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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 by regulating transcription, creating multiple mRNAs via regulated splicing, and controlling the stability, cellular localization and translation of mRNAs produced.  While a lot is known about RNPs in Saccharomyces cerevisiae, we aim to better understand the RNPs in mammalian cells that participate in gene expression, and are key gatekeepers of complexity.  To understand what RNPs do and how they function, we have to understand how they are constructed and the function of each of their components at the molecular level.  To accomplish this, students in our stream study proteins that have been implicated in RNA processing and metabolism, but whose functions are not well understood.  To begin investigating the function of a novel protein we seek to find out what other proteins it interacts with to form complexes, and to map the interactome or “social network” of the nucleus. 

We use molecular cloning and site directed recombination to build DNA molecules 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 purify.  Sometimes we alter the amino acid sequences of the proteins to be expressed to “break” them in specific ways.  These synthetic DNAs are introduced into cultured cells, where they direct the production of proteins that interact with their normal macromolecular partners.  Using the associated affinity tag, the protein can be purified, and any proteins (or other macromolecules) that were associated with it in the cell will remain attached and will be co-purified.  These proteins can be separated and identified by mass spectroscopy.  This will provide clues as to the function of specific proteins, and the mechanisms by which they perform those functions.

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Biology