This volume presents detailed laboratory protocols for in vitro synthesis of mRNA with favorable properties, its introduction into cells by a variety of techniques, and the measurement of physiological and clinical consequences such as protein replacement and cancer immunotherapy. Synthetic techniques are described for structural features in mRNA that provide investigational tools such as fluorescence emission, click chemistry, photo-chemical crosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reduced activation of the innate immune response. Protocols are described for clinical applications such as large-scale transfection of dendritic cells, production of GMP-grade mRNA, redirecting T cell specificity, and use of molecular adjuvants for RNA vaccines. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resource for both laboratory investigators and clinicians interested in this powerful and rapidly evolving technology.

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.

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."

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 publication of this book has required the cooperation of many people along the way. From its very conception, the project of bringing together experiences from ongoing Farming Systems Research projects has faced a problem of communication due to the dispersal of the participants. Dr. William Partridge and Lynne Goldstein were instrumental in the initial presentation of the symposium on Social Science participation in Farming Systems Research at the 83rd Annual Meeting of the American Anthropological Association. Ben Wallace has done an admirable job not only as editor but 8s a ilpoint m-an"- throughout the process or organizirig the conference and preparing the manuscript. He deserves credit tor expediting countless actMties that could never have otherwise been accomplished because of the vagaries or international mails and telecommunications.

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."

A diversity of stressful conditions cause rapid and severe inhibition of protein synthesis in eukaryotic ceIls, in some cases resulting in cell death by apopto sis (programmed cell death). Apoptosis has come to be recognised as an impor tant physiological process in the regulation of growth, development and differentiation. Until recently, relatively little attention had been paid to the changes in protein synthesis during the various phases of apoptosis. The arti des in the present volume are by major contributors to our understanding of signaling pathways that result in the inhibition of protein synthesis. These authors trace the downstream consequences of such stress conditions as virus infection, heat shock, nutrient starvation, release of intracellular calcium ions, and treatment with the immunosuppressant rapamycin. Understanding the mechanisms by which the biosynthesis of pro teins is regulated is important for several reasons. Pro tein synthesis consumes a major portion of the cellular ATP that is generated. Therefore, small changes in pro tein 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."

The publication of this book has required the cooperation of many people along the way. From its very conception, the project of bringing together experiences from ongoing Farming Systems Research projects has faced a problem of communication due to the dispersal of the participants. Dr. William Partridge and Lynne Goldstein were instrumental in the initial presentation of the symposium on Social Science participation in Farming Systems Research at the 83rd Annual Meeting of the American Anthropological Association. Ben Wallace has done an admirable job not only as editor but 8s a ilpoint m-an"- throughout the process or organizirig the conference and preparing the manuscript. He deserves credit tor expediting countless actMties that could never have otherwise been accomplished because of the vagaries or international mails and telecommunications.

This volume presents detailed laboratory protocols for in vitro synthesis of mRNA with favorable properties, its introduction into cells by a variety of techniques, and the measurement of physiological and clinical consequences such as protein replacement and cancer immunotherapy. Synthetic techniques are described for structural features in mRNA that provide investigational tools such as fluorescence emission, click chemistry, photo-chemical crosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reduced activation of the innate immune response. Protocols are described for clinical applications such as large-scale transfection of dendritic cells, production of GMP-grade mRNA, redirecting T cell specificity, and use of molecular adjuvants for RNA vaccines. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resource for both laboratory investigators and clinicians interested in this powerful and rapidly evolving technology.