PUM1 and PUM2 exhibit different modes of regulation for SIAH1 that involve cooperativity with NANOS paralogues.

Pumilio (PUM) proteins are RNA-binding proteins that posttranscriptionally regulate gene expression in many organisms. Their PUF domain recognizes specific PUM-binding elements (PBE) in the 3' untranslated region of target mRNAs while engaging protein cofactors such as NANOS that repress the expression of target mRNAs through the recruitment of effector complexes. Although the general process whereby PUM recognizes individual mRNAs has been studied extensively, the particulars of the mechanism underlying PUM-NANOS cooperation in mRNA regulation and the functional overlap among PUM and NANOS paralogues in mammals have not been elucidated. Here, using the novel PUM1 and PUM2 mRNA target SIAH1 as a model, we show mechanistic differences between PUM1 and PUM2 and between NANOS1, 2, and 3 paralogues in the regulation of SIAH1. Specifically, unlike PUM2, PUM1 exhibited PBE-independent repression of SIAH1 3'UTR-dependent luciferase expression. Concordantly, the PUF domains of PUM1 and PUM2 showed different EMSA complex formation patterns with SIAH1 3'UTRs. Importantly, we show direct binding of NANOS3, but not NANOS2, to SIAH1 3'UTR, which did not require PBEs or the PUF domain. To the best of our knowledge, this is the first report, showing that an NANOS protein directly binds RNA. Finally, using NANOS1 and NANOS3 constructs carrying mutations identified in infertile patients, we show that these mutations disrupt repression of the SIAH1-luciferase reporter and that the central region in NANOS1 appears to contribute to the regulation of SIAH1. Our findings highlight the mechanistic versatility of the PUM/NANOS machinery in mammalian posttranscriptional regulation.

Human fertility protein PUMILIO2 interacts in vitro with testis mRNA encoding Cdc42 effector 3 (CEP3).

PUMILIO protein regulates translation of specific mRNAs in morphogenesis and in development of the germ-line of model organisms such as flies and worms. Given that a human homologue (PUMILIO2) was recently identified in the germ-line stem cells, the question was raised whether it regulates translation of fertility mRNAs similarly to Drosophila Pumilio. Here, we describe a candidate mRNA encoding Cdc42 effector protein 3 (CEP3), however, a function for this protein in reproduction has previously not been reported. We detected three CEP3 transcripts in the testis tissue including one which was highly expressed and testis specific by northern blotting. We found that CEP mRNA contains GUUGU (A) and AUUGUA (B) motifs (ABB) within the 3' untranslated region (3'UTR), which are also present in mRNA targets of Pumilio in Drosophila. Interaction of PUMILIO2 with the fragment of CEP3 transcript containing the ABB array was tested by mobility shift assay and we found that PUMILIO2 binds the 3' untranslated region of the CEP3 mRNA. These results support the hypothesis that CEP3 mRNA may be a target of PUMILIO2 protein in the human male gonad and be under translational control mediated by specific nucleotide motifs within the 3'UTR.

Candidate mRNAs interacting with fertility protein PUMILIO2 in the human germ line.

Pumilio protein regulates translation of specific mRNAs in morphogenesis and germ-line development of the flies by binding nucleotide motifs GUUGU (A) and AUUGUA (B) in 3'untranslated regions. A human homologue, PUMILIO2 has been recently identified in the germ-line stem cells and the question was raised whether it regulates translation. We designed software to screen the GeneBank for A and B motifs and found that they are not uncommon in the human genome. Moreover, some of the genes containing motifs A and B are germ cell specific, but some others are expressed in a number of other tissues. This may indicate that PUMILIO mediated translational regulation is universally used in developmental processes of the human body.