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Abstract

Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism in many biological processes, including immunity. Viral and bacterial infections have been shown to affect polyA site (PAS) selection of mRNAs. In the first part of my thesis the I investigated the role of the cleavage factor Im (CFIm) complex for the translational regulation of the Nfkbid mRNA. The CFIm complex mediates PAS selection in favour of long isoforms. Previously, it has been shown that CFIm binds to the constitutive decay element (CDE) of Nfkbid mRNA, a sequence motif that induces mRNA decay and mediates translational regulation of Nfkbid. I addressed the hypothesis that translational regulation of Nfkbid is mediated by CFIm binding and APA. I used isoform-specific primers for the “common” and “extended” regions of the Nfkbid mRNA to assess APA. To measure translation, of the Nfkbid mRNA I performed polysome profiling of RAW264.7 macrophages after CFIm knockdown and 1 h lipopolysaccharide (LPS) stimulation. I could not observe any changes in translation of Nfkbid isoforms upon CFIm knockdown. Since the CDE is known as a decay element, I also measured stability of the Nfkbid mRNA in LPS stimulated RAW264.7 macrophages upon CFIm knockdown. I could not observe any isoform specific changes in the stability of the Nfkbid mRNA upon CFIm knockdown. Using northern blotting, I demonstrated that the Nfkbid mRNA employs only one PAS. In the second part of my thesis, I investigated the general role of APA after 1 h and 16 h of LPS stimulation using 3’ end sequencing to detect PASs. While after 1 h, only a few APA events could be observed, massive shortening of mRNAs was observed after 16 h of LPS. Among the shortened mRNAs, many immune related mRNAs of signalling molecules such as Nfkb1 and Nfkb2 were detected. Many mRNAs subjected to APA events were also found to either lose or gain an AU-rich element, which regulates stability and decay of mRNAs. I could not find any evidence that APA correlates with mRNA levels. After 16 h of LPS, I also observed reduced levels of CFIm25 mRNA and protein. Therefore, I performed CFIm25 knockdown combined with 3’ end sequencing. CFIm25 knockdown led to massive shortening of mRNAs and around 80% of mRNAs shortened after 16 h LPS were also shortened after CFIm25 knockdown. Among the affected mRNAs, I found Nfkb1, which encodes the Nf-B components p105/p50. CFIm25 knockdown led to increased levels of p50 and p105. Furthermore, the secretion of cytokines and chemokines was affected by the knockdown. For example, increased levels of Tnf, Cxcl2 and Ccl5 protein were observed. Taken together, my analysis indicates that APA plays an important role in macrophage polarization, and that loss of CFIm activity due to LPS treatment or knockdown leads to deregulated production of pro-inflammatory cytokines.