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Regulation of RNA 3' end formation/ polyadenylation. RNA alternative processing. Immunoglobulin heavy chain mRNA. mouse B cell linesThe major source of diversity in metazoans is the ability to process RNA into alternative mRNA's via the selection of alternative splice sites and alternative 3' ends. giving rise to a range of proteins encoded by the same gene which are differentially expressed during growth and development (1). An important pattern of alternative processing involves a competition between splicing on of extra exons and 3' end cleavage at an upstream site which cuts off these exons. This pattern is used by a number of receptors involved in growth and differentiation (2). The best characterized model for this pattern is the IgM heavy chain gene which is alternatively processed into mRNA's encoding a membrane receptor or secreted antibody during differentiation by regulation of the expression of the secretory poly(A) site. Crucial to the activation of the secretory poly(A) site are changes in the binding of Cleavage stimulatory Factor 64K (CstF64K) to GU-rich elements downstream of the poly(A) site which are regulated by differentiation induced transacting factors. In addition. to prevent inappropriate expression of secreted antibody in undifferentiated cells. the stability of secretory mRNA is highly controlled. Changes in stability of the secretory mRNA during differentiation play a major role in the dramatic burst of production of secreted antibody once B cells differentiate into immunoglobulin secreting cells. We showed that the regulated poly(A) site contained dual upstream and downstream core polyadenylation elements and put forward a model to explain it weakness and regulatability (3,4). A key player is multiple U1A molecules which I showed bound both upstream and downstream of the cleavage site to regulate multiple levels of control: Downstream U1A binds between the two crucial GU-rich elements to inhibit cleavage and upstream it regulates post-cleavage poly(A) tail addition and stability (5. 6). U1A levels are regulated during B cell differentiation. so that the amount of U1A available to inhibit secretory poly(A) site expression decreases upon differentiation. Thus U1A is a/the key factor in the regulation of secretory poly(A) site expression. We are investigating the mechanism by which U1A levels are regulated during differentiation. We are now collecting evidence that U1A regulates other genes in the same way. An examination of sequences of other genes which follow the same pattern of regulation reveals clusters of U1A binding motifs surrounding poly(A) sites in 8 of 13 examined. These include the EGF-receptor and Her2. In addition. we are undertaking microarray analysis to identify other targets of U1A regulation. Preliminary results suggest that this is a general mechanism for regulating alternative processing of pre-mRNA. This work would therefore have a significant impact on the understanding of this type of processing and provide targets for drug discovery in the treatment of diseases including autoimmunity and cancer. Selected PublicationsMa J, Gunderson SI, Phillips C. (2006) Non-snRNP U1A levels decrease during mammalian B-cell differentiation and release the IgM secretory poly(A) site from repression. RNA. 12(1):122-32. Phillips. C.. Pachikara. N. and Gunderson. S.I. (2004) U1A inhibits cleavage at the IgM heavy chain secretory poly(A) by binding between the two downstream GU-rich regions. Molecular and Cellular Biology 24:6162-6171. Phillips. C. and Gunderson S.I. (2003) Sequences adjacent to the 5' splice site control U1A binding upstream of the IgM heavy chain secretory poly(A) site. Journal of Biological Chemistry 278:22102-22111. Phillips. C.. Jung S and Gunderson S.I. (2001) Regulation of nuclear poly(A) addition controls the expression of the secretory form of immunoglobulin M-mRNA. EMBO J. 20:1-10. Phillips. C. . Kyriakopoulou. C. B. and Virtanen. A. (1999) Identification of a stem-loop structure important for polyadenylation at the murine IgM secretory poly(A) site. Nucleic Acids Research 27:429-438. Phillips. C. and Virtanen. A. (1997) The murine IgM secretory poly(A) site contains dual upstream and downstream elements which affect polyadenylation. Nucleic Acids Research 25:2344-2351. Phillips. C. and Virtanen. A. (1997) Multiple sequence elements involved in polyadenylation at the murine IgM secretory poly(A) site. Nucleosides and Nucleotides 16:737-742. Phillips. C. . Schimpl. A.. Dietrich-Goetz. W.. Clements. J.B.. and Virtanen. A. (1996) Inducible nuclear factors binding the IgM heavy chain pre-mRNA secretory poly(A) site. European Journal of Immunology 26:3144-3152. Phillips. C. and Klaus. G.G.B. (1993) Crosslinking of sIgM. but not sIgD receptors. by soluble monoclonal antibodies prime murine B cells to secrete Ig in response to lymphokines. European Journal of Immunology 23:574-577. Phillips. C. and Klaus. G.G. B. (1992) Soluble anti-µ monoclonal antibodies prime resting B cells to differentiate in response to Interleukin 4 and Interleukin 5. European Journal of Immunology 22:1541-1546. Phillips. C. (1991) Induction of leukotriene production before antigen challenge enhances antibody affinity in genetically selected affinity non-maturing mice. Cellular Immunology. 136:173-184. Phillips. C. (1991) Prostaglandin E2 controls antibody affinity in genetically selected mice. in Advances in Prostaglandin. Thomboxane and Leukotriene Research Vol 21. Samuelson. B.. Ramwell. P.W.. Paoletti. R.. Folco. G. and Granstrom. E. (eds.) p.497. Raven Press. N.Y. Phillips. C. (1989) Prostaglandin E2 production is enhanced in mice genetically selected to produce high affinity antibody responses. Cellular Immunology 119:382-392. |
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