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Full Stream Name: Vertebrate Interactome Mapping

Research Educator:  Al MacKrell

Principal Investigator: Scott Stevens

Credit Options: Spring & Fall 

How do complexes of macromolecules control gene expression?

Students in the Vertebrate Interactome Mapping (VIM) Stream investigate the ribonucleoprotein complexes (RNPs) that facilitate mammalian gene expression.  RNPs can regulate splicing to create multiple mRNAs from a single gene, and control the stability, cellular localization and translation of mRNAs produced.  We want to understand what RNPs do and how they function.  To do this, we are studying the function of DExH box RNA helicases, proteins that function in gene expression by remodeling ribonucleoproteins.  We are also studying the diverse collection of G patch domain containing proteins found in human cells.  Proteins in this family are frequently observed interacting with DExH proteins and regulating their specificity and activity.  Our primary approach involves the expression of affinity tagged proteins in mammalian cells, which permits the tag-mediated purification of complexes that contain the protein.  We use this approach to understand the functions of these proteins in normal cells, and their role in the pathology of cancer and developmental defects.

Students in the lab use molecular cloning techniques to build recombinant DNA plasmids in which the sequences encoding a protein have been engineered to add an affinity tag – an extra protein segment that makes the protein easy to both identify and to purify.  Using our knowledge of function of these proteins we introduce mutations into the proteins that should block their catalytic activity without altering their folding or interactions with other molecules.  We also introduce mutations that are associated with human genetic diseases and cancer.  Following expression, effects on cell function, proliferation and apoptosis are determined, shedding light on the role the protein plays in these processes.

These proteins are expressed in cultured mammalian cells, where they interact with their normal protein and RNA partners to form complexes.  RNPs including both normal and mutated proteins can be purified using the fused affinity tag.  As long as expression of the protein is not toxic to the cells, large quantities of cells stably expressing the tagged protein can be produced, facilitating this process.  Insight into the function(s) of the protein under study can be gained by the identity of RNAs that co-purify with it.  Proteins isolated in this process can be separated and identified by mass spectroscopy.  This information will provide clues as to the function of specific proteins, and the mechanisms by which their malfunction leads to disease.

 interactome1
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Biochemistry, Biology