Robert E. Rhoads - Böcker

This volumepresents detailed laboratory protocols for invitro synthesis of mRNA with favorable properties, its introduction intocells by a variety of techniques, and the measurement of physiological andclinical consequences such as protein replacement and cancer immunotherapy. Synthetictechniques are described for structural features in mRNA that provide investigationaltools such as fluorescence emission, click chemistry, photo-chemicalcrosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reducedactivation of the innate immune response. Protocols are described for clinicalapplications such as large-scale transfection of dendritic cells, production ofGMP-grade mRNA, redirecting T cell specificity, and use of molecularadjuvants for RNA vaccines. Writtenin the highly successful Methods inMolecular Biology series format, chapters include introductions to theirrespective topics, lists of the necessarymaterials and reagents, step-by-steplaboratory protocols, and tips on troubleshooting and avoiding known pitfalls.SyntheticmRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resourcefor both laboratory investigators and clinicians interested in this powerfuland rapidly evolving technology.

This volumepresents detailed laboratory protocols for invitro synthesis of mRNA with favorable properties, its introduction intocells by a variety of techniques, and the measurement of physiological andclinical consequences such as protein replacement and cancer immunotherapy.

This book deals with anabolic mechanisms which regulate eukaryotic protein synthesis, with particular emphasis on insulin and amino acids. Six chapters written by leading experts in the field provide both new data and comprehensive literature reviews. The regulation of both the eIF2 and eIF4 groups of initiation factors by signal transduction mechanisms is presented, and translation is related to cellular growth in response to nutrient and hormonal signals.

This volume presents the response of the eukaryotic translational apparatus to cellular stress and apoptosis, including kinases activated through both the ERK and stress-activated pathways. It further explores two agents that inhibit protein synthesis, calcium and the immunosuppressant rapamycin. Six chapters written by leading experts in the field provide both new data and comprehensive literature reviews. Both the regulation of initiation and elongation are discussed, and the mechanisms of apoptosis are related to changes in the protein synthesis machinery.

An odd and unexpected finding was reported by the laboratory of Richard Jorgensen in 1990: expression of extra copies of the gene encoding chalone synthase in petunias turned off the endogenous chalone synthase gene. An observation that appeared totally unrelated was made by the laboratory of Victor Ambrose in 1993: a gene in Caenorhabditis elegans, lin-4, controlled the timing of larval development but did not encode a protein. Rather, it expressed two small RNAs that were complementary to the 3'-untranslated region of the lin-14 gene in a region that had previously been shown to repress expression of the LIN-14 protein. From another quarter, David Baulcombe's laboratory showed in 1997 that plant viruses could induce sequen- specific gene silencing. Then in a landmark paper, Andrew Fire and Craig Mello showed in 1998 that double-stranded RNA (dsRNA) triggers a gene-silencing mechanism that they dubbed RNA interference (RNAi), for which discovery they were awarded the Nobel Prize in Physiology or Medicine in 2006. These diverse findings have triggered an explosion of research around the world in both plants and animals to discover the mechanisms and broader ramifications of RNAi.We now know that there are both exogenous pathways involving formation of siRNA when dsRNA is introduced and endogenous pathways involving miRNA, piwiRNA, and rasiRNAs. All pathways culminate in formation of an RNA-induced silencing complex (RISC) containing a member of the Argonaute protein family bound to a 22-nt RNA strand that interacts with a target mRNA or gene through Watson-Crick base pairing.

This volume presents the response of the eukaryotic translational apparatus to cellular stress and apoptosis, including kinases activated through both the ERK and stress-activated pathways. It further explores two agents that inhibit protein synthesis, calcium and the immunosuppressant rapamycin. Six chapters written by leading experts in the field provide both new data and comprehensive literature reviews. Both the regulation of initiation and elongation are discussed, and the mechanisms of apoptosis are related to changes in the protein synthesis machinery.

An odd and unexpected finding was reported by the laboratory of Richard Jorgensen in 1990: expression of extra copies of the gene encoding chalone synthase in petunias turned off the endogenous chalone synthase gene. An observation that appeared totally unrelated was made by the laboratory of Victor Ambrose in 1993: a gene in Caenorhabditis elegans, lin-4, controlled the timing of larval development but did not encode a protein. Rather, it expressed two small RNAs that were complementary to the 3'-untranslated region of the lin-14 gene in a region that had previously been shown to repress expression of the LIN-14 protein. From another quarter, David Baulcombe's laboratory showed in 1997 that plant viruses could induce sequen- specific gene silencing. Then in a landmark paper, Andrew Fire and Craig Mello showed in 1998 that double-stranded RNA (dsRNA) triggers a gene-silencing mechanism that they dubbed RNA interference (RNAi), for which discovery they were awarded the Nobel Prize in Physiology or Medicine in 2006. These diverse findings have triggered an explosion of research around the world in both plants and animals to discover the mechanisms and broader ramifications of RNAi.We now know that there are both exogenous pathways involving formation of siRNA when dsRNA is introduced and endogenous pathways involving miRNA, piwiRNA, and rasiRNAs. All pathways culminate in formation of an RNA-induced silencing complex (RISC) containing a member of the Argonaute protein family bound to a 22-nt RNA strand that interacts with a target mRNA or gene through Watson-Crick base pairing.

The articles in the present volume are by major contributors to our under­ standing of signaling pathways affecting protein synthesis. They focus pri­ marily on two extracellular anabolic signals, although others are included as well. Insulin is one of the best-studied extracellular regulators of protein syn­ thesis. Several of the known pathways for regulation of protein synthesis were elucidated using insulin-dependent systems. Regulation of protein synthesis by amino acids, by contrast, is an emerging field that has recently received a great deal of attention. The dual role of amino acids as substrates for protein syn­ thesis and regulators of the overall process has only recently been recognized. Since amino acids serve as precursors for proteins, one might expect that with­ holding an essential amino acid would inhibit the elongation phase. Surpris­ ingly, research has shown that it is the initiation phase of protein synthesis that is restricted during amino acid starvation. Understanding the mechanisms by which the biosynthesis of proteins is reg­ ulated is important for several reasons. Protein synthesis consumes a major portion of the cellular ATP that is generated. Therefore, small changes in protein synthesis can have great consequences for cellular energy metabolism. Translation is also a major site for control of gene expression, since messenger RNAs differ widely in translational efficiency, and changes to the protein syn­ thesis machinery can differentially affect recruitment of individual mRNAs.