Uniform TitleChanging U1A levels regulate expression of immunoglobulin M and the transcriptional repressor Zhx1 during B cell differentiation
NameMa, Jianglin (author), Gunderson, Samuel (chair), KILEDJIAN, MEGERDIT (internal member), Phillips, Catherine (internal member), kinzy, Terri (outside member), Rutgers University, Graduate School - New Brunswick,
DescriptionDuring B cell differentiation U1A plays an important role in regulating the expression of the secretory poly(A) site by inhibiting both cleavage and polyadenylation. Previous work demonstrated that the inhibitory effect of U1A is alleviated in differentiated cells, which express the secretory poly(A) site, however, the mechanism underneath was unveiled. Using B cell lines representing different stages of B cell differentiation, here we show that U1A levels are reduced in differentiated cells. Undifferentiated B cells have more total U1A than differentiated cells and a greater proportion of U1A is not associated with the U1snRNP. We demonstrate that this non-snRNP associated U1A is available to inhibit poly(A) addition at the secretory poly(A) site. In addition, endogenous non-snRNP associated U1A--immunopurified from the different cell lines--inhibited poly(A) polymerase activity proportional to U1A recovered, suggesting that available U1A level alone is responsible for changes in its inhibitory effect at the secretory IgM poly(A) site.
It is known that U1A can regulate the expression of its own and IgM gene. Here we report that during mouse B cell differentiation U1A also regulates the expression of the transcriptional repressor, Zhx-1 (zinc fingers and homeoboxes 1), via alternative poly(A) site selection. Using affymetrix microarray analysis combined with RT-PCR techniques, we demonstrate that U1A binds to Zhx-1 mRNA in vivo. We show that the levels of Zhx-1 proteins and mRNA are negatively correlated with U1A levels in B cells and overexpression of U1A in HeLa cells significantly inhibits the expression of Zhx-1. Our in vitro and in vivo assays show that U1A regulates the expression of the upstream poly (A) site of Zhx-1 by binding to the five non-consensus motifs around the poly(A) site and inhibiting both poly(A) addition and cleavage. When the upstream poly(A) site of Zhx-1 is inhibited in mature B cells, the usage of the downstream poly(A) site of Zhx-1 results in the inclusion of ARE elements, which destabilize the mRNA transcript. As a result, less Zhx-1 RNA and protein are produced in mature B cells. We proposed one model about how U1A and ARE coordinately regulate the expression of Zhx-1 during B cell differentiation.
NoteIncludes bibliographical references (p. 124-146).
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.