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Ribonucleoprotein Complexes: Biogenesis, Function, and Roles in Human Disease
Summary: Gideon Dreyfuss is interested in RNA-binding proteins, RNA-protein complexes (RNPs), the transport of RNAs and proteins between the nucleus and cytoplasm, and the molecular functions of SMN, the protein responsible for the neurodegenerative disease spinal muscular atrophy.
From the sites of transcription in the nucleus to the outreaches of the cytoplasm, mRNAs are associated with RNA-binding proteins. These proteins, the hnRNP and mRNP proteins, profoundly influence pre-mRNA processing and the transport, localization, translation, and stability of mRNAs. The complexes they form with mRNAs and their precursors are specific and dynamic. Many RNP proteins bind nascent pre-mRNAs, export with the mRNAs to the cytoplasm, and shuttle back into the nucleus, while others associate later as a consequence of specific processes. We have identified and characterized the major proteins that interact with pre-mRNAs (hnRNAs) and mRNAs. Our studies of these RNP proteins have led to the identification of novel nuclear structures and transport pathways and opened up new areas of research on important human genetic diseases, including spinal muscular atrophy and fragile X syndrome.
The SMN Complex, an Assemblyosome of Ribonucleoproteins
Spinal muscular atrophy (SMA) is a common, often lethal, motor neuron degenerative disease that results from reduced levels of the survival of motor neurons (SMN) protein. We identified SMN in the course of our studies of hnRNP proteins, as it interacts with several of them, and we have gained some insights into the functions of this protein and the molecular mechanism of SMA.
The SMN protein is expressed in all eukaryotic organisms and in all cell types of vertebrate organisms. Particularly high levels of SMN are expressed in neuronal cells, including motor neurons of the spinal cord. SMN is found in both the cytoplasm and the nucleus, where it is concentrated in distinct nuclear structures called Gems that are related to and often associated with Cajal bodies. SMN is part of a large multiprotein complex. To date we have described most, if not all, of the components of the SMN complex, proteins we termed Gemins.
One of our main objectives is to determine the complete composition, interactions, structure, and functions of the SMN complex. This complex plays a critical role in the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and possibly other RNPs, in pre-mRNA splicing, and in the assembly of mRNA factories (transcriptosomes). The SMN complex is a macromolecular assembly machine (an assemblyosome) for RNPs. The need for such an activity was unanticipated because several RNPs have been known to self-assemble from purified components in vitro. In the cell, however, such assembly processes would probably be too inefficient and prone to inaccuracies without the assistance of the SMN complex. To assemble RNPs, the SMN complex must recognize and bring together their protein and RNA components. We found that SMN interacts directly with many protein substrates, most of which contain a domain enriched in arginines and glycines (RG-rich domain), a common motif in RNA-binding proteins. RG-rich domains are often modified post-translationally by the methylosome, an arginine-methyltransferase complex. This modifies specific protein arginines to dimethylarginines, enhancing the binding of the proteins (e.g., Sm snRNP proteins) to the SMN complex. In addition, the SMN complex binds specifically to the appropriate RNAs (e.g., snRNAs) by binding to specific code sequences they contain, and facilitates RNP assembly.
Further studies are aimed at understanding the detailed mechanism of RNP assembly and the role of the SMN complex in SMA pathogenesis.
Another major current effort is a high-throughput screening for small molecules as research tools for the SMN complex as well as potential therapeutics for SMA. Using cell-based assays we developed, we are searching for compounds that may increase the amount of SMN protein. We have also developed in vitro assays for the activity of the SMN complex and are screening for compounds that modulate it. (This work was also supported by a grant from the National Institutes of Health and by the Association Française contre les Myopathies.)
mRNA-Binding Proteins and Post-transcriptional Gene Expression
The major hnRNA- and mRNA-binding proteins in divergent eukaryotes have been identified and characterized, and monoclonal antibodies to many of them have been produced. We have defined the major hnRNP proteins, which in human cells comprise about 20 proteins designated A–U. The hnRNP proteins are RNA-binding proteins, and they are among the most abundant proteins in the nucleus. We have isolated and sequenced many of the hnRNP proteins and delineated several motifs for RNA binding and protein-protein interaction. These include the RNP motif (RBD or RRM), the RGG box (a cluster of arginine-glycine-glycine peptides), and the KH (K-homology) domain. The hnRNP proteins strongly affect the interactions of the RNAs to which they are bound, and they have functions in the intracellular transport and translation of mRNAs. The study of the structure and function of hnRNP and mRNP proteins is one of our long-standing interests.
We found that many of the hnRNP proteins shuttle continuously and rapidly between the nucleus and the cytoplasm. This suggested that they also have functions in the cytoplasm and possibly in the nuclear export of mRNAs. We identified sequences in the hnRNP proteins that specify their localization and transport in cells, including novel nuclear localization signals, NLSs) and several nuclear transport receptors that recognize these signals. Our continuing studies are focused on the pathway of mRNA export and on the specific role of nucleocytoplasmic shuttling RNP proteins in this process.
We are particularly interested in defining the molecular composition of postsplicing, pre-export nuclear mRNA-protein complexes (mRNPs) and in the events that occur in the nucleus immediately prior to mRNA export and up until the exported mRNA emerges in the cytoplasm. Recent discoveries revealed that newly exported mRNPs carry precisely positioned proteins, acquired by splicing, that mark exon-exon junctions (the exon-exon junction complex, EJC). We have recently described four protein components of the EJC, termed Y14, magoh, Upf3, and EIF4A3. These proteins bind preferentially to mRNAs that are produced by pre-mRNA splicing in the nucleus and remain bound to the newly exported mRNAs in the cytoplasm. In addition, the EJC contains Srm160, RNPS1, Aly/REF1, and the mRNA export factor TAP. Thus, the splicing-dependent binding of Y14, magoh, and possibly other EJC proteins provides a position-specific molecular memory that communicates to the cytoplasm the location of exon and intron boundaries. This mechanism likely plays an important role in coupling splicing and postsplicing events, including the surveillance of premature termination (nonsense) codons. EJC proteins also play a role in mRNA export and possibly also in cytoplasmic mRNA localization and translation. Current studies focus on the structure, assembly, and functions of the EJC proteins Y14, magoh, and EIF4A3. (Grants from the National Institutes of Health and the Human Frontier Science Program provided support for this work.)
Last updated May 03, 2007
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