Zissimos Mourelatos - US grants

This GOALI project is a collaborative research and educational effort between the University of Michigan and the General Motors Research and Development Center. It targets the general area of simulating the vibration and noise performance of an operating engine, leading to design changes for noise reduction. The specific research focus is on developing an analytical tool for predicting the dynamic response of an engine crankshaft-block system including the oil-film hydrodynamics. Previous work on dynamic reduction and lubrication constitutes the foundation of this effort. The fundamental research concentrates in the areas of developing a dynamic substructuring methodology for capturing the local flexibility of the cap and bulkhead of the block dynamics, developing a new perturbation technique for solving the oil- film Reynolds equation, developing a computational efficient solution for the electrohydro dynamics (EHD) lubrication problem, and developing a crankshaft-block-oil film coupling algorithm. Applied research is performed in parallel by General Motors Research and Development Center in applying dynamic reduction to crankshaft and block dynamics, including the local flexibility of the bearing caps and engine bulkheads, utilizing a `hourglass control` method for developing the oil-film fluidicity matrices, and performing the numerical implementation and validation for the crankshaft-block-oil film coupling algorithm. The educational objectives are identivied in the ara of joint advising, introducing new technology into the University curriculum, and developing a short course for engineers in the Industry. Substantial investment in the part of General Motors is included in the proposed work in terms of the paralled applied research effort, performing testing for validation, time allocation for joint advising, and collaboration in achieving all the educational objectives.

DESCRIPTION (Adapted From The Applicant's Abstract): This proposal sets in motion a 3 year plan to study the protein network involved in the pathogenesis of Spinal Muscular Atrophy (SMA), as well as establish the applicant as an independent investigator. The applicant has spent the last 8 years preparing for a career as an academic neuropathologist through his previous Ph.D. experience, which focused on mechanisms of neurodegeneration, and through clinical residency training in Anatomic Pathology and Neuropathology. The career development proposed in this application will complement his previous experience, which lacked exposure to biochemistry and advanced molecular biology, and provide him with the proper technical skills and intellectual framework to enable him to study pediatric neurodegenerative diseases. The applicant has chosen Dr. Gideon Dreyfuss as his sponsor. Dr. Dreyfuss is a recognized authority in the field of mRNA processing and a pioneer in the study of pediatric neurological diseases caused by aberrant RNA metabolism, including SMA and the Fragile-X syndrome. SMN (for Survival of Motor Neuron), the gene responsible for SMA, plays a critical role in spliceosomal. biogenesis and in pre-mRNA splicing, as first demonstrated by the sponsor's lab. However, the precise function of SMN is unknown. SMN is in a complex with several proteins. The applicant hypothesizes that by isolating and functionally characterizing these proteins he may uncover the molecular mechanism(s) of SMN function. In addition by studying the association of SNIN mutants, found in SMA patients, with these proteins he may uncover defective interactions which would help elucidate the molecular pathogenesis of SMA. Finally, by analyzing the composition of the SMN complex in neuronal cell lines he may uncover neuronal targets for SMN.

DESCRIPTION (provided by applicant): A new paradigm of gene expression regulation has emerged recently with the discovery of microRNAs (miRNAs), an evolutionary conserved class of small (approximately 22 nucleotide -nt-), regulatory RNAs. miRNAs bind with partial or extensive complementarity to their mRNA targets, miRNAs control gene expression by repressing the translation or by destabilizing, by endonucleolytic cleavage, their mRNA targets, miRNAs may exert profound effects in gene expression regulation as they have the capacity to target numerous mRNAs, miRNAs are functionally equivalent to small interfering RNAs (siRNAs). The use of synthetic siRNAs to knockdown gene expression has already transformed basic biology research and is promising to revolutionize the practice of medicine, if issues regarding delivery and specificity of siRNAs are solved. Despite the recent, explosive growth in the mi/siRNA field, many important questions regarding the function of mammalian miRNAs remain unanswered and form the basis of this proposal. In Aim 1, we will identify and characterize the mi/siRNA-guided endoribonuclease (RNAi endoribonuclease). We present the partial affinity purification and properties of this enzyme and biochemical approaches that will allow us to identify and characterize this important enzyme. We also present results and strategies to elucidate how mi/siRNAs recognize their mRNA targets. In Aim 2, we will analyze the factors and mechanisms underlying miRNA-directed translational repression. We have begun the identification of proteins that miRNAs associate with, when they recognize their cognate mRNA targets in polyribosomes, and we have generated an in vitro system that recapitulates mammalian miRNA-directed translational repression. We present approaches to characterize translational repression mediated by mammalian miRNAs. We expect that our studies will promote our understanding of the molecular mechanisms underlying RNAi and the function of miRNAs in humans.

[unreadable] DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is a common motor neuron disease and one of the leading genetic causes of death of young children. SMA is caused by deletions or loss of function mutations of the Survival of Motor Neurons (SMN) gene. The SMN protein associates with six additional proteins into an SMN complex and functions in the assembly of small nuclear ribonucleoproteins (snRNPs) and possibly other RNPs. MicroRNAs (miRNAs) are a newly discovered class of -22 nucleotide regulatory RNAs that are bound to Argonaute proteins. miRNAs act as specificity determinants to direct destruction or translational repression of their mRNA targets. miRNAs have the capacity to regulate numerous genes and they may exert profound effects in gene expression regulation. We have identified a novel RNP, termed microRNP (miRNP), that contains miRNAs, the Argonaute2 protein and the SMN complex proteins GeminS (an RNA helicase) and Gemin4. We propose to study the role of the SMN complex in the biogenesis and function of miRNAs and miRNPs. We will also study whether the biogenesis or function of miRNAs and miRNPs are dysregulated in SMA. Our work will likely uncover novel functions for the SMN complex and will also shed light in the function of miRNAs. Ultimately, investigations of the function of the SMN complex may lead to better understanding of the pathogenesis of SMA and possibly of other motor neuron diseases. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is a common motor neuron disease and one of the leading genetic causes of death of young children. SMA is caused by deletions or loss of function mutations of the Survival of Motor Neurons (SMN) gene. The SMN protein associates with six additional proteins into an SMN complex and functions in the assembly of small nuclear ribonucleoproteins (snRNPs) and possibly other RNPs. MicroRNAs (miRNAs) are a newly discovered class of -22 nucleotide regulatory RNAs that are bound to Argonaute proteins. miRNAs act as specificity determinants to direct destruction or translational repression of their mRNA targets. miRNAs have the capacity to regulate numerous genes and they may exert profound effects in gene expression regulation. We have identified a novel RNP, termed microRNP (miRNP), that contains miRNAs, the Argonaute2 protein and the SMN complex proteins GeminS (an RNA helicase) and Gemin4. We propose to study the role of the SMN complex in the biogenesis and function of miRNAs and miRNPs. We will also study whether the biogenesis or function of miRNAs and miRNPs are dysregulated in SMA. Our work will likely uncover novel functions for the SMN complex and will also shed light in the function of miRNAs. Ultimately, investigations of the function of the SMN complex may lead to better understanding of the pathogenesis of SMA and possibly of other motor neuron diseases. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a human motor neuron degenerative disease caused by loss-of-function mutations of the immunoglobulin u-binding protein 2 (IGHMBP2), a putative RNA/DNA helicase. The co-investigator Cox previously identified the mouse Ighmbp2 gene as the causative gene of the mouse neuromuscular degeneration (nmd) phenotype by a positional cloning approach. We have also identified a major genetic modifier of its phenotypic expression (Mnm). The function of IGHMBP2 and its role in the motor neuron degeneration that underlies the pathogenesis of SMARD1 are unknown. [unreadable] We propose to investigate the function of IGHMBP2 and to uncover the molecular defect(s) responsible for motor neuron degeneration caused by reduced IGHMBP2 levels, by implementing an inter-disciplinary and inter-institutional collaborative approach that will allow us to combine state of the art biochemical and genetic investigations. Toward this goal, we have isolated IGHMBP2 interacting proteins and small RNAs that associate with this helicase and we have employed powerful genetic approaches to identify the critical cell-types that require nmd gene activity using tissue-specific transgenic rescue. Our ability to manipulate the severity of the disease phenotype genetically with at least one modifier gene suggests that a molecular pathway exists with the potential for genetic or clinical intervention. Thus, the nmd mouse and the Mnm modifier gene provide a unique opportunity to identify the underlying processes of neurodegeneration and provide possible entry points in which to intervene in the disease pathway. Our genetic studies will be complemented by biochemical studies aimed towards characterization of the function of IGHMBP2 in cellular and mouse models of SMARD1 and we will extend our studies in human tissues from SMARD1 patients. [unreadable] These studies will likely uncover an entirely novel pathway of RNA regulation and will advance significantly our understanding of RNA processing in motor neurons and the contribution of RNA dysregulation in motor neuron degeneration.We propose to investigate the pathobiology of an inherited, human neurodegenerative disease. Our studies will shed light on pathogenetic mechanisms of human motor neuron diseases and promote the design of strategies to combat these lethal diseases. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Dysregulation of RNA pathways is emerging as a central feature of degeneration of motor neurons. Amyotrophic lateral sclerosis (ALS) is the most common human motor neuron degenerative disease with a fatal outcome and without a cure. Approximately 20% of ALS patients also develop Frontotemporal Lobar Degeneration (FTLD), characterized by dementia due to neuronal degeneration and often with the presence of ubiquitinated protein aggregates (FTDL-U). Recent studies have shown that mutations of two RNA Binding Proteins (RBPs) known as TDP-43 (or TARDBP) and FUS (or FUS/TLS) cause familial ALS (FALS) and FTLD-U;and protein aggregates containing TDP-43 or TDP-43 carboxy-terminal (C-terminal) fragments are detected in the majority of sporadic ALS cases and in many cases of FTLD-U (FTLD-TDP), further underscoring the paramount importance of RBPs and of RNA dysregulation in neuronal degeneration. The RNA targets and function of TDP43 and FUS in motor neurons are unknown. Furthermore, it is unknown how mutations of TDP-43 and FUS lead to neuronal degeneration. In this application we propose to develop novel experimental systems and strategies to investigate the function of TDP-43 and FUS in RNA pathways and to dissect pathogenetic mechanisms involved in motor neuron degeneration by TDP-43 and FUS patient mutants. In Aim 1, we will establish and characterize embryonic stem cell derived motor neuron culture systems to investigate TDP-43 and FUS. We will characterize the cellular and molecular effects of mutant TDP-43 and FUS in cultured motor neurons and in the motor neurons of ALS patients. In Aim 2, we will identify RNA target for TDP-43 and FUS in human brain and motor neurons and we will investigate whether TDP-43 and FUS patient mutations result in RNA dysregulation in motor neurons. We expect that our studies will lead to the development of novel experimental systems and will provide critical insights in the function of TDP-43 and FUS in motor neurons and the role of patient mutations in neuronal degeneration. We also anticipate that the outcome of this exploratory R21 grant application may provide important insights that will guide further investigations in therapeutic strategies to combat this lethal disease. PUBLIC HEALTH RELEVANCE: In this application we propose to develop novel experimental systems and strategies to investigate how motor neurons degenerate in ALS, a devastating neurodegenerative disease. We anticipate that our studies may provide important insights that will guide further investigations in therapeutic strategies to combat this lethal disease.

DESCRIPTION (provided by applicant): Functional characterization of piRNPs Germ cells carry the essence of most forms of multicellular life by storing, shaping (via meiosis), and transmitting the genetic information that propagates a species. They express Piwi family proteins that bind to small RNAs known as piwi-interacting RNAs (piRNAs) to form pi-RiboNucleoProteins (piRNPs) that silence retrotransposons, a function critical for preserving genome integrity. Primary piRNAs are derived from long, single-stranded transcripts that are processed in the cytoplasmic nuage, an electron-dense structure that is in very close apposition to perinuclear mitochondria. Drosophila melanogaster expresses three Piwi proteins termed Aubergine (Aub), Piwi and Ago3. Aub-piRNAs target and cleave transposons and the piRNA response is amplified by successive rounds of Aub and Ago3 interactions, in a process known as the ping-pong cycle. Genetic studies have shown that Aub has an additional role in specification of Primordial Germ Cells (PGCs) in the embryo, along with Csul (an arginine protein methyltransferase) and Tudor. During Drosophila oogenesis, germline RNPs assemble at the posterior of the oocyte to form germ (pole) plasm. Germ plasm is transmitted to the embryo and it is necessary and sufficient to induce the formation of PGCs from undifferentiated cells. Germ plasm contains RNAs that are essential for PGC specification but the identity of many of these mRNAs and how they are anchored at the posterior of the oocyte are not known. Our laboratory discovered that Csul catalyzes symmetrical dimethylation of arginines of Piwi proteins and we showed that Aub methylation is required for germ plasm assembly, in vivo via its interaction with its receptor protein Tudor. Studies from our lab and subsequent studies from other labs have shown that the formation of RNPs that are assembled by methylated Piwi proteins bound to Tudor-domain containing proteins (Tdrds) are conserved in the germline of all animals. Our proposal will address molecular mechanisms of piRNA biogenesis, and functions of Aub-piRNPs and Aub-mRNPs in two broad Aims. In Aim 1, we will determine molecular mechanisms of piRNA biogenesis. By using multiple approaches, including HITS-CLIP and biochemical isolation of piRNA processing factors, we will examine how piRNA precursors are selected and processed into piRNAs. We will also study the role of Aub arginine methylation in quality control of piRNA processing and transposon silencing. In Aim 2, we will determine molecular mechanisms and functions of Aub piRNPs and Aub mRNPs. We will address how Piwi proteins such as Aub bind piRNAs or longer RNAs and how piRNPs bind their targets. We will identify the composition of Aub mRNPs and molecular mechanisms of how Aub germline mRNPs assemble to specify Primordial Germ Cells. We will investigate the biological significance of our findings using Drosophila genetic and transgenic approaches.

Deciphering pachytene piRNA function PROJECT SUMMARY / ABSTRACT An enigmatic class of small RNAs appears at the pachynema of Meiosis I of mammalian spermatocytes. They are processed from long, non-coding RNAs, bind to Piwil1 (in mouse commonly known as Miwi) protein, and are termed pachytene piwi-interacting (pi) RNAs. Miwi/piRNAs are essential for spermiogenesis and male fertility. Our laboratory discovered that in diverse species, Piwi proteins loaded with piRNAs are symmetrically dimethylated on specific arginines by the methylosome, and mediate interaction with Tudor domain containing (Tdrd) proteins. Miwi binds directly to Tdrd6, a protein that contains six canonical Tudor domains, and together form the core of the chromatoid body, a large, cytoplasmic, non-membrane bound structure that contains numerous mRNAs along with pachytene piRNAs. Pachytene piRNAs are very abundant; they are not conserved even among closely related species; their sequence diversity is enormous; and their function still remains a mystery. Hypotheses about their roles need to reconcile two seemingly contradictory properties: like microRNAs, pachytene piRNAs are loaded to an Argonaute protein, Miwi, and can serve as guides to bind RNA targets. Unlike microRNAs, their sequence diversity is so enormous that they can bind any mRNA at multiple sites, thus losing sequence-driven specificity. In this application we propose a radically new conceptual framework to crack the enigma of pachytene piRNA function. We will test the hypothesis that pachytene piRNAs play a critical role in sorting transcripts, by dynamically trapping non-spermiogenic mRNAs in Miwi-piRNA-Tdrd6 assemblies, which form the core of the chromatoid body. In our model, multivalent interactions between Miwi/piRNAs, which bind with partial complementarity to mRNAs, and between Miwi and the multiple Tudor domains of Tdrd6, nucleate the chromatoid body that sequesters trapped mRNAs for eventual elimination during spermiation. The model predicts that longer mRNAs are preferentially trapped as they contain more binding sites for piRNAs, while spermiogenic mRNAs that need to be translated to drive spermatid differentiation should be shorter to avoid trapping. We will also test whether the multivalent interactions in Tdrd6-Miwi/piRNA-mRNA assemblies lead to liquid-liquid phase separations that underlie the formation of the chromatoid body. We are confident that the multiple, orthogonal, in vitro and in vivo approaches that we propose will illuminate expected and unexpected outcomes and truly uncover the elusive function of mammalian pachytene piRNAs.

Ribothrypsis: mechanisms and implications for gene expression regulation PROJECT SUMMARY / ABSTRACT Messenger RNAs transmit the genetic information that dictates protein production and are a nexus for numerous pathways that regulate gene expression. The prevailing view of canonical mRNA decay is that it is mediated by deadenylation and decapping followed by exonucleolysis from the 3' and 5' ends. We recently described ribothrypsis, an endonucleolytic pathway of cotranslational mRNA decay, mediated by ribosome-phased cleavages of the mRNA as it exits the ribosome channel. We posit that ribothrypsis is a unifying and evolutionary conserved mechanism that underlies cotranslational decay of all mRNAs: canonical and aberrant mRNAs that degrade via surveillance mechanisms, such as No-Go Decay (NGD) or Non-Stop Decay (NSD). In that sense, ribothrypsis may be viewed as ?NGD/NSD on steroids?; or conversely NGD/NSD may be viewed as a subset of ribothrypsis. The central integrator of mRNA decay in ribothrypsis is the translating ribosome that under certain conditions activates or recruits an unknown endonuclease (ribothrypsin) to cleave the mRNA as it exits the ribosome. In this proposal, we will investigate the impact of ribothrypsis in gene expression analysis; and mechanisms and impact of ribothrypsis in gene expression regulation. We discovered that endogenously generated mRNA fragments represent a sizable fraction of the total mRNA pool. This finding complicates interpretation of results obtained with all current methods that assay mRNAs, which do not take ribothrypsis into account, and necessitates the development of new experimental and computational tools for RNA sequencing, which we will develop in Aim1. In Aim2, we will investigate ribothrypsis triggers and unexpected roles of ribothrypsis in gene expression regulation via upstream Open Reading Frames (uORFs), and in the decay of long noncoding RNAs (lncRNAs). We will also study molecular mechanisms of ribothrypsis in vitro and in vivo and identify ribothrypsin.