DEVELOPMENTAL DYNAMICS 241:505–521, 2012 a RESEARCH ARTICLE TEG-1 CD2BP2 Regulates Stem Cell Proliferation and Sex Determination in the C. elegans Germ Line and Physically Interacts With the UAF-1 U2AF65 Splicing Factor Chris Wang,1 Laura Wilson-Berry,2 Tim Schedl,2 and Dave Hansen1* Background: For a stem cell population to exist over an extended period, a balance must be maintained between self-renewing (proliferating) and differentiating daughter cells. Within the Caenorhabditis ele- gans germ line, this balance is controlled by a genetic regulatory pathway, which includes the canonical Notch signaling pathway. Results: Genetic screens identified the gene teg-1 as being involved in regulat- ing the proliferation versus differentiation decision in the C. elegans germ line. Cloning of TEG-1 revealed that it is a homolog of mammalian CD2BP2, which has been implicated in a number of cellular processes, including in U4/U6.U5 tri-snRNP formation in the pre-mRNA splicing reaction. The position of teg-1 in the genetic pathway regulating the proliferation versus differentiation decision, its single mutant phenotype, and its enrichment in nuclei, all suggest TEG-1 also functions as a splicing factor. TEG-1, as well as its human homolog, CD2BP2, directly bind to UAF-1 U2AF65, a component of the U2 auxiliary factor. Conclusions: TEG-1 functions as a splicing factor and acts to regulate the proliferation versus meiosis decision. The interaction of TEG-1 CD2BP2 with UAF-1 U2AF65, combined with its previously described function in U4/U6.U5 tri-snRNP, suggests that TEG-1 CD2BP2 functions in two distinct locations in the splicing cascade. Developmental Dynamics 241:505–521, 2012. VC 2012 Wiley Periodicals, Inc. Key words: CD2BP2; TEG-1; stem cell proliferation; pre-mRNA splicing; UAF-1; U2AF65 Key findings: Developmental Dynamics TEG-1 inhibits stem cell proliferation in the C. elegans germ line. TEG-1 likely functions as a splicing factor. TEG-1 CDBP2 physically interacts with UAF-1 U2AF65. Accepted 3 January 2012 INTRODUCTION is tied to their ability to produce to make the desired tissue. Maintain- both self-renewing and differentiating ing the balance between self-renewing Stem cells provide the material neces- sary for tissue generation during devel- daughter cells. Self-renewing daughter and differentiating daughter cells is opment, and tissue replacement and cells retain the stem cell characteristics key to proper stem cell function since a repair in adult animals. The capacity of of the parental cell; therefore, a pool of disruption in this balance prevents stemcellstocontributetotheseproc- stem cells is maintained over time. The long term tissue generation; too little esses over an extended time period differentiating daughter cells are used self-renewal results in the eventual 1University of Calgary, Department of Biological Sciences, Alberta, Calgary, Canada 2Department of Genetics, Washington University School of Medicine, St. Louis, Missouri Grant sponsor: Canadian Institutes of Health Research (CIHR); Natural Sciences and Engineering Research Council of Canada (NSERC); National Institutes of Health (NIH); Grant number: GM63310. *Correspondence to: Dave Hansen, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4. E-mail: [email protected] DOI 10.1002/dvdy.23735 Published online 30 January 2012 in Wiley Online Library (wileyonlinelibrary.com). VC 2012 Wiley Periodicals, Inc. 506 WANG ET AL. depletion of the stem cell population, decrease, allowing cells to enter mei- proliferative fate, resulting in a com- while too little differentiation results in otic prophase. The first obvious indica- plete germline tumor (Berry et al., not enough cells being made for proper tion that cells have entered into mei- 1997; Hansen et al., 2004a). Other tissue generation. Understanding how otic prophase is when the DNA takes genes have been identified that func- this balance between self-renewal and on a crescent moon shape as chromo- tion with the GLP-1/Notch signaling differentiation is maintained is an somes pair and synapse (Francis et al., and gld-1/gld-2 pathways, or parallel essential step in understanding stem 1995a; Dernburg et al., 1998; Mac- to them, to regulate the balance cell biology and function. Queen and Villeneuve, 2001). Cells between proliferation and differentia- The germ line of the nematode continue to move proximally while tion. These genes function in many Caenorhabditis elegans has emerged transiting through the various stages different cellular processes. For exam- as a powerful model to study how a of meiotic prophase, eventually form- ple, mutations that decrease the func- stem cell population maintains the ing fully differentiated gametes tion of the proteasome enhance over- balance between self-renewal and dif- (sperm or oocytes depending on the proliferation mutants, indicating a ferentiation (Hansen and Schedl, age of the hermaphrodite). function to promote meiotic develop- 2006; Kimble and Crittenden, 2007). The GLP-1/Notch signaling path- ment (MacDonald et al., 2008). As In the C. elegans hermaphrodite, the way promotes the proliferative fate, another example, the cell-cycle regu- germ line is contained in two gonad at least in part, by inhibiting the lators Cyclin E (CYE-1) and CDK2 arms that meet at a common uterus. activities of two redundant meiotic (CDK-2) promote the proliferative Populations of mitotically dividing promoting genetic pathways, referred fate (Fox et al., 2011; Jeong et al., (proliferating) cells exist at the ends to as the gld-1 and gld-2 pathways 2011). A number of splicing factors of the two gonad arms distal from the (Kadyk and Kimble, 1998) (Fig. 1B). have also been identified as being uterus (Fig. 1A). In the adult, each Within the gld-1 pathway are the gld- involved in regulating the prolifera- population consists of 200–250 cells 1 and nos-3 genes (Francis et al., tion versus differentiation decision (Lamont et al., 2004; Killian and Hub- 1995b; Hansen et al., 2004b); with (Belfiore et al., 2004; Mantina et al., bard, 2005), with cells progressing gld-1 encoding a KH domain contain- 2009; Kerins et al., 2010; Zanetti through the cell cycle approximately ing RNA binding translational regula- et al., 2011). Genetically, these splic- every 6.5–8 hr (Fox et al., 2011). At tor (Jones and Schedl, 1995), and nos- ing factors appear to function largely least some of these proliferating cells 3 encoding a protein with similarity in the gld-1 pathway, functioning are considered stem cells. Cells are to Drosophila Nanos (Kraemer et al., downstream of GLP-1/Notch signal- kept in the proliferative state due to 1999), also a translational regulator. ing (Fig. 1B). A screen of splicing fac- their close proximity to the Distal Tip Within the gld-2 pathway are the gld- tors found that proteins functioning Cells (DTCs), with a single DTC cap- 2 and gld-3 genes, encoding the cata- throughout the splicing cascade par- ping the distal end of each gonad arm. lytic portion of a poly(A) polymerase ticipate in the proliferation/differen- The proliferation promoting influence (Kadyk and Kimble, 1998), and a tiation decision, suggesting that an of the DTC is primarily accomplished homolog to Drosophila BicC, respec- overall decrease in splicing or spliceo- through the conserved GLP-1/Notch tively (Eckmann et al., 2002). The some activity contributes to over-pro- Developmental Dynamics signaling pathway (Hansen and spatial organization of the activity of liferation (Kerins et al., 2010). Schedl, 2006; Kimble and Crittenden, the GLP-1/Notch signaling pathway Pre-mRNA splicing involves the re- 2007; Racher and Hansen, 2010). The relative to the activities of the gld-1 moval of introns from the pre-mRNA DSL (Delta/Serrate/LAG-2) LAG-2 and gld-2 pathways is key to main- between the exons prior to nuclear and APX-1 ligands are expressed on taining the balance between prolifera- export and translation. The splicing the surface of the DTC (Henderson tion and differentiation. Loss or reaction is accomplished through the et al., 1994; Tax et al., 1994; Nadara- reduction of GLP-1/Notch signaling in activity of the spliceosome, which is a jan et al., 2009). When they bind the the distal end results in ectopic acti- large RNA-Protein complex that is GLP-1/Notch receptor (Yochem and vation of the gld-1 and gld-2 path- assembled in a stepwise manner Greenwald, 1989), which is expressed ways in this region, causing cells to (Wahl et al., 2009; Valadkhan and on the surface of the germ cells (Crit- enter meiosis prematurely and a loss Jaladat, 2010). Some of the earliest tenden et al., 1994), the cleaved intra- of the proliferative population (Austin steps in spliceosome assembly include cellular portion of GLP-1 (referred to and Kimble, 1987; Eckmann et al., recognition of the 50 and 30 splice sites as GLP-1(INTRA)) is thought to enter 2004; Hansen et al., 2004b). Con- on the pre-mRNA by protein or RNA/ the nucleus and bind to the CSL versely, ectopic activation of GLP-1/ protein complexes. The U1 snRNP (CBF1/Suppressor of Hairless/LAG-1) Notch signaling beyond its normal particle, consisting of the U1 snRNA LAG-1 transcription factor (Christen- region of activity, or a combined and a number of proteins, recognizes sen et al., 1996; Mumm and Kopan, reduction in the activities of the and binds to the 50 splice site (Sera- 2000) and the SEL-8/LAG-3 co-activa- redundant gld-1 and gld-2 pathways, phin and Rosbash, 1989; Michaud tor (Doyle et al., 2000; Petcherski and results in excess proliferation (Berry and Reed, 1991). The U2 Auxiliary Kimble, 2000). This complex then acti- et al., 1997; Kadyk and Kimble, 1998; Factor (U2AF) recognizes and binds vates genes necessary for the prolifer- Pepper et al., 2003; Hansen et al., to the 30 splice site (Wahl et al., 2009; ative fate.