The Role of p53-dependent Histone mRNA Processing in the Development of Cancer
Final Report Abstract
The initial step of gene expression for protein-coding genes involves the transcription of a target gene by RNA Polyerase II followed by the further modification of the primary transcript by various mRNA processing events such as 5’ end capping, splicing and 3’ end processing. The mRNAs for most protein-coding genes have a number of adenosines added to their 3’ end in a process called polyadenylation, which serves to stabilize the mRNA and improve its ability to be translated into a functional protein by ribosomes. In contrast to all other mRNAs, the mRNAs encoding histone proteins which are produced during cellular proliferation in order to repackage newly synthesized DNA (“replication-dependent histones”) are not polyadenylated, but instead contain a stem-loop structure with a well-defined sequence which is recognized by the stem-loop binding protein (SLBP). This specialized form of mRNA 3’ end processing helps to ensure that these histone proteins are only produced at the precise time when they are needed (i.e., while DNA is being newly synthesized). In our previous results, we discovered that a number of genes encoding “replication-dependent histones” are capable of producing polyadenylated mRNAs under certain conditions such as depletion of epigenetic regulatory proteins. In this project, we sought to build upon these findings by determining whether the production of these mRNAs also occurs under physiological conditions such as the activation of the p53 tumor suppressor pathway, during DNA damage (such as chemo- or radiotherapy) and during cellular differentiation. Moreover, we sought to determine the importance of this regulation during tumorigenesis. The findings of this project resulted in the publication of a number of manuscripts directly related to these hypotheses as well as several others which serendipitously benefitted from the findings of this project. In particular, we could show that stabilization of the tumor suppressor p53, resulting in an arrest in proliferation prior to the begin of new DNA synthesis (a “G1 cell cycle arrest”), leads to decreased recruitment of the Nuclear protein of the ataxia telangiectasia mutated locus (NPAT) to the genes encoding the histone proteins. In addition, we identified Cyclin-Dependent Kinase-9 (CDK9) as a direct interaction partner of NPAT and showed NPAT is required for the recruitment of CDK9 to the histone genes. This in turn has further effects on the epigenetic status of the histone genes by resulting in decreased recruitment of the Polymerase Associated Factor-1 (PAF1) protein, which is required for the epigenetic modification of histone H2B via monoubiquitination (H2Bub1). Our further studies show that many, but not all genes encoding “replication-dependent” histone proteins, are also able to produce polyadenylated mRNAs under certain conditions such as cell cycle arrest, DNA damage or cellular differentiation. Moreover, in contrast to the previous dogma that polyadenylated histone mRNAs produced from otherwise replication-dependent histone genes are non-functional, we have shown that the polyadenylated histone mRNAs produced under physiological conditions are transported from the nucleus to the cytoplasm and are found in polyribosomes, suggesting that they are translated and likely produce fully functional histone proteins. Additional future work will be necessary to further test the necessity and function of these polyadenylated histone mRNAs. However, this represents a significant challenge due to the large number of histone genes capable of producing polyadenylated mRNAs and to the difficulty in specifically targeting these mRNAs by classical approaches such as RNA interference. In order to more fully address the function of these mRNAs, more sophisticated approaches will be necessary. Nevertheless, the work performed in this project provides a basis for such future studies and demonstrates a novel regulatory mechanism for alternative 3’ end processing of mRNAs, which is regulated in a specific manner under various cellular conditions.
Publications
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2009. Insights into the function of the human P-TEFb component CDK9 in the regulation of chromatin modifications and cotranscriptional mRNA processing. Cell Cycle 8: 3636-3642
Pirngruber,J., A.Shchebet, and S.A.Johnsen
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2010. Induced G1 cell-cycle arrest controls replicationdependent histone mRNA 3' end processing through p21, NPAT and CDK9. Oncogene 29: 2853-2863
Pirngruber,J. and S.A.Johnsen
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2012. CDK9 and H2B Monoubiquitination: A Well-Choreographed Dance. PLoS Genet. 8(8): e1002860
Johnsen,S.A.
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2012. The enigmatic role of H2Bub1 in cancer. FEBS Lett. 586: 1592-1601
Johnsen,S.A.
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2012. The histone H2B monoubiquitination regulatory pathway is required for differentiation of multipotent stem cells. Mol. Cell 46: 705-713
Karpiuk,O., Z.Najafova, F.Kramer, M.Hennion, C.Galonska, A.König, N.Snaidero, T.Vogel, A.Shchebet, Y.Begus-Nahrmann, M.Kassem, M.Simons, H.Shcherbata, T.Beissbarth, and S.A.Johnsen
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2013. A subset of histone H2B genes produce polyadenylated mRNAs under a variety of cellular conditions. PLoS One 8:e63745
Kari,V., O.Karpiuk, B.Tieg, M.Kriegs, E.Dikomey, H.Krebber, Y.Begus-Nahrman, S.A.Johnsen
