Stem cell factor (SCF), erythropoietin (Epo), and GATA-1 play an essential role(s) in erythroid development. We examined how these proteins interact functionally in G1E cells, a GATA-1 ؊ erythroblast line that proliferates in an SCF-dependent fashion and, upon restoration of GATA-1 function, undergoes GATA-1 proliferation arrest and Epo-dependent terminal maturation. We show that SCF-induced cell cycle progression is mediated via activation of the Src kinase/c-Myc pathway. Restoration of GATA-1 activity induced G 1 cell cycle arrest coincident with repression of c-Kit and its downstream effectors Vav1, Rac1, and Akt. Sustained expression of each of these individual signaling components inhibited GATA-1-induced cell cycle arrest to various degrees but had no effects on the expression of GATA-1-regulated erythroid maturation markers. Chromatin immunoprecipitation analysis revealed that GATA-1 occupies a defined Kit gene regulatory element in vivo, suggesting a direct mechanism for gene repression. Hence, in addition to its well-established function as an activator of erythroid genes, GATA-1 also participates in a distinct genetic program that inhibits cell proliferation by repressing the expression of multiple components of the c-Kit signaling axis. Our findings reveal a novel aspect of molecular cross talk between essential transcriptional and cytokine signaling components of hematopoietic development.Receptor tyrosine kinases (RTKs) trigger a multitude of cellular events, including proliferation, survival, differentiation, and migration. These functions are modulated in hematopoietic stem and progenitor cells by the essential RTK c-Kit (8,11,43). The expression of c-Kit is downregulated as progenitors mature to their respective lineages, with the exception of mast cells, which rely on c-Kit for survival, proliferation, and function throughout their life span (20). Unrestrained c-Kit activity contributes to several neoplastic disorders, including gastrointestinal stromal tumors (GIST), mastocytosis, and leukemia (5,12,21,37,46,55). In GIST, somatic kinase-activating Kit mutations result in malignant transformation. In the hematopoietic system, similar activating Kit mutations occur in stem/ progenitor cells and mast cells, causing mastocytosis and acute myelogenous leukemia, respectively (45, 54).Mutant mice without c-Kit (dominant white spotting, or W, mutants) demonstrate severe deficiencies in erythroid development with reduced CFU-erythroid progenitors in the fetal liver and embryonic death from anemia at around day 16 of gestation (11, 57). Erythropoietin (Epo) receptor (Epo-R)-deficient mice demonstrate a similar decrease in CFU-erythroid progenitors and die of anemia between days 13 and 15 of gestation, suggesting that erythroid progenitors cannot survive, proliferate, or differentiate unless both the c-Kit and Epo-R signal transduction pathways are functional. Recent studies suggest that Epo and Epo-R interactions contribute to this process by preventing apoptosis through activation of the survival factor...

eWe have previously demonstrated that the KH-domain protein ␣CP binds to a 3= untranslated region (3=UTR) C-rich motif of the nascent human alpha-globin (h␣-globin) transcript and enhances the efficiency of 3= processing. Here we assess the genomewide impact of ␣CP RNA-protein (RNP) complexes on 3= processing with a specific focus on its role in alternative polyadenylation (APA) site utilization. The major isoforms of ␣CP were acutely depleted from a human hematopoietic cell line, and the impact on mRNA representation and poly(A) site utilization was determined by direct RNA sequencing (DRS). Bioinformatic analysis revealed 357 significant alterations in poly(A) site utilization that could be specifically linked to the ␣CP depletion. These APA events correlated strongly with the presence of C-rich sequences in close proximity to the impacted poly(A) addition sites. The most significant linkage was the presence of a C-rich motif within a window 30 to 40 bases 5= to poly(A) signals (AAU AAA) that were repressed upon ␣CP depletion. This linkage is consistent with a general role for ␣CPs as enhancers of 3= processing. These findings predict a role for ␣CPs in posttranscriptional control pathways that can alter the coding potential and/or levels of expression of subsets of mRNAs in the mammalian transcriptome.

G-proteins play critical roles in many cellular processes and are regulated by accessory proteins that modulate the nucleotide-bound state. Such proteins, including eukaryotic translation elongation factor 1A (eEF1A), are frequently reactivated by guanine nucleotide exchange factors (GEFs). In the yeast Saccharomyces cerevisiae, only the catalytic subunit of the GEF complex, eEF1Ba, is essential for viability. The requirement for the TEF5 gene encoding eEF1Ba can be suppressed by the presence of excess substrate, eEF1A. These cells, however, have defects in growth and translation. Two independent unbiased screens performed to dissect the cause of these phenotypes yielded dominant suppressors that bypass the requirement for extra eEF1A. Surprisingly, all mutations are in the G-protein eEF1A and cluster in its GTP-binding domain. Five mutants were used to construct novel strains expressing only the eEF1A mutant at normal levels. These strains show no growth defects and little to no decreases in total translation, which raises questions as to the evolutionary expression of GEF complexity and other potential functions of this complex. The location of the mutations on the eEF1A-eEF1Ba structure suggests that their mechanism of suppression may depend on effects on the conserved G-protein elements: the P-loop and NKXD nucleotide-binding element.

Post-transcriptional control of mRNA stability and translation is central to multiple developmental pathways. This control can be linked to cytoplasmic polyadenylation in certain settings. In maturing Xenopus oocytes, specific mRNAs are targeted for polyadenylation via recruitment of the Cytoplasmic Polyadenylation Element (CPE) binding protein (CPEB) to CPE(s) within the 39 UTR. Cytoplasmic polyadenylation is also critical to early embryonic events, although corresponding determinants are less defined. Here, we demonstrate that the Xenopus ortholog of the poly(rC) binding protein aCP2 can recruit cytoplasmic poly(A) polymerase activity to mRNAs in Xenopus post-fertilization embryos, and that this recruitment relies on cis sequences recognized by aCP2. We find that the ha-globin 39 UTR, a validated mammalian aCP2 target, constitutes an effective target for cytoplasmic polyadenylation in Xenopus embryos, but not during Xenopus oocyte maturation. We further demonstrate that the cytoplasmic polyadenylation activity is dependent on the action of the C-rich aCP-binding site in conjunction with the adjacent AAUAAA. Consistent with its ability to target mRNA for poly(A) addition, we find that XaCP2 associates with core components of the Xenopus cytoplasmic polyadenylation complex, including the cytoplasmic poly(A) polymerase XGLD2. Furthermore, we observe that the C-rich aCP-binding site can robustly enhance the activity of a weak canonical oocyte maturation CPE in early embryos, possibly via a direct interaction between XaCP2 and CPEB1. These studies establish XaCP2 as a novel cytoplasmic polyadenylation trans factor, indicate that C-rich sequences can function as noncanonical cytoplasmic polyadenylation elements, and expand our understanding of the complexities underlying cytoplasmic polyadenylation in specific developmental settings.

the vector by a pathogen, providing a theoretical framework for its impact on human health and disease. These and related findings demonstrate how relevant ecology and vector biology are in protecting human and animal health, especially for emerging zoonotic and vector-borne diseases. AcknowledgmentsThis work was supported in part by NIH/ NIAID R01 AI44102 and R21 AI080911.