Human Pumilio-2 is expressed in embryonic stem cells and germ cells and interacts with DAZ (Deleted in AZoospermia) and DAZ-like proteins

Abstract

Early in development, a part of the embryo is set aside to become the germ cell lineage that will ultimately differentiate to form sperm and eggs and transmit genetic information to the next generation. Men with deletions encompassing the Y-chromosome DAZ genes have few or no germ cells but are otherwise healthy, indicating they harbor specific defects in formation or maintenance of germ cells. A DAZ homolog, DAZL (DAZ-Like), is found in diverse organisms, including humans and is required for germ cell development in males and/or females. We identified proteins that interact with DAZ proteins to better understand their function in human germ cells. Here, we show that PUM2, a human homolog of Pumilio, a protein required to maintain germ line stem cells in Drosophila and Caenorhabditis elegans, forms a stable complex with DAZ through the same functional domain required for RNA binding, protein-protein interactions and rescue of Pumilio mutations in flies. We also show that PUM2 is expressed predominantly in human embryonic stem cells and germ cells and colocalizes with DAZ and DAZL in germ cells. These data implicate PUM2 as a component of conserved cellular machinery that may be required for germ cell development.

Figure 1

Alignment of RNA-binding domains of Pumilio. Sequences are from human (hPUM1 and hPUM2), mouse (mPum1 and mPum2), frog (XPum), and flies (DPum).

Figure 2

Interaction of PUM2 and DAZ proteins. (A) The DAZ protein was coimmunoprecipitated with PUM2 when HA beads were incubated with supernatant from yeast that expressed both DAZ and PUM2 fused to an HA tag; shown is DAZ protein detected by Western analysis (Left). DAZ protein was not coimmunoprecipitated with HA beads from yeast that expressed DAZ or PUM2 alone. Right lane shows yeast supernatant that expressed DAZ as a positive control. (Center) PUM2 protein coimmunoprecipitated with DAZ protein from primate COS-7 cells. Western analysis showed that PUM2 protein formed a complex with S-tag:DAZ protein captured from COS-7 cells that expressed PUM2 and DAZ. PUM2 protein was not coimmunoprecipitated from supernatant of COS-7 cells when S-beads were incubated with supernatants from cells with PUM2 or DAZ alone. Cell supernatant that expressed PUM2 was included as a positive control (right-most lane in Center). PUM2 protein was coimmunoprecipitated with DAZ from testis extract (Right). Western analysis showed that PUM2 proteins formed a complex with S-tag:DAZ protein when incubated in mouse testis extract. No PUM2 was detected when S-beads were incubated in mouse testis extract that did not contain the S-tag:DAZ fusion protein. Mouse testis extract was included as a positive control (right-most lane in Right). (B) Deletions of the PUM2 protein defined minimal domain of PUM2 required for interaction with DAZ. Eight deletions of PUM2:GAL4 protein were assayed for interaction with intact DAZ in the yeast two-hybrid system. Positions of amino acids on PUM2 constructs are as in GenBank accession no. AF272350. (C) Eleven deletions of DAZ defined minimal region required for PUM2 interaction via the yeast two-hybrid assay. Several constructs truncated the protein near the RRM domain (RNP-2 and -1) and interacted only weakly with PUM2, even though they contain the minimal interaction region. This weak interaction may have been caused by improper folding of DAZ due to disruption of RRM structure. +++, Maximal activity equivalent to that of intact interacting proteins; −, no interaction; ±, weak interaction; +, strong interaction; ++, nearly wild-type interaction.

Figure 3

Expression of human PUMILIO mRNAs. (A) Northern analysis of adult tissues indicated PUM2 expression in ovary and testis. Two transcripts of ≈5.5 and 6.5 kb were detected. (B) RT-PCR expression analysis of PUM1 and PUM2 in ES cells, fetal ovary and testis, adult brain and heart, and adult ovary and testis. (C) RT-PCR expression analysis of PUM1 and PUM2 in various fetal tissues and from men with normal germ cells (GCs) or no germ cells present in their testis. Germ cell number varied between biopsies.

Figure 4

PUM2 expression mirrored that of DAZ and DAZL. (A) Western analysis with PUM2 antisera (lanes 1–3), PUM2 preimmune antisera (lanes 4–6), and PUM2 antisera preincubated with PUM2 protein competitor (lanes 7–9). Cell extracts were from human testis (lanes 1, 4, and 7), mouse testis (lanes 2, 5, and 8), and mouse ES cells (lanes 3, 6, and 9). The major PUM2 protein band was ≈95 kDa in extracts from human testis, mouse testis, and mouse ES cells. Antisera specific to both DAZ and DAZL proteins (4) were used to stain human sections of testis (B), ovary (C), and fetal testis (D). Antisera that recognized a PUM2-specific epitope were used to stain human sections of testis (E) (Inset is a magnified cross section of stained tissue), ovary (F), and fetal testis (G). Preimmune sera were used to stain human sections of adult testis (H), adult ovary (I), and fetal testis (J). Sections B–J were counterstained with hematoxylin/eosin and stained for specific proteins with horseradish peroxidase-conjugated secondary antibodies (brown staining). Magnification: ×200. Spg, spermatogonial cells; Spc, spermatocytes; Spd, spermatids; SC, somatic cells; Oc, oocyte; RS, round spermatid.

Figure 5

Expression of Pum2 in mouse. Pum2 expression was observed in adult ovary (A) and adult testis (B) (Inset is a magnified cross section of stained tissue). No staining was observed with PUM2 preimmune sera on adult ovary (C) or testis (D). The pattern of PUM2 expression was also compared with that of mouse Boule (25). Boule protein was not present in adult ovary (E) and demonstrates a meiosis-specific expression pattern in adult testis (F). A–F were counterstained with hematoxylin/eosin and stained for specific proteins with horseradish peroxidase-conjugated secondary antibodies (brown staining). Magnification: ×200. Abbreviations are as in Fig. 4.

Figure 6

Interaction of PUM2, DAZL, and NRE RNA. (A) Schematic of components used to detect PUM2/NRE interaction. Positive RNA–protein interaction was detected when the NRE and PUM2:AD were present as assayed by 5bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal) assay (Right). No interaction was detected with GAL4 activation domain (AD) or PUM2:AD without the NRE or with NRE and AD alone. (B) Schematic of components used to detect NRE/PUM2/DAZL complex. A ternary complex was formed with NRE, PUM2, and DAZL:AD present (Left). No interaction was detected with the NRE and the AD or DAZL:AD alone or with the NRE with PUM2 and the AD alone. No interaction was detected in the absence of the NRE, or with PUM2 and DAZL:AD present. Positive control interaction of the NRE with PUM2:AD is shown.