Phillip A. Sharp

Bio: Phillip A. Sharp is an academic researcher from Massachusetts Institute of Technology. The author has contributed to research in topics: RNA & RNA splicing. The author has an hindex of 172, co-authored 614 publications receiving 117126 citations. Previous affiliations of Phillip A. Sharp include McGovern Institute for Brain Research & Medical Research Council.

Harnessing RNA interference for therapy: the silent treatment.

Abstract: RNA interference (RNAi) is a ubiquitous pathway that regulates gene expression. It uses small, imperfectly paired, double-stranded RNAs approximately 21 nucleotides long, called microRNAs, that are processed from longer stem-loop transcripts.1 MicroRNAs are taken up by the cytoplasmic RNA-induced silencing complex (RISC), which removes 1 strand, leaving an unpaired strand that binds to messenger RNAs (mRNAs) with a partially complementary sequence. RISC suppresses the expression of bound mRNAs by accelerating their degradation and suppressing their translation into protein. MicroRNAs and the RNAi gene-silencing pathway were first discovered in the 1990s in plants, worms, and flies. In those organisms, microRNAs play an important role in regulating changes in gene expression that occur in development and in protection from viruses. In 2001, Elbashir et al2 discovered that the RNA interference (RNAi) pathway could be directly accessed in mammals. They showed that transfection of small doublestranded RNAs (called small interfering RNAs [siRNAs]), which are exactly complementary to sequences of a cellular mRNA, cause the targeted mRNA to be degraded— selectively knocking down or silencing the expression of the gene. These siRNAs enter the microRNA pathway by binding to the RISC, which directs cleavage of the target mRNA. This discovery led to the development of therapies designed to knock down disease-causing genes. Researchers quickly demonstrated that knocking down HIV genesorHIVreceptorscouldinhibitHIVreplicationinvitro, and injection of siRNAs targeting the Fas cell surface death receptor (FAS [NCBI Entrez Gene 14 102]) could protect mice from lethal hepatitis.3 In principle, RNAi could be harnessed to knock down any mRNA, expanding the universe of drug targets beyond the enzymes and receptors targeted by conventional small molecules. Although gene knockdown was not as specific as initially thought, because of incremental silencing of genes possessing partially complementary sequences and stimulation of innate immune viral RNA receptors that induce interferons (and indeed the promising clinical results of early siRNA trials were probably due to off-target interferons), soon chemical modifications of siRNAs were identified. These chemical modifications virtually eliminated these off-target effects without sacrificing on-target silencing of the intended target gene. Taken together, these results spurred some to hail siRNAs as the next new class of drugs. Very quickly hundreds of biotech companies were started and major pharmaceutical companies prepared to develop RNAi-based drugs. However, enthusiasm waned when delivering small RNAs into the cytoplasm of cells to the RISC proved difficult. There are 2 bottlenecks to delivering negatively charged RNAs into cells—crossing the plasma membrane and, once in the cell, getting out of endosomes. These barriers also plague development of other small oligonucleotide drugs that use other mechanisms, such as antisense inhibition or inhibition of splicing, to modulate gene expression. However, siRNAs have advantages over other antisense oligonucleotide strategies. Once taken up into the RISC, the active strand of the siRNA is protected from digestionbycellularRNA-degradingenzymes(RNases)and is typically stable and active in the cell for weeks. More importantly, the same siRNA complex catalytically cleaves multiple mRNAs. Because of the catalytic nature of RNAi, it is estimated that less than a few hundred siRNAs per cell are needed to silence a target gene. Because the liver is the main blood-filtering organ that separates useful metabolites from harmful toxins and particulates, it is easier to deliver RNAi-based therapeutics to liver cells than to other internal organs. Accordingly, development of siRNA drugs has mostly focused on targets in the liver. Since the liver is also the body’s major producer of many metabolites and secreted proteins, gene knockdown in the liver can potentially be used to treat many diseases. These include rare genetic diseases caused by mutations of genes encoding proteins produced in the liver, such as enzymes needed for heme biosynthesis, cholesterol metabolism, clotting proteins and complement, common metabolic syndromes in which proteins synthesized in the liver play a critical role, and diseases primarily involving the liver such as infectious hepatitis and nonalcoholic fatty liver. Lipid nanoparticles (LNPs), incorporating cationic lipids that bind negatively charged RNAs, lodge in the liver where they are taken up by hepatocytes to knock down gene expression. The earliest clinical trials targeting the liver used first-generation LNPs that had a low therapeutic index. By modifying their lipid composition and the chemistry of the RNAs they carry, currentgeneration LNPs are able to knock down expression of a target gene transthyretin (mutation of this gene causes amyloidosis) in the liver by 80% to 95% for 1 month— without any serious adverse events.4 Currently transthyretin-targeting LNPs (patisiran, 0.3 mg/kg given every 3 weeks) are in phase 3 studies for familial amyloidotic polyneuropathy, a progressive fatal disease. An openlabel extension study of patisiran showed an encouraging slight improvement in neurological score in patients with this disorder when compared with the decline expected (based on historical control patients). Another strategy for hepatocyte delivery uses siRNAs conjugated at one end with a polyvalent sugar VIEWPOINT

Iron elevates mesenchymal and metastatic biomarkers in HepG2 cells.

Abstract: Liver iron excess is observed in several chronic liver diseases and is associated with the development of hepatocellular carcinoma (HCC). However, apart from oxidative stress, other cellular mechanisms by which excess iron may mediate/increase HCC predisposition/progression are not known. HCC pathology involves epithelial to mesenchymal transition (EMT), the basis of cancer phenotype acquisition. Here, the effect of excess iron (holo-transferrin 0-2 g/L for 24 and 48 h) on EMT biomarkers in the liver-derived HepG2 cells was investigated. Holo-transferrin substantially increased intracellular iron. Unexpectedly, mRNA and protein expression of the epithelial marker E-cadherin either remained unaltered or increased. The mRNA and protein levels of metastasis marker N-cadherin and mesenchymal marker vimentin increased significantly. While the mRNA expression of EMT transcription factors SNAI1 and SNAI2 increased and decreased, respectively after 24 h, both factors increased after 48 h. The mRNA expression of TGF-β (EMT-inducer) showed no significant alterations. In conclusion, data showed direct link between iron and EMT. Iron elevated mesenchymal and metastatic biomarkers in HepG2 cells without concomitant decrement in the epithelial marker E-cadherin and altered the expression of the key EMT-mediating transcription factors. Such studies can help identify molecular targets to devise iron-related adjunctive therapies to ameliorate HCC pathophysiology.

tRNAs Marked with CCACCA Are Targeted for Degradation

Abstract: Transfer RNAs with unstable acceptor stems can be tagged by CCA-adding enzymes and targeted for destruction by 3′-5′ exonucleases. The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] adds CCA to the 3′ ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking structurally unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3′ ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. In addition, hypomodified mature tRNAs are subjected to CCACCA addition as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addition is a universal mechanism for controlling tRNA levels and preventing errors in translation.

A Novel 50-Kilodalton Fragment of Host Cell Factor 1 (C1) in G0 Cells

Abstract: Upon herpes simplex virus (HSV) infection, the viral protein VP16 (also called αTIF and vmw65) is released from the virion particle and forms a complex with two cellular proteins, Oct-1 and host cell factor 1 (HCF-1; also called C1 and VCAF) (2, 7, 16, 19). This complex, termed the C1 complex, directs specific transcription from the alpha/immediate-early (α/IE) promoter element of the viral genome (8). Oct-1 is a member of the POU domain family of proteins and normally regulates transcription from octamer and related elements (5′-ATGCAAAT-3′) found in the promoters of a diverse array of cellular genes (5, 21). In contrast, the complex of Oct-1 and VP16 has specificity for the viral α gene promoters, with Oct-1 contacting the 5′ half of the α/IE promoter element (5′-ATGCTAAT-3′), and VP16 making contacts in the 3′ half (8). HCF-1 stabilizes the interaction between VP16 and Oct-1 and may also contact the DNA (8, 23). HCF-1 consists of an array of polypeptides, varying in length from 100 to 230 kDa (6, 9, 23). All of the observed peptides are derived from the proteolysis of a single 2,035-amino-acid, 230-kDa protein (6, 23). After translation, HCF-1 is imported into the nucleus, where it is cleaved at one or more of the six near-perfect 26-amino-acid repeats found near the center of the protein. The cleavage is specific and occurs between a glutamic acid and a threonine (6, 25). Neither this 26-amino-acid sequence nor a related motif has been identified as a cleavage site in other proteins, but this repeat can direct the cleavage of a heterologous protein when inserted into its coding sequence (25). After cleavage, the N and C termini of HCF-1 remain strongly associated (6, 25). The N terminus of HCF-1 contains a series of six kelch repeats (named after the Drosophila egg chamber protein kelch, in which they were originally recognized). These repeats are found in other unrelated proteins and are predicted to fold into a barrel-like β-propeller structure (22). The first 360 amino acids of HCF-1 (HCF1–360), encompassing the kelch repeat region, are sufficient for binding to VP16 and for formation of a fast-mobility C1-like complex in vitro (12, 22). The cellular protein luman, or LZIP, a member of the basic leucine zipper family of DNA-binding proteins, has also been shown to bind HCF-1 in this region (1, 13). VP16 and luman share a short region of amino acid homology that, when mutated, greatly reduces both proteins' ability to bind HCF-1 (1, 14). A proline-to-serine mutation in the third kelch repeat (P134S) confers a temperature-sensitive phenotype on HCF-1. BHK cells carrying this mutation in their only copy of HCF-1 arrest in a G0-like state. These cells divide for 36 h after a shift to the nonpermissive temperature, then arrest and remain highly viable for several days. If they are shifted back to the permissive temperature during this time, they will reenter the G1 phase of the cell (3). This suggests that a function of HCF-1 is required for cells to cycle. Consistent with this hypothesis, HCF-1 is most abundant in cycling cells, including fetal tissue and immortalized cell lines (6, 24). It has therefore been proposed that VP16 interacts with HCF-1 to allow the infecting virus to sense the proliferative state of the host cell (3). At the nonpermissive temperature, the expression levels and proteolytic profiles of the P134S mutant HCF-1 are identical to those of the wild-type protein, and the mutant N terminus is still able to associate with the cleaved C terminus. However, mutant HCF-1 is unable to support VP16-mediated transcription from an α/IE reporter element in vivo and does not support C1 complex formation in vitro (3, 22). Nearly the entire N terminus of HCF-1 (HCF1–902) is required for complementation of the temperature-sensitive phenotype; deletion of as few as 66 additional amino acids from the C terminus of this fragment renders it unable to prevent G0 arrest at the nonpermissive temperature (22). The role of HCF-1 in controlling HSV entry into either a replicative or latent state is not known. The amount of VP16 that enters the nucleus may influence the balance toward one type of infection or the other (20). La Boissiere et al. recently reported that VP16 entry into the nucleus depends on the HCF-1 nuclear localization signal (NLS), which is found within the C terminus of HCF-1. When the NLS was deleted, transfected HCF-1 remained in the cytoplasm, along with a large portion of the cotransfected VP16 (11). In extracts from dorsal root ganglia, a cell type in which HSV is able to establish latency, a faster-mobility form of the C1 complex has been observed, suggesting that an altered form of HCF-1 may be present (4). Interestingly, immunofluorescent staining of HCF-1 in trigeminal ganglia shows the protein to be sequestered in cytoplasmic granules, and conditions which activate HSV from latency rapidly induce the appearance of HCF-1 in the nucleus of these cells (10). We have investigated the structure of HCF-1 in primary cells and describe a truncated form of HCF-1 that is present in resting, or G0, cells. This N-terminal fragment is able to bind VP16 and is present in cytoplasmic extracts from these cells.

Initial sequencing and analysis of the human genome.

Abstract: The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Abstract: A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.