o n R leo sp in intissu o f lic g e d v lo mn ee p et & R c n p o e tiv s in e e t r sp c e e ig n tic p ee s

Nm a e R ll o Ya er S ssio e n : D u aA in ilr b fr :1 2 8 2 0102 : 2 nd : 2 1 -1 00 1 D p o G n tic E g e r g& e t. f e e n in e in B te h o g io c n lo y S b itte toD . A ifu H q e um d r r l au A ssista t P o sso n r fe r Mle u r P th lo y o c la a o g In stitu o B lo ic l S ie c s te f io g a c n e S b issio D te um n a : 2 -1 -2 1 . 0 1 01

R le o sp in intissu o f lic g e d v lo mn ee p et
Splicing is the process through which introns from the RNA transcript are removed and exons are joind together. Alternative splicing is one of the typical splicing types where splicing can occur in different patterns so that a particular group of exons forms one mRNA and a different group from the same gene transcript forms another mRNA. This leads to different proteins, thus a single gene may code for multiple proteins. This mechanism may be employed to produce variant forms of protein required in diffent tissues of their development. Alternative splicing is one of several exceptions to the original idea that one DNA sequence codes for one polypeptide (the one gene one enzyme hypothesis) It might be more correct now to say'One gene many polypeptides'. It has been proposed that for eukaryotes altenative splicing was a very important step towards higher efficiency because information can be stored much more economically. Several proteins can be encoded by a single gene, rather than requiring a separate gene for each, and thus allowing a more varied proteome from a genome of limited size. It also provides evolutionary flexibility. A single piont mutation may cause a given exon to be occasionally excluded or included from a transcript during splicing, allowing production of a new protein isoform without loss of the original protein. Comparative studies indicate that alternative splicing preceded multicellularity in evolution, and suggest that this mechanism might have been co-opted to assist in the development of multicellular organisms.

Chages in the RNA processing mechinery may lead to missplicing of multiple transcript, while single nucleotide alernations in splice sites or cis-acting splicing regulatory sites may lead to differences in splicing of a single gene, and thus in the mRNA produced from a mutant gene's transcripts. A probabilistic analysis indicates that over 60% human disease-causing mutations affect splicing rather than directly affecting coding sequences.Abnormally spliced mRNAS are also found in a high proportion of cancerous cells. Until recently, it was unclear whether such aberrant patterns of splicing played a role in causing cancerous growth or were merely a consequence of cellular abnormalities associated with cancer. It has been shown that ther is actually a reduction of alternative splicing in cancerous cells compared to normal ones and the types of splicing differ, for instance, cancerous cells show higther levels of intron retention the normal cells, but low levels of exon skipping. One study found that a relatively small percentage ( 303 out of over 26000) of alternative splicing variants were significantly higher in frequency in tumor eclls than normal cells, suggesting that there is a limited set of genes which when mis-spliced, contribute to tumor development.Some of the differences in splicing in cancerous cells may result from changes in phosphorylation of trans-acting splicing factors. Others may be produced by changes in the relative amounts of splicing factors produced for instance, breast cancer cells have been shown to have increased levels of the splicing factor SF2/ASF. One example of a specific splicing variant assoceated with cancers is in one of the human DNMT genes. There DNMT genes encode enzymes that add methyl groups to DNA, a modification that often has reglatory effects. Several abnormally spliced DNMT3B mRNAS are found in tumors

and caner cell lines. In two seperate studies, expression of two of these abnormally spliced mRNAS in mammalian cells caused changes in the DNA methylation patterns in those cells. Cells with one of the abnormal mRNAS also grew twice as fast as control cells, indicating a direct cortribution to tumor development by this product. Another example is the Ron (MST1R) proto-oncogene. An important property of cancerous cells is their ablity to move and invade normal tissue, production of an abnormally spliced transcript of Ron has been found to be associated with increased levels of the SF2/ASF in breast cancer cells.The abnormal isoform of the Ron protein encoded by this mRNA leads to cellmotility.Genome-wide analysis of alternative splicing is a challenging task. Typically, alternatively spliced transcripts have been found by comparing EST sequences, but this requires sequencing of very large numbers of ESTs. Most EST libraries come from a very limited number of tissues, so tissue-specific splice variants are likely to be missed in any case. High-throughput approaches to investigate splicing fall into three categories DNA microarray-based analyses, CLIP, and in vivo reporter gene assays. In microarray analysis, arrays of DNA fragments representing individual exons (e.g. Affymetrix exon microarray) or exon/exon boundaries (e.g. arrays from ExonHit or Jivan) have been used. The array is then probed with labeled cDNA from tissues of interest. The probe cDNAs bind to DNA from the exons that are included in mRNAs in their tissue of origin, or to DNA from the boundary where two exons have been joined. This can reveal the presence of particular alternatively spliced mRNAs. Deep sequencing technologies are also being used to perform genome-wide studies of transcript variation.CLIP (Cross-linking and immunoprecipitation) uses UV radiation to link proteins to

RNA molecules in a tissue during splicing. A trans-acting splicing regulatory protein of interest is then precipitated using specific antibodies. When the RNA attached to that protein is isolated and cloned, it reveals the target sequences for that protein. Finally, it is possible to find the splicing proteins involved in a specific alternative splicing event by constructing reporter genes that will express one of two different fluorescent proteins depending on the splicing reaction that occurs. This method has been used to isolate mutants affecting splicing and thus to identify novel splicing regulatory proteins inactivated in those mutants.

R fe e c s e r ne
1. 2. 3.
4.

5.
6.

7.

8.

9. 10.

11.

12. 13. 14.

15.

(Web): RNA Synthesis and RNA Processinghttp:///www.ncbi.nlm.nih.gov/bookshelf/brfegi?book=cell&part=A1682. Black, Douglas L. (2003). Mechanisms of alternative premessenger RNA splicing. Annual Reviews of Biochemistry72(1):291-336. Pan, Q; Shai O, Lee LJ, Frey BJ, Blencowe BJ (Dec 2008). "Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing". Nature Genetics 40 (12): 14131415. David, C. J.; Manley, J. L. (2008). "The search for alternative splicing regulators: new approaches offer a path to a splicing code". Genes & Development 22 (3): 279 85. Skotheim, R I and Nees, M (2007). "Alternative splicing in cancer: noise, functional, or systematic?". The international journal of biochemistry & cell biology 39 (7-8): 143249. Bauer, Joseph Alan; He, Chunjiang; Zhou, Fang; Zuo, Zhixiang; Cheng, Hanhua; Zhou, Rongjia (2009). Bauer, Joseph Alan. ed. "A Global View of Cancer-Specific Transcript Variants by Subtractive Transcriptome-Wide Analysis". PLoS ONE 4 (3): e4732. Fackenthal, Jd; Godley, La (2008). "Aberrant RNA splicing and its functional consequences in cancer cells" (Free full text). Disease models & mechanisms 1 (1): 3742. Danckwardt S, Neu-Yilik G, Thermann R, Frede U, Hentze MW, Kulozik AE (2002). "Abnormally spliced beta-globin mRNAs: a single point mutation generates transcripts sensitive and insensitive to nonsense-mediated mRNA decay". Blood 99 (5): 18116. Wang, Z; Burge, Cb (2008). "Splicing regulation: from a parts list of regulatory elements to an integrated splicing code" (Free full text). RNA 14 (5): 80213. "HHMI Bulletin September 2005: Alternative Splicing". www.hhmi.org. http://www.hhmi.org/bulletin/sept2005/features/splicing.html. Retrieved 2009-0526. Irimia, Manuel; Rukov, Jakob; Penny, David; Roy, Scott (2007). "Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing". BMC Evolutionary Biology 7: 188. Kim E, Goren A, Ast G (2008). "Insights into the connection between cancer and alternative splicing". Trends Genet. 24 (1): 710. Ghigna C, Giordano S, Shen H, et al. (2005). "Cell motility is controlled by SF2/ASF through alternative splicing of the Ron proto-oncogene". Mol. Cell 20 (6): 88190. Hui L, Zhang X, Wu X, et al. (2004). "Identification of alternatively spliced mRNA variants related to cancers by genome-wide ESTs alignment". Oncogene 23 (17): 301323. Luco, RF; Allo, M; Schor, IE; Kornblihtt, AR; Misteli, T. (2011). "Epigenetics in alternative pre-mRNA splicing". Cell 144 (1): 1626.

R c n p o e tiv s in e e t r sp c e e ig n tic p ee s
In genctics, epigenetics is the study of heritable change in gene expession or cellular phenotype caused by mechanisms other than changes in the under-lying DNA sequence.Examples of such changes might be DNA methylation or histone deacetylatim, both of which serve to supperss gene expressim without altering the sequence of the silenced genes. In 2011, it was proved that the methylation of mRNA has a critical role in human homeostasis. This opened the field of RNA epigenetics. Epigenetics was coined by C.H. Waddington in 1942 as a portmanteau of the words genetics and epigenesis. Epigenesis is an old word which has more recently been used to describe the differentiation of cells from their initial totipotent state in embryonic development. Robin Holliday defined epigenetics as "the studay of the mechanisms of temporal and spatial control of gene activity during the devlopment of complex organisms. Thus epigenetic can be used to describe anything other than DNA sequence that influences the development of an organism. Though the topic of epigenetics was once confined to the field of molecular biology, in the past 5 year there have been exciting new applications of an epigenetic perspective to the study of development and behavior with increasing appreciation of the plaslticity that these mechanisms can confer. The term'epigenetic' can be used broadly to refer variation in phenotype that is not exclusively related to genetic variation or more specifically to refer to the molecular mechanisms that regulate gene transcription

without modifying gene sequence.In development, somatic epigenetic inheritance,Particularly through DNA methylatim and chromatin remodeling, is very important in the develpment of multicellular eukarytic organism. The genome sequence is static but cells differentiate into many different types, which perform different functions and differently to the environment and intercellular signaling. Thus, as individuals develop, morphogens activate or silence genes in an epigenetically heritable fashion, giving cells a memory. In mammals, most cells terminally differentiate, with only stem cells retaing the ability to differentiate into several cell types. In mammals. some stem cells continue producing new differentiated cells throughout life, but mammals are not able to respond to loss of some tissues, for example, the inability to regenerate limbs,which some other animals are capable of. Unlike animals, plant cells do not terminally dofferentiate, remaining to totipotent with the ability to give rise to a new individual plant, While plants do utilise many of the same epigenetic mechanisms as animals, such as chromatin remodeling, it has been hypothesised that plant cells do not have "memories" resetting their gene expression patterns at each cell division using positional information from the environment and surrounding cells to determine their fate. Epigenetics has many and varied potential medical applications as it tends to be mutidimentional in nature. Congital genetic disease in well understood and it is also clear that epigenetics can play a role for example, in the case Angelman syndrome and Prader-Wlli syndrome. These are normal genetic diseases caused by gene deletions or inactivation of the genes, but are unusually common because individuals are essentially hemizygous because of genomic imprinting and therefore a single gene knock out is sufficient to cause the disease,

where most cases would require both copies to be knocked out.Epigenetic features may play a role in short-term adaption of species by allowing for reversible phenotype variability.The modification of epigenetic features associated with a region of DNA allows organisms, on a multigenerational time scale, to swithch between phenotypes that express and repress that particular gene. When the DNA sequence of the region is not mutated, this change is reversible. It has also been speculated that organisms may take advantage of differential mutation rates associated with epigenetic features to control the mutation rates of paritcular genes. Interestingly recent analysis have suggested that members of the APOBEC/AID family of cytosine deaminases are capable of simultaneouly mediating genetic and epigenetic inheritance using similar molecular mechanisms. Some human disorders are associated with genomic inprinting, a phenomenon in mammals where the father and mother contribute different epigenetic patterns for specific genomic loci in their germ cells. The best known case of imprinting in human disorders is that of Angelman syndrome and Prader-Willi syndrom both can be praduced by the some genetic mutation, chromosome 15q partial deletion and particular syndrome that will develop depends or whether the mutation is inherited from the child`s mother or from their father. Marcus Pembrey and colleagues also observed in the overkalix study that the paternal (but not maternal) grandsons of Swedish men who were exposed during preadolescence to famine in the 19th century were less likely to die of cardiovascular disease; if food was plentiful then diabetes mortality in the grandchildren increased, suggesting that this was a transgenerational epigenetic inheritance. The opposite effect was observed for femalesthe paternal (but not maternal)

granddaughters of women who experienced famine while in the womb (and therefore while their eggs were being formed) lived shorter lives on average. A variety of compounds are considered as epigenetic carcinogensthey result in an increased incidence of tumors, but they do not show mutagen activity (toxic compounds or pathogens that cause tumors incident to increased regeneration should also be excluded). Examples include diethylstilbestrol,arsenite, hexachlorobenzene, and nickel compounds. Recent studies have shown that the Mixed Lineage Leukemia (MLL) gene causes leukemia by rearranging and fusing with other genes in different chromosomes, which is a process under epigenetic control.Other investigations have concluded that alterations in histone acetylation and DNA methylation occur in various genes influencing prostate cancer. Gene expression in the prostate can be modulated by nutrition and lifestyle changes.In 2008, the National Institutes of Health announced that $190 million had been earmarked for epigenetics research over the next five years. In announcing the funding, government officials noted that epigenetics has the potential to explain mechanisms of aging, human development, and the origins of cancer, heart disease, mental illness, as well as several other conditions. Some investigators, like Randy Jirtle, PhD, of Duke University Medical Center, think epigenetics may ultimately turn out to have a greater role in disease than genetics. DNA methylation is an important regulator of gene transcription and a large body of evidence has demonstrated that aberrant DNA methylation is associated with unscheduled gene silencing, and the genes with high levels of 5-methylcytosine in their promoter region are transcriptionally silent. DNA methylation is essential during embryonic development, and in somatic cells, patterns of

DNA methylation are generally transmitted to daughter cells with a high fidelity. Aberrant DNA methylation patterns have been associated with a large number of human malignancies and found in two distinct forms: hypermethylation and hypomethylation compared to normal tissue. Hypermethylation is one of the major epigenetic modifications that repress transcription via promoter region of tumour suppressor genes. Hypermethylation typically occurs at CpG islands in the promoter region and is associated with gene inactivation. Global hypomethylation has also been implicated in the development and progression of cancer through different mechanisms.The histone variants of the H2A family are highly conserved in mammals, playing critical roles in regulating many nuclear processes by altering chromatin structure. One of the key H2A variants, H2A.X, marks DNA damage, facilitating the recruitment of DNA repair proteins to restore genomic integrity. Another variant, H2A.Z, plays an important role in both gene activation and repression. A high level of H2A.Z expression is ubiquitously detected in many cancers and is significantly associated with cellular proliferation and genomic instability. Current research has shown that epigenetic pharmaceuticals could be a putative replacement or adjuvant therapy for currently accepted treatment methods such as radiation and chemotherapy, or could enhance the effects of these current treatments. It has been shown that the epigenetic control of the proto-onco regions and the tumor suppressor sequences by conformational changes in histones directly affects the formation and progression of cancer. Epigenetics also has the factor of reversibility, a characteristic that other cancer treatments do not offer.Drug development has mainly focused on Histone Acetyltransferase (HAT) and Histone

Deacetylase (HDAC), including the introduction of the new pharmaceutical Vorinostat, a HDAC inhibitor, to the market. HDAC specifically has been shown to play an integral role in the progression of oral squamous cancer. Current front-runner candidates for new drug targets are Histone Lysine Methyltransferases (KMT) and Protein Arginine Methyltransferases (PRMT).Recent studies involving both dizygotic and monozygotic twins have produced some evidence of epigenetic influence in humans.

References

1. 2.

http://www.physorg.com/news/2011-10-links-common-rna-modification-obesity.html

C.H. Waddington (1942) (1977). "The epigenotype". Endeavour 1: 1820. doi:10.1016/01609327(77)90005-9. 3. Holliday, R., 1990. Mechanisms for the control of gene activity during development. Biol. Rev. Cambr. Philos. Soc. 65, 431-471. 4. Griffith, J.S., & Mahler, H.R (1969). DNA ticketing theory of memory. Nature, 223 (5206), 580582. 5. Costa, Silvia; Shaw, Peter (2006). "'Open Minded' cells: how cells can change fate". Trends in Cell Biology 17 (3): 101106. 6. Chahwan R, Wontakal SN, Roa S (March 2011). "The multidimensional nature of epigenetic information and its role in disease". Discovery medicine 11 (58): 23343. 7. O.J. Rando and K.J. Verstrepen (2007). "Timescales of Genetic and Epigenetic Inheritance". Cell 128 (4): 655 668. 8. Chahwan R., Wontakal S.N., and Roa S. (2010). "Crosstalk between genetic and epigenetic information through cytosine deamination". Trends in Genetics 26 (10): 443448. 9. A.J. Wood and A.J. Oakey (2006). "Genomic imprinting in mammals: Emerging themes and established theories". PLOS Genetics 2 (11): 16771685. 10. J.H.M. Knoll, R.D. Nicholls, R.E. Magenis, J.M. Graham Jr, M. Lalande, S.A. Latt (1989). "Angelman and Prader-Willi syndromes share a common chromosome deletion but differ in parental origin of the deletion". American Journal of Medical Genetics 32 (2): 285290. 11. Pembrey ME, Bygren LO, Kaati G, et al.. Sex-specific, male-line transgenerational responses in humans. Eur J Hum Genet 2006; 14: 15966. 12. http://www.pbs.org/wgbh/nova/transcripts/3413_genes.html 13. Bishop, JB; Witt KL and Sloane RA (December 1997). "Genetic toxiticities of human teratogens". Mutat Res 396 (12): 943. 14. Gurvich, N; Berman MG, Wittner BS et al. (July 2004). "Association of valproate-induced teratogenesis with histone deacetylase inhibition in vivo". FASEB J 19 (9): 11661168. 15. Friedler, G (December 1996). "Paternal exposures: impact on reproductive and developmental outcome. An overview". Pharmacol Biochem Behav 55 (4): 691700. 16. Cicero, TJ; Adams NL, Giodarno A et al. (March 1991). "Influence of morphine exposure during adolescence on the sexual maturation of male rats and the development of their offspring". J Pharmacol Exp Ther. 256 (3): 10861093. 17. Newbold, RR; Padilla-Banks E and Jefferson WN (June 2006). "Adverse effects of the model environmental estrogen diethylstilbestrol are transmitted to subsequent generations". Endocrinology 147 (6 Suppl): S11S17. 18. Mandall, SS (2010). "Mixed lineage leukemia: versatile player in epigenetics and human disease". The FEBS Journal 227 (8): 1789. 19. Li, LC; Carroll, PR; Dahiya, R (2005). "Epigenetic changes in prostate cancer: implication for diagnosis and treatment". Journal of the National Cancer Institute 2 (97): 10315. 20. Ornish, D; Magbanua, MJ; Weidner, G; Weinberg, V; Kemp, C; Green, C; Matttie, MD; Marlin, R et al. (2008). "Changes in prostate gene expression in men undergoing an intensive nutrition and lifestyle intervention". Proceedings of the National Academy of Sciences in the United States of America 105 (24): 83698374. 21. Beil, Laura (Winter, 2008). "Medicine's New Epicenter? Epigenetics: New field of epigenetics may hold the secret to flipping cancer's "off" switch.". CURE (Cancer Updates, Research and Education). http://www.curetoday.com/index.cfm/fuseaction/article.show/id/2/article_id/949. 22. Craig, JM; Wong, NC (editor) (2011). Epigenetics: A Reference Manual. Caister Academic Press. ISBN 978-1-904455-88-2. 23. Wang, LG; Chiao, JW (2010). "Prostate cancer chemopreventive activity of phenethyl isothiocyanate through epigenetic regulation (review)". International Journal of Oncology 3 (37): 5339. 24. Iglesias-Linares, A; Yaez-Vico, RM; Gonzlez-Moles, MA (2010). "Potential role of HDAC inhibitors in cancer therapy: insights into oral squamous cell carcinoma". Oral Oncology 5 (46): 3239. 25. Spannhoff, A; Sippl, W; Jung, M (2009). "Cancer treatment of the future: Inhibitors of histone methyltransferases". The International Journal of Biochemistry & Cell Biology 1 (41): 411. 26. Toward the development of potent and selective bisubstrate inhibitors of protein arginine methyltransferases". Bioorganic & Medicinal Chemistry Letters 7 (20): 21035. April 2010. 27. O'Connor, Anahad (2008-03-11). "The Claim: Identical Twins Have Identical DNA". New York Times. http://www.nytimes.com/2008/03/11/health/11real.html. Retrieved 2010-05-02. 28. Kaminsky, Zachary A; Tang, T; Wang, SC; Ptak, C; Oh, GH; Wong, AH; Feldcamp, LA; Virtanen, C et al. (2009). "DNA methylation profiles in monozygotic and dizygotic twins". Nature Genetics 41 (2): 2405. 29. Fraga, M. F.; Ballestar, E; Paz, MF; Ropero, S; Setien, F; Ballestar, ML; Heine-Suer, D; Cigudosa, JC et al. (2005). "Epigenetic differences arise during the lifetime of monozygotic twins". Proceedings of the National Academy of Sciences 102 (30): 106049.