No abstract

Posttranscriptional regulation plays a fundamental role in the biology of embryonic stem cells (ESCs). Many studies have demonstrated that multiple mRNAs are coregulated by one or more RNA-binding proteins (RBPs) that orchestrate mRNA expression. A family of RBPs, which is known as the Pumilio-FBF (PUF) family, is highly conserved among different species and has been associated with the undifferentiated and differentiated states of different cell lines. In humans, two homologs of the PUF family have been found: Pumilio 1 (PUM1) and Pumilio 2 (PUM2). To understand the role of these proteins in human ESCs (hESCs), we first assessed the influence of the silencing of PUM1 and PUM2 on pluripotency genes and found that the knockdown of Pumilio genes significantly decreased the OCT4 and NANOG mRNA levels and reduced the amount of nuclear OCT4, which suggests that Pumilio proteins play a role in the maintenance of pluripotency in hESCs. Furthermore, we observed that PUM1-and-PUM2-silenced hESCs exhibited improved efficiency of in vitro cardiomyogenic differentiation. Through an in silico analysis, we identified mRNA targets of PUM1 and PUM2 that are expressed at the early stages of cardiomyogenesis, and further investigation will determine whether these target mRNAs are active and involved in the progression of cardiomyogenesis. Our findings contribute to the understanding of the role of Pumilio proteins in hESC maintenance and differentiation.

19Posttranscriptional regulation plays a fundamental role in the biology of embryonic stem 20 cells (ESCs). Many studies have demonstrated that multiple mRNAs are coregulated by 21 one or more RNA binding proteins (RBPs) that orchestrate the expression of these 22 molecules. A family of RBPs, known as PUF (Pumilio-FBF), is highly conserved among 23 species and has been associated with the undifferentiated and differentiated states of 24 different cell lines. In humans, two homologs of the PUF family have been found: 25 Pumilio 1 (PUM1) and Pumilio 2 (PUM2). To understand the role of these proteins in 26 human ESCs (hESCs), we first demonstrated the influence of the silencing of PUM1 and 27 PUM2 on pluripotency genes. OCT4 and NANOG mRNA levels decreased significantly 28 with the knockdown of Pumilio, suggesting that PUMILIO proteins play a role in the 29 maintenance of pluripotency in hESCs. Furthermore, we observed that the hESCs 30 silenced for PUM1 and 2 exhibited an improvement in efficiency of in vitro 31 cardiomyogenic differentiation. Using in silico analysis, we identified mRNA targets of 32 PUM1 and PUM2 expressed during cardiomyogenesis. With the reduction of PUM1 and 33 2, these target mRNAs would be active and could be involved in the progression of 34 cardiomyogenesis.35 36 Keywords: human embryonic stem cells, Pumilio 1, Pumilio 2, cardiomyogenesis, 37 RNA-binding proteins.38 39 2 40 42inner cell mass of the blastocyst that have potential for differentiation into three germ 43 layers (1-3). In an undifferentiated state, hESCs are characterized by the expression of 44 stemness factors such as OCT4 (POU5F1), SOX2 and NANOG (4). These three 45 transcription factors, which are positively regulated, are responsible for pluripotency 46 maintenance and contribute to the repression of lineage-specific genes (reviewed by 5). 47When hESCs are stimulated to initiate the differentiation process, expression of genes 48 associated with pluripotency is negatively regulated and genes associated with the germ 49 layer begin to be positively regulated (6). 50A complex network of gene expression underlies the molecular signaling that 51 will give rise to the adult heart. Cardiomyogenic differentiation is a highly regulated 52 process that depends on the fine regulation of gene expression (7). In vitro 53 cardiomyogenic differentiation of hESCs mimics embryonic development and can be 54 used as a model for cardiac development studies per se and as a model for research 55 ranging from tissue electrophysiology to drug screening (reviewed by 8). 56 RNA-binding proteins (RBPs) are proteins that have RNA-binding domains and 57 form ribonucleoprotein complexes in association with RNAs (RNPs). These proteins 58 play a critical role in the posttranscriptional regulation of gene expression. The 59 dynamics and function of these complexes depend on their composition, targets and 60 cofactors (9). The PUF (Pumilio-FBF) family of RBPs is highly conserved among 61 species and is found in plants, insects, nematodes and mammals...