Molecular Biology and Biochemistry Department Faculty Profile


	Catherine Phillips, Ph.D.
	Assistant Research Professor Back to Faculty Page	Dept. of Molecular Biology and Biochemistry Rutgers University Nelson Biological Laboratories, Room A322 604 Allison Road Piscataway, NJ 08854 (732) 445-1017 fax: (732) 445-4213 catphill@rci.rutgers.edu
Research Summary
The secretion of immunoglobulin during B cell development is tightly controlled. If this control is lost the resulting excessive antibody production leads to profound pathological effects including autoimmunity and immune complex disease which are major diseases the US as a Western nation with an aging affluent population. The production of secreted antibodies is controlled at the level of RNA processing of the Immunoglobulin M (µ) pre-mRNA and the activation of an alternative upstream secretory poly(A) site. The activation of alternative poly(A) sites is one of the basic mechanisms regulating alternative gene expression during cell development and differentiation. The µ-secretory poly(A) site represents the best characterized model of regulated poly(A) site usage. Furthermore, the pattern of gene expression used by the immunoglobulin gene locus is the classic model for a class of gene that follows the same pattern of processing which includes many receptors controlling cell growth and differentiation. Thus elucidation of this mechanism will have implications for a whole class of genes and provide avenues of treatment for a whole range of diseases. Furthermore, the study of a naturally regulated endogenous mammalian gene yields vital information not obtainable from the study of viral or synthetic poly(A) sites. In order to understand the regulation of the usage of the µ-secretory poly(A) site during B cell development it is crucial to have a precise understanding of the biochemical interactions involved. We have taken the approach to precisely define the cis- and trans-acting factors involved in this process on the molecular level using in vitro assays and recombinant and purified components. This is coupled with an understanding of the physiological significance of these elements in vivo using transfection of mutated genes or reporter constructs into B cell lines representing different stages of B cell development. Using this approach we have made a number of important contributions to the understanding of gene expression at the Immunoglobulin M locus. We identified the cis-acting elements at the immunoglobulin secretory poly(A) site and showed that they consist of bi-partite elements upstream and downstream of the site of cleavage and 3' end formation. This feature makes this poly(A) site inherently weak and regulatable during development. We showed that the distal downstream element is linearly placed too far downstsream of the cleavage site to optimally bind the polyadenylation/cleavage complex but is brought into close proximity via RNA structure which presents these sequences on the 5' surface a bent stem-loop. The full function of this poly(A) site is dependent on the intact structure of this stem loop in vivo and in vitro, demonstrating the importance of secondary RNA structure in the functioning of polyadenylation sites. We have showed that the secretory poly (A) site consists of a clover leaf of interconnected stem-loops presenting transacting factors in a "lazy susan" fashion that facillitates interactions between them. Two upstream stem-loops bind U1A, a component of the splicing aparatus, via novel suboptimal binding sites which we identified. Multiple U1A molecules act in unison to prevent poly(A) addition to the secretory poly(A) site in the nucleus and control expression of the secretory form of µ-mRNA at at the level of stability and nuclear to cytoplasmic transport. This is the first demonstration of the physiological importance of regulation of nuclear poly(A) addition in regulation of gene expression during development. We are now in the process of purifying a novel 30kD polypeptide whose induction in primary cells correlates with the switch to the usage of the secretory poly(A) site. It binds the downstream stem-loop necessary for the full function of the secretory poly(A) site and to maintain the secondary structure of the upstream stem loop that binds the distal U1A molecule. It thus represents one of the best candidates for a regulatory molecule for the µ-secretory poly(A) site.