Here I report the profiling of the transcriptome at single nucleotide resolution through two continuous mitotic cycles with a comprehensive analysis. I identified more than 1,000 cell cycle-dependent transcripts, including 40 long non-coding RNA that are periodically expressed during the cell cycle. Furthermore, I found widespread coordination of periodic alternative splicing with cell division, a new observation that presents a novel regulatory mode for cell cycle progression. Mechanistically, I determined how cell cycle-dependent alternative splicing is regulated by a protein kinase CLK1. In addition, inhibition of CLK1 led to inhibition of mitotic progression and cellular proliferation, suggesting that the temporal regulation of splicing during cell cycle is critical for progression through the cell division cycle. Our work is summarized in the model below (Figure 6.1). In addition, I developed mitotic trait, a computational method for inferring cell cycle stage from gene expression data. Mitotic trait analysis of normal and tumor tissues demonstrates the robustness of this approach in inferring cell cycle differences across these samples. I also noted loss of G1 state in p53-null tumors, again suggesting the power of this tool in corroborating previously known information.
Still many questions remain: Why is the cell cycle-dependent transcriptome so cell type-dependent? This question emanates from the very modest overlap of periodic genes across multiple cell lines (only 67 of close to 3,000 identified). It would be of great value to the cell cycle field if a single laboratory could measure the periodic transcriptome
103
across a panel of cell lines utilizing the same techniques. This approach should eliminate the experimental differences across laboratories. This is especially interesting when it comes to the differences between normal and cancer cell lines, as tumor cells have poorly regulated cell cycle. Furthermore, if inherent cell line (or tissue)-dependent differences are observed, the underlying regulatory factors accounting for these changes could be studied.
Is periodic AS a general feature of cell cycle regulation in other cell types and organisms? How frequently does periodic AS alter protein function? Alternative splicing is undoubtedly a critical component of gene expression and is highly tissue-specific. My work opens a new area of study during cell cycle. It will be important to see how general periodic splicing is, and if the regulated genes are conserved from cell-to-cell. The overall consequence of periodic splicing on protein sequence and function can be very difficult to assess, however I do know that at least a subset of the identified periodic events lead to loss of protein production (greater than 30%). A future direction is to annotate how subtle changes in the amino acid composition of polypeptides as a consequence of AS alters their function.
What are the other regulators of periodic AS besides CLK1? I demonstrated that hundreds of RNA-binding protein show periodic fluctuations at the RNA level, however if these changes are also observed at the protein level is still unclear. The presence of cis sequence motifs in periodic exons leads me to hypothesize that there is a complex regulatory network that controls periodic AS. In the future it will be beneficial to determine how the cell coordinates this network to achieve a faithful cell division.
104
CLK1 emerged as an important molecule that controls cell cycle via AS regulation, a function that had not yet been assigned to this protein. How is CLK1 protein degraded during cell cycle (E3 ligase)? I can clearly show that an auto-inhibitory loop exists to control CLK1 periodic expression, but the rest of the molecular machinery that controls CLK1 is still unknown. In preliminary experiments, I identified the Skp2-Cul1-F-box complex as a putative E3 ligase. This complex seems like an ideal candidate because of the timing CLK1 protein turnover and the pre-requisite for phosphorylation. Can CLK1 substrates whose phosphorylation depends on cell cycle stage be identified? Using mass spectrometry I identified a set of proteins whose phosphorylation changed upon treatment with TG003, although most these proteins are indeed RNA-binding factors these results remain preliminary and have not yet been validated. From a cancer and therapeutic perspective, are CLK1 inhibitors effective in blocking tumor cell proliferation in animal models and, if so, does the mechanism involve splicing regulation and cell cycle?
Together this work delineates a new regulatory pathway involved in cell cycle control. This work opens a new area of study in cell cycle regulation. To the best of our knowledge this represents the first demonstration that alternative splicing is cell cycle- dependent.
105
Figure 6.1. Model. Periodic expression of lncRNAs and periodic alternative splicing occurs during cell cycle. These mechanisms complement the classical regulators of cell cycle progression shown in gray. CLK1 was shown to auto-regulate its own protein levels during cell cycle via the ubiquitin-proteasome pathway. I propose that CLK1 differentially phosphorylates SR proteins during cell cycle to control a splicing network of genes with cell cycle functions, many of which are periodically spliced. Disruption of CLK1 activity leads to cell pronounced cycle defects.
106
109
REFERENCES
Acquaviva, C. and J. Pines (2006). "The anaphase-promoting complex/cyclosome: APC/C." J Cell Sci 119(Pt 12): 2401-2404.
Ahn, E. Y., R. C. DeKelver, et al. (2011). "SON controls cell-cycle progression by coordinated regulation of RNA splicing." Mol Cell 42(2): 185-198.
Anczukow, O., A. Z. Rosenberg, et al. (2012). "The splicing factor SRSF1 regulates apoptosis and proliferation to promote mammary epithelial cell transformation." Nat Struct Mol Biol 19(2): 220-228.
Bahler, J. (2005). "Cell-cycle control of gene expression in budding and fission yeast." Annual review of genetics 39: 69-94.
Baltz, A. G., M. Munschauer, et al. (2012). "The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts." Molecular cell 46(5): 674-690. Bar-Joseph, Z., Z. Siegfried, et al. (2008). "Genome-wide transcriptional analysis of the
human cell cycle identifies genes differentially regulated in normal and cancer cells." Proceedings of the National Academy of Sciences of the United States of America 105(3): 955-960.
Barash, Y., J. A. Calarco, et al. (2010). "Deciphering the splicing code." Nature 465(7294): 53-59.
Bartek, J., J. Bartkova, et al. (2007). "DNA damage signalling guards against activated oncogenes and tumour progression." Oncogene 26(56): 7773-7779.
Bartek, J. and J. Lukas (2003). "Chk1 and Chk2 kinases in checkpoint control and cancer." Cancer Cell 3(5): 421-429.
Bartek, J. and J. Lukas (2007). "DNA damage checkpoints: from initiation to recovery or adaptation." Curr Opin Cell Biol 19(2): 238-245.
Batista, P. J. and H. Y. Chang (2013). "Long noncoding RNAs: cellular address codes in development and disease." Cell 152(6): 1298-1307.
110
Ben-David, Y., K. Letwin, et al. (1991). "A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators." The EMBO journal 10(2): 317-325.
Ben-Yehuda, S., I. Dix, et al. (2000). "Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae." Genetics 156(4): 1503-1517.
Bertoli, C., J. M. Skotheim, et al. (2013). "Control of cell cycle transcription during G1 and S phases." Nature reviews. Molecular cell biology 14(8): 518-528.
Bettencourt-Dias, M., R. Giet, et al. (2004). "Genome-wide survey of protein kinases required for cell cycle progression." Nature 432(7020): 980-987.
Blaustein, M., F. Pelisch, et al. (2005). "Concerted regulation of nuclear and cytoplasmic activities of SR proteins by AKT." Nat Struct Mol Biol 12(12): 1037-1044.
Blencowe, B. J. (2003). "Splicing regulation: the cell cycle connection." Curr Biol 13(4): R149-151.
Blencowe, B. J., S. Ahmad, et al. (2009). "Current-generation high-throughput sequencing: deepening insights into mammalian transcriptomes." Genes Dev 23(12): 1379-1386.
Braude, P., V. Bolton, et al. (1988). "Human gene expression first occurs between the four- and eight-cell stages of preimplantation development." Nature 332(6163): 459-461.
Brugiolo, M., L. Herzel, et al. (2013). "Counting on co-transcriptional splicing." F1000prime reports 5: 9.
Bullock, A. N., S. Das, et al. (2009). "Kinase domain insertions define distinct roles of CLK kinases in SR protein phosphorylation." Structure 17(3): 352-362.
Cappell, K. M., B. Larson, et al. (2010). "Symplekin specifies mitotic fidelity by
supporting microtubule dynamics." Molecular and cellular biology 30(21): 5135- 5144.
111
Castello, A., B. Fischer, et al. (2012). "Insights into RNA biology from an atlas of mammalian mRNA-binding proteins." Cell 149(6): 1393-1406.
Chen, T. W., T. H. Wu, et al. (2011). "Interrogation of alternative splicing events in duplicated genes during evolution." BMC Genomics 12 Suppl 3: S16. Chibon, F. (2013). "Cancer gene expression signatures - the rise and fall?" Eur J
Cancer 49(8): 2000-2009.
Cho, R. J., M. J. Campbell, et al. (1998). "A genome-wide transcriptional analysis of the mitotic cell cycle." Molecular cell 2(1): 65-73.
Cho, R. J., M. Huang, et al. (2001). "Transcriptional regulation and function during the human cell cycle." Nature genetics 27(1): 48-54.
Christensen, J., P. Cloos, et al. (2005). "Characterization of E2F8, a novel E2F-like cell- cycle regulated repressor of E2F-activated transcription." Nucleic Acids Res 33(17): 5458-5470.
Ciriello, G., M. L. Miller, et al. (2013). "Emerging landscape of oncogenic signatures across human cancers." Nat Genet 45(10): 1127-1133.
Clift, D. and M. Schuh (2013). "Restarting life: fertilization and the transition from meiosis to mitosis." Nature reviews. Molecular cell biology 14(9): 549-562. Coller, H. A., L. Sang, et al. (2006). "A new description of cellular quiescence." PLoS
Biol 4(3): e83.
Colwill, K., L. L. Feng, et al. (1996). "SRPK1 and Clk/Sty protein kinases show distinct substrate specificities for serine/arginine-rich splicing factors." J Biol Chem 271(40): 24569-24575.
Colwill, K., T. Pawson, et al. (1996). "The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution." Embo J 15(2): 265- 275.
Cooper, T. A. (2005). "Alternative splicing regulation impacts heart development." Cell 120(1): 1-2.
112
Cramer, P., J. F. Caceres, et al. (1999). "Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer." Mol Cell 4(2): 251-258.
Diehl, J. A., M. Cheng, et al. (1998). "Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization." Genes Dev 12(22): 3499-3511. Duncan, P. I., D. F. Stojdl, et al. (1997). "In vivo regulation of alternative pre-mRNA
splicing by the Clk1 protein kinase." Molecular and cellular biology 17(10): 5996- 6001.
Duncan, P. I., D. F. Stojdl, et al. (1998). "The Clk2 and Clk3 dual-specificity protein kinases regulate the intranuclear distribution of SR proteins and influence pre- mRNA splicing." Exp Cell Res 241(2): 300-308.
Dyson, N. (1998). "The regulation of E2F by pRB-family proteins." Genes Dev 12(15): 2245-2262.
Edmond, V., G. Merdzhanova, et al. (2013). "A new function of the splicing factor
SRSF2 in the control of E2F1-mediated cell cycle progression in neuroendocrine lung tumors." Cell cycle 12(8): 1267-1278.
Edmond, V., E. Moysan, et al. (2011). "Acetylation and phosphorylation of SRSF2 control cell fate decision in response to cisplatin." The EMBO journal 30(3): 510- 523.
Engelmann, D. and B. M. Putzer (2012). "The dark side of E2F1: in transit beyond apoptosis." Cancer Res 72(3): 571-575.
Escudero-Paunetto, L., L. Li, et al. (2010). "SR proteins SRp20 and 9G8 contribute to efficient export of herpes simplex virus 1 mRNAs." Virology 401(2): 155-164. Eser, U., M. Falleur-Fettig, et al. (2011). "Commitment to a cellular transition precedes
genome-wide transcriptional change." Mol Cell 43(4): 515-527.
Fairbrother, W. G., R. F. Yeh, et al. (2002). "Predictive identification of exonic splicing enhancers in human genes." Science 297(5583): 1007-1013.
113
Fedorov, O., K. Huber, et al. (2011). "Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing." Chem Biol 18(1): 67-76.
Foley, E. A. and T. M. Kapoor (2013). "Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore." Nat Rev Mol Cell Biol 14(1): 25-37. Fregoso, O. I., S. Das, et al. (2013). "Splicing-factor oncoprotein SRSF1 stabilizes p53
via RPL5 and induces cellular senescence." Molecular cell 50(1): 56-66. Fu, X. D. (2004). "Towards a splicing code." Cell 119(6): 736-738.
Fukuoka, M., A. Uehara, et al. (2013). "Identification of preferentially reactivated genes during early G1 phase using nascent mRNA as an index of transcriptional activity." Biochem Biophys Res Commun 430(3): 1005-1010.
Gabut, M., P. Samavarchi-Tehrani, et al. (2011). "An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming." Cell 147(1): 132-146.
Gardina, P. J., T. A. Clark, et al. (2006). "Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array." BMC Genomics 7: 325.
Geng, Y., Y. M. Lee, et al. (2007). "Kinase-independent function of cyclin E." Mol Cell 25(1): 127-139.
Ghigna, C., S. Giordano, et al. (2005). "Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene." Mol Cell 20(6): 881-890. Ghosh, A., D. Stewart, et al. (2004). "Regulation of human p53 activity and cell
localization by alternative splicing." Mol Cell Biol 24(18): 7987-7997.
Ghosh, G. and J. A. Adams (2011). "Phosphorylation mechanism and structure of serine-arginine protein kinases." FEBS J 278(4): 587-597.
Glotzer, M., A. W. Murray, et al. (1991). "Cyclin is degraded by the ubiquitin pathway." Nature 349(6305): 132-138.
114
Grant, G. D., L. Brooks, 3rd, et al. (2013). "Identification of cell cycle-regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors." Mol Biol Cell 24(23): 3634-3650.
Grant, G. D., J. Gamsby, et al. (2012). "Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control." Mol Biol Cell 23(16): 3079-3093.
Gui, J. F., W. S. Lane, et al. (1994). "A serine kinase regulates intracellular localization of splicing factors in the cell cycle." Nature 369(6482): 678-682.
Han, H., M. Irimia, et al. (2013). "MBNL proteins repress ES-cell-specific alternative splicing and reprogramming." Nature 498(7453): 241-245.
Harbour, J. W., R. X. Luo, et al. (1999). "Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1." Cell 98(6): 859-869.
Hayashi, M. T., A. J. Cesare, et al. (2012). "A telomere-dependent DNA damage checkpoint induced by prolonged mitotic arrest." Nat Struct Mol Biol 19(4): 387- 394.
Holley, S. L., J. Heighway, et al. (2005). "Induced expression of human CCND1 alternative transcripts in mouse Cyl-1 knockout fibroblasts highlights functional differences." Int J Cancer 114(3): 364-370.
Huang, Y., W. Wang, et al. (2012). "CENP-E kinesin interacts with SKAP protein to orchestrate accurate chromosome segregation in mitosis." The Journal of biological chemistry 287(2): 1500-1509.
Hughes, M. E., G. R. Grant, et al. (2012). "Deep sequencing the circadian and diurnal transcriptome of Drosophila brain." Genome Res 22(7): 1266-1281.
Hwang, H. C. and B. E. Clurman (2005). "Cyclin E in normal and neoplastic cell cycles." Oncogene 24(17): 2776-2786.
Hwang, L. H., L. F. Lau, et al. (1998). "Budding yeast Cdc20: a target of the spindle checkpoint." Science 279(5353): 1041-1044.
115
Iyer, V. R., M. B. Eisen, et al. (1999). "The transcriptional program in the response of human fibroblasts to serum." Science 283(5398): 83-87.
Jiang, K., N. A. Patel, et al. (2009). "Akt2 regulation of Cdc2-like kinases (Clk/Sty), serine/arginine-rich (SR) protein phosphorylation, and insulin-induced alternative splicing of PKCbetaII messenger ribonucleic acid." Endocrinology 150(5): 2087- 2097.
Johnson, D. G. and R. Schneider-Broussard (1998). "Role of E2F in cell cycle control and cancer." Front Biosci 3: d447-448.
Johnson, K. W. and K. A. Smith (1991). "Molecular cloning of a novel human
cdc2/CDC28-like protein kinase." The Journal of biological chemistry 266(6): 3402-3407.
Kaida, D., H. Motoyoshi, et al. (2007). "Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA." Nat Chem Biol 3(9): 576-583. Kalnina, Z., P. Zayakin, et al. (2005). "Alterations of pre-mRNA splicing in cancer."
Genes Chromosomes Cancer 42(4): 342-357.
Karlas, A., N. Machuy, et al. (2010). "Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication." Nature 463(7282): 818-822.
Karni, R., E. de Stanchina, et al. (2007). "The gene encoding the splicing factor SF2/ASF is a proto-oncogene." Nat Struct Mol Biol 14(3): 185-193.
Karni, R., Y. Hippo, et al. (2008). "The splicing-factor oncoprotein SF2/ASF activates mTORC1." Proc Natl Acad Sci U S A 105(40): 15323-15327.
Kastan, M. B. and J. Bartek (2004). "Cell-cycle checkpoints and cancer." Nature 432(7015): 316-323.
Katz, Y., E. T. Wang, et al. (2010). "Analysis and design of RNA sequencing experiments for identifying isoform regulation." Nature methods 7(12): 1009- 1015.
116
Kim, S. H., D. P. Lin, et al. (1998). "Fission yeast Slp1: an effector of the Mad2- dependent spindle checkpoint." Science 279(5353): 1045-1047.
Kim, Y., J. E. Heuser, et al. (2008). "CENP-E combines a slow, processive motor and a flexible coiled coil to produce an essential motile kinetochore tether." The Journal of cell biology 181(3): 411-419.
Kim, Y., A. J. Holland, et al. (2010). "Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E." Cell 142(3): 444-455. Koboldt, D. C., K. M. Steinberg, et al. (2013). "The next-generation sequencing
revolution and its impact on genomics." Cell 155(1): 27-38.
Kornblihtt, A. R. (2007). "Coupling transcription and alternative splicing." Adv Exp Med Biol 623: 175-189.
Kornblihtt, A. R., I. E. Schor, et al. (2013). "Alternative splicing: a pivotal step between eukaryotic transcription and translation." Nature reviews. Molecular cell biology 14(3): 153-165.
Kpebe, A. and L. Rabinow (2008). "Dissection of darkener of apricot kinase isoform functions in Drosophila." Genetics 179(4): 1973-1987.
Kurokawa, K., Y. Akaike, et al. (2013). "Downregulation of serine/arginine-rich splicing factor 3 induces G1 cell cycle arrest and apoptosis in colon cancer cells." Oncogene.
Lane, K. R., Y. Yu, et al. (2013). "Cell cycle-regulated protein abundance changes in synchronously proliferating HeLa cells include regulation of pre-mRNA splicing proteins." PloS one 8(3): e58456.
Laoukili, J., M. R. Kooistra, et al. (2005). "FoxM1 is required for execution of the mitotic programme and chromosome stability." Nat Cell Biol 7(2): 126-136.
Lee, S. B., S. H. Kim, et al. (2001). "Destabilization of CHK2 by a missense mutation associated with Li-Fraumeni Syndrome." Cancer Res 61(22): 8062-8067.
117
Leva, V., S. Giuliano, et al. (2012). "Phosphorylation of SRSF1 is modulated by replicational stress." Nucleic Acids Res 40(3): 1106-1117.
Li, J., G. Li, et al. (2013). "Histone deacetylase inhibitors: an attractive strategy for cancer therapy." Curr Med Chem 20(14): 1858-1886.
Li, P., G. Carter, et al. (2013). "Clk/STY (cdc2-like kinase 1) and Akt regulate alternative splicing and adipogenesis in 3T3-L1 pre-adipocytes." PloS one 8(1): e53268. Li, X. and J. L. Manley (2005). "Inactivation of the SR protein splicing factor ASF/SF2
results in genomic instability." Cell 122(3): 365-378.
Li, X., J. Wang, et al. (2005). "Loss of splicing factor ASF/SF2 induces G2 cell cycle arrest and apoptosis, but inhibits internucleosomal DNA fragmentation." Genes Dev 19(22): 2705-2714.
Lindqvist, A., V. Rodriguez-Bravo, et al. (2009). "The decision to enter mitosis: feedback and redundancy in the mitotic entry network." J Cell Biol 185(2): 193-202.
Loomis, R. J., Y. Naoe, et al. (2009). "Chromatin binding of SRp20 and ASF/SF2 and dissociation from mitotic chromosomes is modulated by histone H3 serine 10 phosphorylation." Molecular cell 33(4): 450-461.
Luco, R. F., M. Allo, et al. (2011). "Epigenetics in alternative pre-mRNA splicing." Cell 144(1): 16-26.
Malumbres, M. (2011). "Physiological relevance of cell cycle kinases." Physiol Rev 91(3): 973-1007.
Malumbres, M. and M. Barbacid (2001). "To cycle or not to cycle: a critical decision in cancer." Nat Rev Cancer 1(3): 222-231.
Malumbres, M. and M. Barbacid (2005). "Mammalian cyclin-dependent kinases." Trends Biochem Sci 30(11): 630-641.
Malumbres, M. and M. Barbacid (2009). "Cell cycle, CDKs and cancer: a changing paradigm." Nat Rev Cancer 9(3): 153-166.
118
Malumbres, M. and A. Pellicer (1998). "RAS pathways to cell cycle control and cell transformation." Front Biosci 3: d887-912.
McLaughlin, F., P. Finn, et al. (2003). "The cell cycle, chromatin and cancer:
mechanism-based therapeutics come of age." Drug Discov Today 8(17): 793- 802.
Michlewski, G., J. R. Sanford, et al. (2008). "The splicing factor SF2/ASF regulates translation initiation by enhancing phosphorylation of 4E-BP1." Mol Cell 30(2): 179-189.
Mocciaro, A. and M. Rape (2012). "Emerging regulatory mechanisms in ubiquitin-