Showing papers by "Phillip A. Sharp published in 1999"

Targeted mRNA degradation by double-stranded RNA in vitro

Abstract: Double-stranded RNA (dsRNA) directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. The biochemical mechanisms underlying this dsRNA interference (RNAi) are unknown. Here we report the development of a cell-free system from syncytial blastoderm Drosophila embryos that recapitulates many of the features of RNAi. The interference observed in this reaction is sequence specific, is promoted by dsRNA but not single-stranded RNA, functions by specific mRNA degradation, and requires a minimum length of dsRNA. Furthermore, preincubation of dsRNA potentiates its activity. These results demonstrate that RNAi can be mediated by sequence-specific processes in soluble reactions.

20 Splicing of Precursors to mRNAs by the Spliceosomes

Abstract: The splicing of precursors to mRNAs occurs in two steps, both involving single transesterification reactions (Fig. 1). The first step generates a 2′ – 5′ bond at the branch site upstream of the 3′ splice site and a free 3′ hydroxyl group on the 5′ exon. The resulting lariat RNA intermediate, with its slow migration in gels, is the most common assay for splicing in vitro. In the second step, attack of the 3′ hydroxyl on the phosphodiester bond at the 3′ splice site displaces the lariat intron with a 3′ hydroxyl group and results in joining of the two exons. The bimolecular nature of the intermediate in splicing indicated that the reaction must occur within a stable splicing body or spliceosome. Surprisingly, assembly and functioning of the spliceosome requires approximately 100 polypeptides and five small nuclear RNAs (snRNAs), not considering gene-specific RNA-binding factors. There are two distinct types of spliceosomes in most cells. The major class or U2-type spliceosome is universal in eukaryotes, whereas the minor class or U12-type spliceosome may not be present in some organisms. The evolutionary relationship of these two spliceosomes is uncertain. The sequence specificity for the splicing of introns must be encoded within the gene. In vertebrate genes, particularly for U2-type introns, the sequence specificity for splicing is not determined solely by the consensus sequences at the intron boundaries but is more broadly distributed within the gene. In contrast, the consensus sequences of the introns in the yeast Saccharomyces cerevisiae are generally adequate to specify...

RNAi and double-strand RNA.

Abstract: The σ subunit of eubacterial RNA polymerase is required throughout initiation, but how it communicates with core polymerase (α2ββ′) is poorly understood. The present work addresses the location and function of the interface of σ with core. Our studies suggest that this interface is extensive as mutations in six conserved regions of σ70 hinder the ability of σ to bind core. Direct binding of one of these regions to core can be demonstrated using a peptide-based approach. The same regions, and even equivalent residues, in σ32 and σ70 alter core interaction, suggesting that σ70 family members use homologous residues, at least in part, to interact with core. Finally, the regions of σ that we identify perform specialized functions, suggesting that different portions of the interface perform discrete roles during transcription initiation.

PUF60: a novel U2AF65-related splicing activity.

Abstract: We have identified a new pyrimidine-tract binding factor, PUF, that is required, together with U2AF, for efficient reconstitution of RNA splicing in vitro. The activity has been purified and consists of two proteins, PUF60 and the previously described splicing factor p54. p54 and PUF60 form a stable complex in vitro when cotranslated in a reaction mixture. PUF activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA. This reaction is dependent upon the presence of the large subunit of U2AF, U2AF65, but not the small subunit U2AF35. PUF60 is homologous to both U2AF65 and the yeast splicing factor Mud2p. The C-terminal domain of PUF60, the PUMP domain, is distantly related to the RNA-recognition motif domain, and is probably important in protein-protein interactions.

The SRm160/300 splicing coactivator is required for exon-enhancer function

Abstract: Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. We have recently identified a splicing “coactivator,” SRm160/300, which contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. In the present study, we show that SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independently of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound “activators” and the snRNP machinery of the spliceosome.

Ubiquitination of RNA polymerase II large subunit signaled by phosphorylation of carboxyl-terminal domain

Abstract: A sensitive assay using biotinylated ubiquitin revealed extensive ubiquitination of the large subunit of RNA polymerase II during incubations of transcription reactions in vitro. Phosphorylation of the repetitive carboxyl-terminal domain of the large subunit was a signal for ubiquitination. Specific inhibitors of cyclin-dependent kinase (cdk)-type kinases suppress the ubiquitination reaction. These kinases are components of transcription factors and have been shown to phosphorylate the carboxyl-terminal domain. In both regulation of transcription and DNA repair, phosphorylation of the repetitive carboxyl-terminal domain by kinases might signal degradation of the polymerase.

Promoter-specific hypoacetylation of X-inactivated genes

Abstract: The histone H4 acetylation status of the active X (Xa) and inactive X (Xi) chromosomes was investigated at the level of individual genes. A moderate level of acetylation was observed along the lengths of genes on both the Xi and Xa, regardless of their X inactivation status. However, this moderate level of acetylation was modified specifically in promoter regions. Transcriptionally active genes showed elevated levels of acetylation at their promoters on both the Xi and Xa. In contrast, promoters of X-inactivated genes were markedly hypoacetylated, which coincided with the methylation of adjacent CG dinucleotides. This promoter-specific hypoacetylation may be a key component of an X inactivation machinery that operates at the level of individual genes.

Crystal structure of an OCA-B peptide bound to an Oct-1 POU domain/octamer DNA complex: specific recognition of a protein-DNA interface

Abstract: We have determined the crystal structure, at 3.2 Å, of a ternary complex containing an OCA-B peptide, the Oct-1 POU domain, and an octamer DNA site. The OCA-B peptide binds in the major groove near the center of the octamer site, and its polypeptide backbone forms a pair of hydrogen bonds with the adenine base at position 5 of the octamer DNA. Numerous protein–protein contacts between the OCA-B peptide and the POU domain are also involved in the ternary complex. In particular, the hydrophobic surface from a short α-helix of OCA-B helps to stabilize the complex by binding to a hydrophobic pocket on the POU-specific domain. The structure of this ternary complex is consistent with previous biochemical studies and shows how peptide–DNA and peptide–protein contacts from OCA-B provide structural and functional specificity in the regulation of immunoglobulin transcription.

Tat-SF1 Protein Associates with RAP30 and Human SPT5 Proteins

Abstract: The potent transactivator Tat recognizes the transactivation response RNA element (TAR) of human immunodeficiency virus type 1 and stimulates the processivity of elongation of RNA polymerase (Pol) II complexes. The cellular proteins Tat-SF1 and human SPT5 (hSPT5) are required for Tat activation as shown by immunodepletion with specific sera and complementation with recombinant proteins. In nuclear extracts, small fractions of both hSPT5 and Pol II are associated with Tat-SF1 protein. Surprisingly, the RAP30 protein of the heterodimeric transcription TFIIF factor is associated with Tat-SF1, while the RAP74 subunit of TFIIF is not coimmunoprecipitated with Tat-SF1. Overexpression of Tat-SF1 and hSPT5 specifically stimulates the transcriptional activity of Tat in vivo. These results suggest that Tat-SF1 and hSPT5 are indispensable cellular factors supporting Tat-specific transcription activation and that they may interact with RAP30 in controlling elongation.

Science over politics.

Abstract: Last month, 70 members of the U.S. Congress, including Henry Hyde, Chairman of the House Judiciary Committee, and J. C. Watts Jr. Republican Conference Chairman, signed a letter urging the federal government to ban all research on stem cells obtained from human embryos and fetuses. The letter calls upon the U.S. Department of Health and Human Services (DHHS) to reverse National Institutes of Health (NIH) Director Harold Varmus's decision to allow funding of pluripotent stem cell research. The lawmakers object “in the strongest possible terms” to Varmus's decision, as well as to the memorandum issued in January by DHHS General Counsel Harriet Rabb, which served as the legal basis for Varmus's position. In their letter, the members of Congress state, “Any NIH action to initiate funding of such research would violate both the letter and spirit of the federal law banning federal support for research in which human embryos are harmed or destroyed.” Federal laws and regulations, they claim, have protected human embryos and fetuses “from harmful experimentation at the hands of the Federal government” for more than two decades. “This area of law has provided a bulwark against government's misuse and exploitation of human beings in the name of medical progress. It would he a travesty for this Administration to attempt to unravel this accepted ethical standard.”