Gideon Dreyfuss - US grants

Messenger RNAs (mRNAs), the intermediates of gene expression which direct protein synthesis, are found in eukaryotic cells associated with a specific subset of cytoplasmic proteins. The mRNA-protein complexes, termed mRNP particles (mRNPs), are distinct structural entitles different in protein composition, structure, function and subcellular compartmentalization from nuclear pre-mRNA-protein (hnRNP) particles. A plethora of recent observations indicate that mRNP proteins play a cardinal role in mRNA transport, translation, stability and localization. The mRNP proteins can be photochemically crosslinked to the mRNA in intact cells and the crosslinked complexes can be readily isolated. We have studied these complexes in normal and virus-infected cells across eukaryotes. In all of these, the most abundant protein is a 72,000 dalton protein which is crosslinked to the poly(A) tail of the mRNA. The poly(A) binding protein is a highly conserved, poorly immunogenic protein that has been the focus of much interest because it appears to play a key role in mRNA metabolism. We have immunized mice with crosslinked purified mRNP complexes to produce antibodies to the mRNP proteins. Antibodies were obtained to the poly(A) binding protein from yeast, and we have begun to characterize the protein and have isolated the gene encoding it. The antibodies and the gene are the first probes for this protein from any organism and they open the way of its molecular and genetic characterization. We shall investigate the structure, function, properties and localization of the mRNP proteins and the mRNP complexes with particular emphasis on the poly(A) binding protein. Additional antibodies to mRNP proteins will be produced. The cDNAs encoding these proteins will be isolated by immunological screening of expression vector libraries. The complete amino acid sequence of the proteins will be determined by nucleotide sequencing of the cDNAs, and their mRNAs and genes will be characterized. The interaction of the proteins with RNA will be studied in detail. The subcellular localization of the proteins will be examined by immunocytochemical techniques. The functions of the proteins will be investigated in vivo by gene disruption and mutagenesis in yeast and by introduction of antibodies into living animal cells, and in vitro using cell free systems for protein synthesis and mRNA transcription, splicing and polyadenylation. From these studies a better picture of ho mRNA is formed, maintained and functions in animal cells is likely to emerge.

Messenger RNA (mRNA) in eukaryotic cells appears to be associated with a set of proteins to form ribonucleoprotein (RNP) complexes. These complexes are considered to be the true functional units of mRNA and their proteins are widely believed to play a central role in mRNA synthesis, processing, storage and translation. However, little is known about the proteins which are in direct contact with mRNA in vivo. Recent observations also suggest that most of the mRNA is associated with a complex cytoplasmic proteinacious network of fiber-forming and contractile proteins referred to as the cytoskeleton. This association seems to be important for mRNA translation and suggests there may be a link between cellular skeletal structures and regulation of gene expression. This hypothesis must be tested through detailed molecular examination of the proteins which are associated with mRNA. The study of the complexes of mRNA with specific cytoplasmic proteins is the focal point of this proposal. To identify mRNP proteins in vivo, UV irradiation of intact cells is used to induce crosslinking of mRNA to its associated proteins. The attributes of this technique make it extremely suitable for this purpose. Preliminary experiments enable the identification of a small subset of proteins which become crosslinked to poly(A)+mRNA in HeLa cells. The crosslinked complex will be used to raise monoclonal antibodies to the major proteins of the bulk of the cytoplasmic mRNPs. These will be used to study the intracellular localization of mRNP proteins, to obtain additional information about them and to begin to examine their possible function. The possible association of mRNPs with cytoskeletal elements will also be investigated. The proteins which are associated with a small, distinct set of mRNAs--VSV mRNAs--will also be determined.

Messenger RNAs (mRNAs) are formed in the nuclei of eukaryotic cells by extensive post-transcriptional processing of primary RNA polymerase II transcripts. These transcripts are termed heterogeneous nuclear RNAs (hnRNAs) and they are associated with proteins throughout the time they are in the nucleus. The collective term for the proteins that bind hnRNAs, and which are not stable components of other classes of RNP complexes such as snRNPs, is hnRNP proteins. The hnRNP proteins are avid RNA-binding proteins and the processing of hnRNA in the nucleus takes place in hnRNA-protein (hnRNP) complexes. The significance of hnRNP proteins is that they influence the structure of hnRNAs and therefore their fate and processing into mRNAs. HnRNP proteins may also play crucial roles in the interaction of hnRNP complexes with other nuclear structures, in nucleo-cytoplasmic transport of mRNA, and they may have biochemical activity relevant to hnRNA metabolism. In addition, the hnRNP proteins are among the most abundant proteins in the nucleus in growing vertebrate cells, and hnRNP complexes are thus also of interest because they are major nuclear structures. Our goal is to understand in detail how the post-transcriptional portion of the pathway of gene expression operates in the cell. To do so we investigate the structure, function, and localization of the hnRNP and cytoplasmic mRNA-binding (mRNP) proteins and RNP complexes. Much progress has been made in these areas over the past several years but there is still a great deal of fundamental importance that remains to be learned. The focus of the research proposed in this grant application is the characterization of the hnRNP proteins, including their sequence, structure, RNA-binding activity, protein-protein interactions, assembly into complexes, biochemical activities, and function in mRNA biogenesis.

DESCRIPTION (Adapted from the Investigator's Abstract): Spinal Muscular Atrophy (SMA) is a common, devastating and often fatal motor neuron degenerative disease. SMA results from reduced levels of, or mutations in, the SMN (survival of motor neurons) protein. Work on the molecular characterization of the SMN protein led to the discovery of several of its functions, revealing novel cellular pathways and proteins. The investigators found that SMN is part of a complex that contains several proteins. All the components of the complex are novel proteins and they have already characterized several of them. Because they are tightly associated with SMN, the other proteins of the complex must be considered to be collaborators or modifiers of SMN function, and thus play a role in the course and severity of SMA. They also are candidate disease genes for other neuronal diseases. The SMN complex is expressed in all cells, but particularly high levels are found in neurons. It is present in both the cytoplasm and in the nucleus where it is concentrated in nuclear bodies, they termed gems. SMN functions in the biogenesis of snRNPs, the essential building blocks of the pre-mRNA splicing machinery, and in the pre-mRNA splicing process itself. Much remains to be learned about the structure, functions, and mechanism of action of SMN and the SMN complex. Studies will be carried out to understand in details the molecular functions and interactions of the SMN protein and of the SMN complex. From these studies approaches to therapy of SMA are likely to emerge.

DESCRIPTION (provided by applicant): This proposal's overarching goal is to understand the mechanism of telescripting, a new and major gene expression process recently discovered in my laboratory. Telescripting is essential for full length RNA polymerase II (pol II) transcription from the majority of protein-coding genes in eukaryotes necessary for messenger RNA (mRNA) synthesis. It relies on U1 snRNP (U1), an abundant non-coding 11-subunit ribonucleoprotein particle, to protect nascent pol II transcripts from early termination by cleavage and polyadenylation (CPA) in introns, which is highly destructive. U1 snRNP is well characterized for its role in 5' splices site (ss) recognition, a key and the first step in splicing of introns. Howeer, we found that U1 has an additional non-splicing function as a suppressor of premature CPA (PCPA) from cryptic polyadenylation signals (PASs) that are stochastically present in large introns. We refer to U1's PCPA suppression also as telescripting, as it is necessary for allowing transcription to go farther. In contrast, nascent upstream antisense transcripts from divergent polII promoters are relatively unprotected due to an inverse PAS to U1 binding ratio and are rapidly degraded, indicating telescripting's general role in shaping the transcriptome. Furthermore, telescripting activity, which can be finely modulated by slight changes in U1 level also determines mRNA length. For example, slight U1 decrease causes widespread shortening due to usage of more proximal PASs in the 3' untranslated region (3'UTR), thereby removing mRNA-regulating elements such as translation repressing microRNA binding sites. Based on our previous studies we proposed a model to explain how U1 suppresses PASs in introns, including the hypothesis that it binds in introns and not only to 5'ss. However, basic information needed to test this hypothesis and for understanding telescripting mechanism is lacking. To address this, my laboratory established tools to precisely manipulate PCPA and its suppression by U1, and assays to probe it in detail. We propose to pursue three specific aims: 1) To generate genome wide map of U1 snRNP binding sites on nascent transcripts in live cells, which will be interpreted relative to genome wide map of PCPA sites that we recently completed. 2) To determine the role of U1 snRNP proteins and essential U1 snRNA domain(s) for telescripting. 3) To identify CPA complex and/or other targets of U1's PCPA suppression using a comprehensive RNP interactome discovery approach pioneered in my laboratory. Together, these aims will provide important insights into mechanism of U1 snRNP telescripting, and advance understanding and potential applications of this new dimension in gene regulation to biology and medicine.

PROJECT SUMMARY This proposal?s overall goal is to understand the molecular mechanism of telescripting, a new and major gene expression process that is crucial for full-length transcription of most protein-coding genes and regulates messenger RNAs (mRNAs) isoforms and mRNA length in humans and other complex organisms. mRNAs are processed from nascent RNA polymerase II (PolII) transcripts, which generally includes the removal of introns (splicing) and transcription-terminating 3?-end cleavage and polyadenylation (CPA). Splicing and CPA are specified by splice sites and CPA signals (PASs), respectively. However, numerous PASs indistinguishable from the ultimate, gene ends? PASs are scattered throughout pre-mRNAs, especially in introns and 3? untranslated regions (3?UTRs), and can trigger premature CPA (PCPA). PCPA is suppressed by U1 snRNP (U1), human cells? most abundant small non-coding nuclear RNA-protein particle. For brevity, and to distinguish it from U1?s role in splicing, we call U1 suppression of PCPA, telescripting (as it is necessary for long-distance transcription). Like U1 function in splicing, telescripting also depends on U1 snRNA base-pairing to nascent transcripts, which can be abrogated with U1 antisense oligonucleotides (U1 AMO), causing PCPA. Recent studies revealed that even slight changes in the balance between U1 and PASs has great impact on gene expression and can profoundly alter the mRNAs and proteins cells produce. Such changes occur naturally, for example rapid transcription up-regulation during cell stimulation, and create transient U1 deficit relative to transcription output, causing PCPA that produces shorter mRNA isoforms needed to respond to acute environmental changes. Importantly, U1 AMO recapitulates the same mRNA isoform shifts and U1 over- expression can prevent their production in stimulated cells. U1 AMO also elicits widespread 3'UTR shortening, which occurs in and contributes to cell proliferation and cancer. U1 telescripting?s overarching role in transcriptome regulation impacts transcription, splicing, CPA and thereby all downstream events in the life of mRNAs. It has numerous potential applications in biology and medicine. Realizing them requires detailed understanding of the molecular mechanism by which U1 suppresses PASs and the factors that regulate it, which remain unknown. We have made significant progress towards that, including mapping the transcriptome binding locations of U1 and cleavage and polyadenylation factors (CPAFs), and interpreted them in relation to PCPA locations. We have also captured U1 and CPAFs complexes in cells, determined their compositions and stoichiometries, and determined how cells produce the great U1 abundance required for telescripting. These advances lay the foundation for future studies. I anticipate the mechanistic studies and new information will identify potential points of intervention, including druggable targets, and will advance the prospects of harnessing them for novel therapies.