Clinton C. MacDonald, Ph.D.
Tissue-specific mechanisms of mRNA processing, polyadenylation, control of RNA processing during spermatogenesis and brain development.
Role of τCstF-64 (gene symbol Cstf2t) in polyadenylation of germ cell mRNAs.
Mechanisms of polyadenylation are different in male germ cells, and we investigate the consequences of those differences. One of those differences is that testes express a variant form of the CstF-64 polyadenylation protein that we named τCstF-64 (Cstf2t). We have found that male Cstf2t knockout mice are infertile due to incorrect expression of key spermatogenesis genes including intronless small genes and LINE repetitive elements. Current projects are to determine how τCstF-64 controls polyadenylation of those mRNAs.
Role of CstF-64 splice variants in brain function.
There is a spliced variant of CstF-64 that is expressed only in neurons, which introduces a new domain in CstF-64. We call this spliced variant ßCstF-64. We are trying to determine the how this variant functions in brains and why it is important.
The functions of CstF-64 in control of polyadenylation.
Discovered in 1988, CstF-64 has been shown to be the central regulatory protein in polyadenylation. Despite its importance, many of its functions remain mysteries. We have developed several assays for CstF-64 and are using them to test functions of its individual domains.
- Grozdanov, P. N., Amatullah, A., Graber, J. H., and MacDonald, C. C. (2016). “τCstF-64 mediates correct mRNA polyadenylation and splicing of activator and repressor isoforms of the cyclic AMP-responsive element modulator (CREM) in mouse testis.” Biology of Reproduction, in the press.
- Grozdanov, P. N. and MacDonald, C. C. (2015) “Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor–1α promoter using the Gibson assembly cloning.” J. Visual. Exp. in the press (doi: 10.3791/52235).
- Youngblood, B. A. and MacDonald, C. C. (2014) “CstF–64 is necessary for endoderm differentiation resulting in cardiomyocyte defects.” Stem Cell Res. 13(3A), 413–421 (doi: 10.1016/j.scr.2014.09.005). PubMed
- Youngblood, B. A., Grozdanov, P. N., and MacDonald, C. C. (2014) “CstF–64 supports pluripotency and cell cycle progression in embryonic stem cells through histone 3′ end processing.” Nucl. Acids Res. 42(13), 8330–8342 (doi: 10.1093/nar/gku551). PubMed
- Alfano, R., Youngblood, B. A., Zhang, D., Huang, N., and MacDonald, C. C. (2014) “Human Leukemia Inhibitory Factor produced by the ExpressTec method from rice (Oryza sativa L.) is active in human neural stem cells and mouse induced pluripotent stem cells,” Bioengineered 5(3), 180–185 (doi: 10.4161/bioe.28996). PubMed
- Youngblood, B. A., Alfano, R., Pettit, S., Zhang, D., Dallmann, H. G., Huang, N., and MacDonald, C. C. (2014) “Application of recombinant human Leukemia Inhibitory Factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells.” J. Biotechnol. 172, 67–72 (doi: 10.1016/j.jbiotec.2013.12.012). PubMed
- Grozdanov, P. N. and MacDonald, C. C. (2014) “High-throughput Sequencing of RNA isolated by Crosslinking and Immunoprecipitation (HITS-CLIP) to determine sites of binding of CstF–64 on nascent RNAs.” Methods in Molecular Biology 1125, 187–208 (doi: 10.1007/978–1–62703–971–0_17). PubMed
- Hockert, J. A. and MacDonald, C. C. (2014) “The Stem-Loop Luciferase Assay for Polyadenylation (SLAP) method for determining CstF–64-dependent polyadenylation activity.” Methods in Molecular Biology 1125, 109–117 (doi: 10.1007/978–1–62703–971–0_9). PubMed
- Shankarling, G. S. and MacDonald, C. C. (2013) “Polyadenylation site-specific differences in the activity of the neuronal βCstF–64 protein in PC–12 cells.” Gene 529, 220–227 (doi: 10.1016/j.gene.2013.08.007). PubMed
- Li, W., Yeh, Hsiang-Jui, Shankarling, G. S., Tian, B., and MacDonald, C. C. (2012) “The τCstF–64 polyadenylation protein controls genome expression in testis.” PLoS ONE 7, e48373 (doi: 10.1371/journal.pone.0048373). PubMed
- Hockert, K. J., Martincic, K., Mendis-Handagama, S. M. L. C., Borghesi, L. A., Milcarek, C., Dass, B., and MacDonald, C. C. (2011) “Spermatogenic but not immunologic defects in mice lacking the τCstF-64 polyadenylation protein,” Journal of Reproductive Immunology, 89, 26–37 (doi: 10.1016/j.jri.2011.01.018). PubMed
- Tardif, S., Akrofi, A., Dass, B., Hardy, D. M., and MacDonald, C. C. (2010) “Infertility with impaired zona pellucida adhesion of spermatozoa from mice lacking τCstF-64.” Biology of Reproduction 83, 464–472. PubMed
- MacDonald, C. C. and McMahon, K. W. (2010) “Tissue-Specific Mechanisms of Alternative Polyadenylation: Testis, Brain and Beyond,” WIREs RNA 1, 494–501 PubMed
- Hockert, J. A., Yeh, H-J. and MacDonald, C. C. (2010). “The hinge domain of the cleavage stimulation factor protein CstF-64 is essential for CstF-77 interaction, nuclear localization, and polyadenylation,” J. Biol. Chem., 285, 695–704. This manuscript was chosen as Paper of the Week by the Editors of the Journal of Biological Chemistry. PubMed
- Shankarling, G., Coates, P. W., Dass, B., and MacDonald, C. C. (2009) “A family of splice variants of CstF-64 expressed in vertebrate nervous systems,” BMC Molecular Biology 10, 22. PubMed
- Dass, B., Tardif, S., Park, J-Y., Tian, B., Weitlauf, H. M., Hess, R. A., Carnes, K., Griswold, M. D., Small, C. L., and MacDonald, C. C. (2007). “Loss of polyadenylation protein τCstF-64 causes spermatogenic defects and male infertility.” Proc. Natl. Acad. Sci., USA 104, 20374–20379. PubMed
- Liu, D., Brockman, J. M., Dass, B., Hutchins, L. N., McCarrey, J. R., MacDonald, C. C., Singh, P., and Graber, J. H. (2007). “Systematic variation in mRNA 3′-processing signals during mouse spermatogenesis.” Nucleic Acids Research 35, 234–246. PubMed
- Monarez, R. R., MacDonald, C. C., and Dass, B. (2007). “Polyadenylation Proteins CstF-64 and τCstF-64 Exhibit Differential Binding Affinities for RNA Polymers.” Biochemical J. 401, 651–658. PubMed
- McMahon, K. W., Hirsch, B. A., and MacDonald, C. C. (2006). “Differences in polyadenylation site choice between somatic and male germ cells.” BMC Mol. Biol. 7, 35. PubMed
- D’mello, V., Lee, J. Y., MacDonald, C. C., and Tian, B. (2006). “Alternative mRNA polyadenylation can potentially affect detection of gene expression by Affymetrix GeneChip® arrays.” Applied Bioinformatics 5, 249–253. PubMed
- Huber, Z., Monarez, R. R., Dass, B., and MacDonald, C. C. (2005). “The mRNA encoding τCstF-64 is expressed ubiquitously in mouse and rat tissues.” Ann. NY Acad. Sci. 1061, 163–172. PubMed
- Wallace, A. M., Denison, T. L., Attaya, E. N. and MacDonald, C. C. (2004). “Developmental differences in expression of two forms of the CstF-64 polyadenylation protein in rat and mouse.” Biol. Reprod. 70, 1080–1087. PubMed
- Dass, B., McDaniel, L., Schultz, R. A., Attaya, E. and MacDonald, C. C. (2002). “The gene CSTF2T encoding the human variant CstF-64 polyadenylation protein τCstF-64 is intronless and may be associated with male sterility.” Genomics 80, 509–514. PubMed
- Dass, B., Attaya, E. N., Wallace, A. M. and MacDonald, C. C. (2001). “Overexpression of the CstF-64 and CPSF-160 polyadenylation protein mRNAs in mouse male germ cells.” Biol. Reprod. 64, 1722–1729. PubMed
- Dass, B., McMahon, K. W., Jenkins, N. A., Gilbert, D. J., Copeland, N. G. and MacDonald, C. C. (2001). “The gene for a variant form of the polyadenylation protein CstF-64 is on chromosome 19 and is expressed in pachytene spermatocytes in mice.” J. Biol. Chem. 276, 8044–8050. PubMed