Marvin Wickens - US grants

[unreadable] DESCRIPTION (provided by applicant): Eukaryotic mRNAs can be controlled at many steps. In the nucleus, transcription and mRNA processing are needed to generate mRNAs that can be translated. In the cytoplasm, mature mRNAs can be regulated at the levels of stability, translation and localization. The objective of the proposed work is to understand the molecular mechanisms that regulate mature mRNAs in animal cells. We focus on controls mediated by sequences that lie beyond the termination codon - in the 3' untranslated region (3'UTR) and the poly(A) tail. Regulated changes in poly(A) length occur throughout development and affect translation and stability of many mRNAs. Here,we focus on a novel form of cytoplasmic poly(A) polymerase in C. elegans, consisting of a catalytic subunit, GLD-2, with distinct RNA-binding protein partners. One partner binds to and antagonizes FBF, a founder of a family of 3'UTR repressors, the PUF proteins. Each protein we focus on - GLD-2, its partners, and FBF - controls key events in development. Our ultimate goals are to understand, in molecular terms, how these proteins control the fate and function of mature mRNAs. [unreadable] [unreadable] The approach taken is first to elucidate how the novel polyadenylation system functions. Using molecular genetics and biochemistry, we identify regions of GLD-2 that promote catalysis and bind partners. Through grafting and mutagenesis, we test the model that GLD-2 lacks RNA-binding activity, but gains it via distinct protein partners. This work is consummated by determining structures of the key components. We elucidate the evolutionary breadth of the polyadenylation system, focusing on vertebrates and in vivo assays, and elucidate how it antagonizes PUF-mediated repression. Finally, combining the yeast three-hybrid system and other methods, we identify mRNA targets of the system. Throughout, we combine the use of Xenopus oocytes, yeast molecular genetics, and in vitro systems to elucidate how these proteins function. We exploit novel methods we developed that have broader utility. The rich genetics and biology of C. elegans development provides a critical biological foundation. In focusing sharply on a few specific examples, we hope to illuminate the broad molecular questions of how 3'UTRs control function, evolve, and coordinate expression of multiple mRNAs.

Eukaryotic mRNAs are not replicas of the genes that encode them. Instead, they are produced by a series of post-transcriptional modifications of a primary transcript. In principle, each maturation step provides a means of regulating gene expression. In the first period of this grant, my lab identified regions of the mRNA precursor that are required for cleavage (formaton of the 3' terminus), and showed that a factor binds stably to one of these regions during the cleavage reaction. From these studies, we propose a model that provides the framework for many of the experiments described here. I plan an intensive analysis of three mRNA processing steps: cleavage, polyadenylation, and transport from the nucleus to the cytoplasm. We will use the mRNA of a tumor virus, SV40, as a model, and will assay maturation both in vivo, using frog oocytes and cultured monkey cells, and in vitro, using a nuclear extract. Sequences in the precursor essential for each of these maturation steps will be identified both by mutation and by direct chemical methods. Our model predicts that any mutations affecting polyadenylation will affect cleavage identically. To further analyze each reaction, we will (1) identify component(s) (enzymes, snRNAs) that bind to the precursor during cleavage, (2) examine the products of the cleavage reaction in biochemical detail, (3) test whether processing requires recognition of the 5' end of the mRNA precursor by using a circular RNA, and (4) determine what features of the precursor are required for transport by injecting mRNA processing intermediates directly into nuclei. The experiments proposed will elucidate fundamental mechanisms of gene expression, and therefore will have important practical applications. Because various organisms use similar but distinct mechanisms to process their mRNAs, the opportunity exists to create a new generation of antibiotics directed against a previously unexploited target - mRNA processing. Our work will provide fundamental knowledge needed for the rational design of these drugs.

Eukaryotic mRNAs are not replicas of the genes that encode them. Instead, they are produced by a series of post- transcriptional modifications of a primary transcript. In principle, each maturation step provides a means of regulating mRNA-formation. In the past four years, using molecular biological approaches, we have identified regions of the mRNA precursor that are required for cleavage (formation of the 3' terminus), and showed that a factor binds stably to one of these regions during the reaction. We propose a model which provides the framework for many of the experiments described here. We plan an intensive analysis of three mRNA processing steps: cleavage, polyadenylation, and transport from the nucleus to the cytoplasm. We will use the mRNA of tumor virus, SV40, as a model and will assay maturation both in vivo and in vitro. Sequences essential for each step will be identified both by mutation and by direct chemical means. To further analyze each reaction, we will: (1) identify those features of each base which are critical for processing; (2) identify those phosphates which participate in processing and determine whether they do so by RNA-protein contacts; (3) identify factors required for processing and the activation of maternal mRNAs in C. elegans; (4) identify component(s) that bind stably to the precursor during cleavage; (5) test whether processing requires recognition of the 5' end of the pre-mRNA by using a circular RNA; and (6) determine what features of the precursor are necessary for transport by injecting processing intermediates into nuclei. The experiments proposed will have important practical applications. Because various organisms use similar but distinct mechanisms to process their mRNAs, the opportunity exists to create a new generation of antibiotics directed against a previously unexploited target - mRNA processing. Our work will provide fundamental knowledge for the rational design of such drugs.

Eukaryotic mRNAs generally are not replicas of the genes that encode them. Instead, mRNA is produced by a series of covalent modifications of a primary transcript including capping, splicing and polyadenylation. In principle, each maturation step provides a means of regulating mRNA formation. The ultimate objective of our work is to understand the molecular mechanism of mRNA synthesis and the means by which it is regulated. In this proposal, we focus on a form of regulated RNA processing that is critical in early development -- the addition and removal of poly (A) from maternal mRNAs. We propose to study these regulated RNA processing reactions in frog oocytes and embryos. We focus on the periods of oocyte maturation and fertilization, and use cyclin and c-mos mRNAs as models. In the last grant period, we laid the groundwork for the work proposed here. We identified sequences that regulate poly (A) addition and removal, and characterized the enzymes involved. We developed assays for poly (A) addition and removal, both in vivo, by microinjection, and in vitro, using extracts of frog eggs. In addition, we elucidated key features of the mechanism by which poly (A) is added in the nucleus of all cells; this knowledge provides an invaluable intellectual framework and critical reagents for the work we now propose In the next grant period, sequences that are critical for poly (A) addition and removal during development will be identified precisely. We will characterize and purify the enzymes involved, both by classical means and molecular cloning, and determine how those activities are regulated during development. We will test our hypothesis that changes in the length of the poly (A) tail of c-mos mRNA help control the embryonic cell cycle. By in vitro complementation tests, we will determine whether factors that direct cytoplasmic polyadenylation during development are functionally interchangeable with those that add poly (A) in the nucleus. The work proposed will elucidate fundamental mechanisms of gene expression, and therefore has important practical applications. Changes in poly (A) length occur throughout development, and in the embryos of all animal species so far examined. Our detailed investigations of the regulation of c-mos, a proto-oncogene normally expressed only in the germ line, bear on how cell division is controlled both in normal and abnormal growth.

Eukaryotic mRNAs can be controlled at many different steps. In the nucleus, transcription and mRNA processing are required to produce an mRNA that can be translated. In the cytoplasm, mature mRNAs can be regulated at the level of stability, transnational activity, and cellular location. The objective of the proposed research is to understand the biochemical mechanisms that control mature mRNAs in animal cells. We focus on the roles of poly (A) addition and removal in the transnational control of mRNAs during early development. These reactions are regulated by sequences that lie between the translation termination codon and the poly (A) tail--in the 3' untranslated region of the mRNA (3'UTR). Our ultimate goals are to understand, in molecular terms how poly (A) and elements in the 3'UTR control translation. We propose to study these reactions in frog oocytes and embryos. We focus on the period of oocyte maturation and use cycline and c-mos mRNAs, as models. We have shown that changes in poly (A) length can regulate translation during early development, and that sequences in the 3"UTR govern the extent and timing of transnational activation and repression. We have developed straightforward in vivo and in vitro assays for the effects of poly (A) and sequences in the 3"UTR. We propose to identify how poly (A) stimulates translation by combining in vivo and in vitro approaches. Using molecular genetics and biochemistry, we will identify the step(s) at which poly (A) exerts its effects and examine the role of the protein to which it binds. By constructing well-defined mutations in synthetic mRNAs, we will precisely identify elements in the 3"UTR that control transnational activity in vivo and determine how they function. We will test our hypothesis that changes in the length of the c-mos mRNA's poly (A) tail, governed by signals in its 3"UTR, are critical for control of the embryonic cell cycle. The work proposed will elucidate fundamental mechanisms of gene expression, and therefore has important practical applications. Regulated changes in poly (A) length occur in many, if not all, animal species, affect many mRNAs in the embryo, and continue throughout life. Our detailed investigations of the regulation of c-mos, a proto-oncogene normally expressed only in the germ line, bear on how cell division is controlled both in normal and abnormal growth.

9318966 Wickens This award will support an annual meeting of researchers studying the processing of RNA molecules. The post transcriptional modification of RNAs by a variety of mechanisms including splicing, polyadenylation, base modification, nuclease trimming, and editing is crucial to their biological activities. The meeting has been held annually for over 10 years and has played a key role in stimulating research in this broad, important field. %%% The biosynthesis of RNA from DNA is the first step in utilizing genetic information. The RNA is almost always modified in one or more ways before the cell can actually use these molecules for the many functions which they serve. This award supports a conference of researchers who study the modifications of RNA molecules which transform them into key components of the cellular machinery. ***

Eukaryotic mRNAs can be controlled at many different steps. In the nucleus, transcription and mRNA processing are required to produce an mRNA that can be translated. In the cytoplasm, mature mRNAs can be regulated at the level of stability, transnational activity, and cellular location. The objective of the proposed research is to understand the biochemical mechanisms that control mature mRNAs in animal cells. We focus on the roles of poly (A) addition and removal in the transnational control of mRNAs during early development. These reactions are regulated by sequences that lie between the translation termination codon and the poly (A) tail--in the 3' untranslated region of the mRNA (3'UTR). Our ultimate goals are to understand, in molecular terms how poly (A) and elements in the 3'UTR control translation. We propose to study these reactions in frog oocytes and embryos. We focus on the period of oocyte maturation and use cycline and c-mos mRNAs, as models. We have shown that changes in poly (A) length can regulate translation during early development, and that sequences in the 3"UTR govern the extent and timing of transnational activation and repression. We have developed straightforward in vivo and in vitro assays for the effects of poly (A) and sequences in the 3"UTR. We propose to identify how poly (A) stimulates translation by combining in vivo and in vitro approaches. Using molecular genetics and biochemistry, we will identify the step(s) at which poly (A) exerts its effects and examine the role of the protein to which it binds. By constructing well-defined mutations in synthetic mRNAs, we will precisely identify elements in the 3"UTR that control transnational activity in vivo and determine how they function. We will test our hypothesis that changes in the length of the c-mos mRNA's poly (A) tail, governed by signals in its 3"UTR, are critical for control of the embryonic cell cycle. The work proposed will elucidate fundamental mechanisms of gene expression, and therefore has important practical applications. Regulated changes in poly (A) length occur in many, if not all, animal species, affect many mRNAs in the embryo, and continue throughout life. Our detailed investigations of the regulation of c-mos, a proto-oncogene normally expressed only in the germ line, bear on how cell division is controlled both in normal and abnormal growth.

DESCRIPTION (provided by applicant): Project Summary - DNA makes RNA makes protein. The second step of that pathway - the critical function of RNA in genetic information transfer - is exquisitely regulated. mRNA activity, stability and location are controlled by factors that recognize specific RNA sequences. We focus on the PUF proteins, a widespread family of mRNA regulatory proteins that control key steps in early development and are required for establishing memory. We propose to determine how these proteins find and recognize specific mRNAs, and how they then execute that decision. PUBLIC HEALTH RELEVANCE: Project Narrative The proposed work will illuminate how a widespread family of mRNA regulatory proteins work. These proteins control stem cells and participate in the formation of memory. Understanding how they work should provide practical opportunities for intervention.

Project Summary /Abstract DNA makes RNA makes protein. The second step of that pathway ? the critical function of RNA in genetic information transfer ? is exquisitely regulated. mRNA activity, stability and location are controlled by factors that recognize specific RNA sequences. The PUF proteins are a widespread family of mRNA regulatory proteins that control key steps in early development and are required for establishing memory. They must find and recognize specific mRNAs, and then execute that mRNA's fate ? activation, repression, destruction, or movement. We elucidate the way in which these proteins form networks of control ? recognizing and controlling a substantial proportion of the mRNAs in human cells. They act through collaborations with protein partners ? interactions that are conserved from yeast to humans. PUF proteins and their partners have important roles in development, hoemostasis, cellular senescence, and human fertility. The networks of RNAs they control are vital in these processes.