REGULATION OF THE 64-kDA SUBUNIT OF CLEAVAGE STIMULATORY FACTOR ACTIVITY IN MACROPHAGE AND B LYMPHOCYTE mRNA 3'-END PROCESSING
Year: 2005
Type: article
Abstract: Eukaryotic pre-mRNA is processed within the 3'-untranslated region (3'-UTR) resulting in cleavage and polyadenylation. The efficiency of the cleavage reaction is dependent on the binding activity of the 64-kDa subunit of CstF, CstF-64, to the pre-mRNA and is increased with elevated levels of CstF-64. There is evidence that alternative polyadenylation occurs in the presence of increased CstF-64. Our results showed that CstF-64 levels increased with LPS stimulation of RAW 264.7 macrophages while the expression of other pre-mRNA processing factors remained unchanged. Because of the evidence that several macrophage genes exhibit alternative polyadenylation and post-transcriptional regulation under LPS stimulation, we used a reporter mini-gene to identify alternative polyadenylation in LPS-stimulated RAW macrophages. Upon LPS stimulation we measured a 2.5-fold increase in proximal poly(A) site selection that correlated with elevated levels of CstF-64. Forced expression of CstF-64 demonstrated similar alternative polyadenylation. Microarray analysis demonstrated 515 genes changed expression with LPS stimulation, 15 of which also changed with CstF-64 over-expression. A closer analysis of 5 of these 15 genes demonstrated alternative polyadenylation within their 3'-UTR. Closer analysis of the 3'-UTRs showed putative AU-rich regulatory elements. There is also evidence that pre-mRNA processing is coupled with transcription. Previous work has shown that the carboxy-terminal domain (CTD) of RNAP-II is necessary for 3'-end processing, that CstF binds to RNAP-II CTD and that this binding is CTD phosphorylation dependent. Because our lab has also shown that increases in CstF-64 binding activity upon B-cell differentiation causes alternative polyadenylation on the Ig heavy chain gene and occurs in the absence of CstF-64 increases, we believe that the local concentration of CstF-64 to the nascent pre-mRNA is increased in plasma cells through the phosphorylation-dependent recruitment of CstF-64 to RNAP-II CTD. Using chromatin immunoprecipitation (ChIP), we measured an increase in Serine-2 and Serine-5 phosphorylation of the RNAP-II CTD at the promoter and variable regions of the Ig heavy chain gene in plasma cells compared to memory B cells. We believe that this increase in RNAP-II CTD phosphorylation plays a role in either increased transcription of the Ig heavy chain gene or recruitment of pre-mRNA processing factors to the transcriptional machinery.
Author: Scott A. Shell
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W636722722