-3(−) double mutant gonads contain far fewer germ cells than both single mutants and wild-type. While these cells lack mitotic, meiotic and sperm markers, they retain the germ cell-specific P granules, and are capable of gametogenesis if GLP-1, which normally blocks meiotic entry, is removed. Significantly, we find that at least one of these two proteins is essential for germ cell proliferation even in meiotic entry-defective mutants, which otherwise produce germ cell tumors. We conclude PUF-8 and MEX-3 contribute to GSC maintenance by promoting mitotic proliferation rather than by blocking meiotic entry.

RNA recognition by the embryonic cell fate determinant and germline totipotency factor MEX-3

“…The unusual transdifferentiated germline teratoma phenotype is only observed in worms with mutations in both mex-3 and gld-1, a second KH-domain RNA binding protein (4). Moreover, recent evidence indicates that MEX-3 and PUF-8, a Puf-domain RNA binding protein, promote mitotic division of germ cells (41). These studies suggest that MEX-3 is playing redundant roles with other RNA binding proteins to ensure preservation and maintenance of germline stem cells.…”

Translational Control in the Caenorhabditis elegans Germ Line

“…Both FBF proteins are essential for actively dividing germ stem cells (Crittenden et al 2002 ) , meiotic entry (Lamont et al 2004 ) , and the sperm-tooocyte switch (Zhang et al 1997 ) . PUF-8 is important for germline proliferation (Ariz et al 2009 ) , the sperm-to-oocyte switch , and male meiotic progression (Subramaniam and Seydoux 2003 ) . While PUF-5, -6, -7 assist oocyte differentiation and maturation (Lublin and Evans 2007 ) , PUF-3 is essential for early embryogenesis (Sonnichsen et al 2005 ) .…”

“…Originally discovered as a maternaleffect lethal gene that regulates early embryonic cell fates, mex-3 is also active in the postembryonic germ line (Draper et al 1996 ;Mootz et al 2004 ;Ciosk et al 2004Ciosk et al , 2006Ariz et al 2009 ) . MEX-3 is a 3 ¢ UTR-associated translational repressor and prevents the premature accumulation of the maternally donated embryonic cell fate determinant PAL-1 in the growing oocytes (Draper et al 1996 ;Mootz et al 2004 ;Hunter and Kenyon 1996 ;Jadhav et al 2008 ) .…”

“…Consistent with the notion that each individual KH domain of MEX-3 may contact one motif to achieve overall high RNA selectivity and af fi nity, the elimination of both motifs is more detrimental to MEX-3 RNA binding than compromising an individual motif of the MRE (Pagano et al 2009 ) . Although numerous candidate MEX-3 target mRNAs were suggested (Pagano et al 2009 ) , little is known about how MEX-3 functions with GLD-1 to maintain totipotency of germ cells (Ciosk et al 2006 ) and functions with PUF-8 in germ cell proliferation (Ariz et al 2009 ) .…”

“…In the case of translational repressors, fi ve distinct protein expression patterns are prevalent in the adult hermaphrodite (Types A-D and U, Fig. 8.3 ): The PUF proteins FBF-1, FBF-2, and PUF-8 are predominantly expressed in the distal most part of the germ line, corresponding to the mitotic region and the initial stages of meiosis (Type A) (Crittenden et al 2002 ;Lamont et al 2004 ;Ariz et al 2009 ) . The expression pattern of the maxi-KH protein GLD-1 covers a larger region of the distal part extending to the germline loop, overlapping with the mitotic region and the early meiotic stages until diplotene (Type B) (Jones et al 1996 ) .…”