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Abstract Introduction The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. Methods We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. Results We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. Conclusions For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.

The 5'→3' exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with crucial functions in RNA metabolism and RNA interference (1-5). XRN2, or Rat1 in yeast (6), functions primarily in the nucleus and also has an important role in transcription termination by RNA polymerase II (refs 7-14). Rat1 exoribonuclease activity is stimulated by the protein Rai1 (refs 15, 16). Here we report the crystal structure at 2.2 Å resolution of Schizosaccharomyces pombe Rat1 in complex with Rai1, as well as the structures of Rai1 and its murine homologue Dom3Z alone at 2.0 Å resolution. The structures reveal the molecular mechanism for the activation of Rat1 by Rai1 and for the exclusive exoribonuclease activity of Rat1. Biochemical studies confirm these observations, and show that Rai1 allows Rat1 to degrade RNAs with stable secondary structure more effectively. There are large differences in the active site landscape of Rat1 compared to related and PIN (PilT N terminus) domain-containing nucleases (17-20). Unexpectedly, we identified a large pocket in Rai1 and Dom3Z that contains highly conserved residues, including three acidic side chains that coordinate a divalent cation. Mutagenesis and biochemical studies demonstrate that Rai1 possesses pyrophosphohydrolase activity towards 5' triphosphorylated RNA. Such an activity is important for messenger RNA degradation in bacteria (21), but this is, to our knowledge, the first demonstration of this activity in eukaryotes and suggests that Rai1/Dom3Z may have additional important functions in RNA metabolism., The XRNs share two regions of sequence conservation, corresponding to residues 1-390 and 590-751 in S. pombe Rat1 (22) (Fig. 1a and Supplementary Fig. 1). The segment between them and [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.07.033 Byline: Ming-Der Lin (a), Xinfu Jiao (b), Dominic Grima (c), Sarah F. Newbury (c), Megerditch Kiledjian (b), Tze-Bin Chou (a) Keywords: Processing bodies; Drosophila decapping protein 1; Drosophila decapping protein 2; Pacman; Me31B; Oogenesis Abstract: Processing bodies (P-bodies) have emerged as important subcellular structures that are involved in mRNA metabolism. To date, a detailed description of P-bodies in Drosophila oogenesis is lacking. To this end, we first demonstrate that Drosophila decapping protein 2 (dDcp2) contains intrinsic decapping activity and its enzymatic activity was not detectably enhanced by Drosophila decapping protein 1 (dDcp1). dDcp1-containing bodies in the nurse cell cytoplasm can associate with the 5' to 3' exoribonuclease, Pacman in addition to dDcp2 and Me31B. The size and number of dDcp1 bodies are dynamic and dramatically increased in dDcp2 and pacman mutant backgrounds supporting the conclusion that dDcp1 bodies in nurse cell cytoplasm are Drosophila P-bodies. In stage 2-6 oocytes, dDcp1 bodies appear to be distinct from previously characterized P-bodies since they are insensitive to cycloheximide and RNase A treatments. Curiously, dDcp2 and Pacman do not colocalize with dDcp1 at the posterior end of the oocyte in stage 9-10 oocytes. This suggests that dDcp1 bodies are in a developmentally distinct state separate from the 5' end mRNA degradation enzymes at later stages in the oocyte. Interestingly, re-formation of maternally expressed dDcp1 with dDcp2 and Pacman was observed in early embryogenesis. With respect to developmental switching, the maternal dDcp1 is proposed to serve as a marker for the re-formation of P-bodies in early embryos. This also suggests that a regulated conversion occurs between maternal RNA granules and P-bodies from oogenesis to embryogenesis. Author Affiliation: (a) Institute of Molecular and Cellular Biology, College of Life Science, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan (b) Department of Cell Biology and Neuroscience, Rutgers University, 604 Allison Road, Piscataway, NJ 08854-8082, USA (c) Brighton and Sussex Medical School, University of Sussex, Falmer, Brighton, BN1 9QG, UK Article History: Received 27 February 2008; Revised 14 July 2008; Accepted 15 July 2008

Research has been conducted on the mammalian mRNA turnover. Results indicate that this turnover can proceed via the coupled 3' to 5' exoribonuclease degradation-dependent decapping pathway.

Two Distinct Transcription Factors That Bind the Immunoglobulin Enhancer μE5/κE2 Motif THE IMMUNOGLOBULIN HEAVY chain (IgH) enhancer activates transcription of rearranged heavy chain genes [1]. In transfection experiments it stimulates [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.11.035 Byline: Vincent Shen (1), Megerditch Kiledjian (1) Abstract: The exosome is a 3' to 5' exoribonuclease central to many cellular processes, including mRNA decay. now present the biochemical reconstitution and crystal structure of the eukaryotic exosome. This remarkable achievement provides key insights into the composition and assembly of the human and yeast exosomes, revealing functions of individual subunits. Author Affiliation: (1) Rutgers University, Department of Cell Biology and Neuroscience, 604 Allison Road, Piscataway, NJ 08854, USA