Non Coding RNAs in replication
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The document summarizes the role of non-coding RNAs in DNA replication initiation. It discusses how the Tetrahymena 26T RNA interacts with the origin recognition complex (ORC) to recruit it specifically to the rDNA origin for replication initiation. It also describes how the Epstein-Barr virus encodes a G-rich RNA that recruits the human ORC complex through its interaction with EBNA1. Additionally, the document outlines the essential role of vertebrate Y RNAs in mammalian DNA replication, particularly in the initiation step of the process.
Non Coding RNAs in replication
- 2. CONTENTS…… Overview of replication Players in replication initiation Small non coding RNAs RNAs involved in replication Tetrahymena 26T RNA EBV G rich RNA Vertebrate Y RNA Concluding remarks
- 3. Bidrectional DNA synthesis Replication forks will merge Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
- 4. DNA Replication Initiation 1. Recognition: label the origin with the ORC 2. Initiative assembly or licensing: load the DNA helicase to form the pre-RC 3. Unwinding: activate the DNA helicase 4. Elongative assembly: load the replisome including DNA polymerase (POL) holoenyzmes and SSB (single-stranded DNA binding protein).
- 5. ORC-origin interaction – key to choosing the start site? ORC- 6 subunit Missing link-the mechanism that recruits ORC to Cdc6 chromatin G1 ORCA/LRWD1 Cdt1 HBO1 14-3-3 MCM Cdt1-Geminin associated proteins Dpb11, GINS complex, HOX etc... MCM10, Cdc45, S Non-coding RNAs and DNA polymerase
- 6. NON CODING RNAs DNA replication 26T Y RNA G rich RNA RNA
- 7. A review on ROLE OF NON CODING RNA IN REPLICATION INITIATION
- 8. INTEGRAL PART OF TETRAHYMENA ORC- 26T RNA
- 9. Tetrahymena thermophila • Unicellular ciliate • Model organism • Nuclear dimorphism -germline micronuclie, vegetative macronuclie. • Macronuclie- 5 monocentric chromosome-exceptional case rDNA chromosome-highly amplified. • cis-acting replication determinants, type I elements that associate with sequence-specific, single-stranded binding factors, TIF1 through TIF4. • TIF4- tetrahymena ORC. (Mohammad et. Al, 2003,2007). • Binds to T rich strand. • RNase treatment eliminates binding, its an RNP (Mohammad et. al,2009) • ORC binds to rDNA origin throughout the cell cycle. HOW?
- 10. Why rDNA ORC is stable? 26T RNA Binding of TtORC to rDNA ori is regulated by a noncoding RNA. (Mohammad et al.,2007)
- 11. 26T RNA • Isolated and sequenced from TIF4 • Corresponds to 282 nts of 3’ terminus of mature 23S rRNA. (Mohammad et al., 2007) • Hence the name • Sequence specifically interact with rDNA origin. • Confirmed by pull down assay and complementary mutations in 26TRNA. • Molecular mechanism- specific base pairing.
- 12. Proposed sequence of 26T RNA Characterization of 26T RNA Sequence alignment with rDNA ori Mohammad et al., 2007
- 13. • ChIP studies shows that ORC binds specifically to rDNA origin, but not other sites on the rDNA macronuclear chromosome. • Recently, another ori-ARS 1 was identified, to which ORC binds independent of 26TRNA.( Donti et al., 2009) • But, the binding is on random sites. • Concluding, 26T RNA needed for differential binding of ORC to rDNA origin.
- 14. • In conclusion, the key role of 26T RNA in the control of rDNA origin is linked to the efficient and site specific recruitment of ORC to this region, which is highly amplified during development. • Give selective advantage under competitive demand for limited initiation factors. • A new role for ribosomal RNA in chromosome biology
- 15. Unanswered Question • ORC-26T RNA does not bind to the perfectly complementary rDNA region • Does not bind to any other type 1 Element • Something beyond the specific base pairing????????
- 16. Viral encoded G rich RNA recruits human ORC to EBV genome.
- 17. Epstein- Barr Virus • Oncogenic human gamma herpes virus. • EBV origin of plasmid replication (OriP) provides an attractive model to study ORC recruitment in human cells. • EBNA 1 , a virus encoded protein recruits host ORC to the particular element of Ori P, the dyad symmetry element. (Lindner and Sugden, 2007)
- 18. ORC binding to the viral origin EBNA 1 ORC EBV episomal Ori P minichromoso me Norseen et al., 2009
- 19. • Human ORC does not have any sequence specificity, then how it is targeted to a particular sequence in EBV OriP? • A structured non coding RNA plays key role in this sequence specific interaction. (Norseen et al., 2008) • EBNA1 have amino terminal linking regions LR1 and LR2 binding to ORC. • Further study revealed LR contains RGG motif. • RGG motif is known for RNA binding. Mutation analysis confirmed this motif is essential for ORC binding.
- 20. • EBNA 1 binds to RNA through RGG motif.(Snudden et al., 1994, Lu et al., 2004) suggesting that ORC binding via these motif may influenced by RNA. • Confirmed by treatment of immuno precipitants by RNase treatment- disrupted the interaction between EBNA and ORC. (Norseen et al., 2008)
- 21. • EBNA1 binds to its own mRNA. • This RNAs are heterogeneous in size, EBNA1 shows preference to G rich RNA with particular secondary structures.(Lu et al., 2004, Norseen et al., 2009)) • G rich RNA can form G quadruplexes, suggesting G quadruplex mediate interaction of ORC and EBNA. • EBNA1 mRNA have G rich motif.
- 22. Model of G rich RNA mediating ORC recruitment Norseen et al., 2008
- 23. What’s next? • Exact mechanism unknown. • FMRP and HMGA1a proteins also contains RGG motifs • MNase treatment of nuclear extracts released 40% ORC, Rnase treatment, 20%. (Norseen et al., 2008) • Involved in cellular replication also??? • G-quadruplex-interacting compounds may be useful for the pharmacological inhibition of EBNA1-dependent replication.
- 24. Vertebrate Y RNA is essential for DNA replication
- 25. Y RNA • First identified in 80’s from the patients with autoimmune diseases. (Lerner et al, • 1981) • Component of Ro ribonucleoprotein. • Found in all vertebrates, C.elegans and D.radiodurans. • In humans, Located in chromosome 7,transcribed by RNA polymerase III , 85-112 nucleotides. • most abundant in heart and brain tissue
- 26. Y RNAs are highly conserved among vertebrates Teunissen et al. Nuc Acids Res. 2000
- 27. Specific secondary structure is responsible for function
- 28. Cellular functions • Effect on Ro 60 well understood • UV resistance in mammalian cells and bacteria • Localization of Ro 60 • Inhibit chaperone activities of La, hn RNP I and K • Essential role in DNA replication
- 29. • Required for the functional Cytosolic extract reconstitution of mammalian chromosomal replication in cell free system. (Christov et al.,2006) (RPA)QA QB(PCNA+X) • Isolated from cellular extract of S phase cell. • Interacts with two proteins, RPA and PCNA. ArFT(Protein) ArE (NA) • All vertebrate YRNAs are DNase I RNase A functionally redundant with RNA each other. Digestion • Non vertebrate Y RNAs could not replace function.
- 30. •Depletion of hY RNA inhibits chromosomal DNA replication in late G1 template nuclei. •Other hY RNAs could reconstitute the function •Y RNA is not priming the replication Christov et al.,2006
- 31. Functional substitution of hY1 RNA with other Y RNAs
- 32. • Upper stem contains conserved motif for function in replication • Non vertebrate Y RNAs do not have this motif. • Only this motif is needed. • two mutant Y RNAs with shuffled nucleotide sequences.SH and SH:US SH SH:US Replication Gardiner et al. 2009
- 33. What’s the Y doing? ? Y ?
- 34. Replication of chromosomal DNA in isolated G1- and S-phase nuclei. Krude T et al. J Cell Sci 2009;122:2836-2845 ©2009 by The Company of Biologists Ltd
- 35. Y RNA is not needed in extension DNA combing assay Nascent DNA assay • aligns DNA fibres on glass cover • Nascent DNA was released and slips in a parallel fashion analyzed on alkaline agarose gel • individual tracks of replicated • For elongation studies S phase DNA can be visualised by cells pre-labelled in vitro, then fluorescent microscopy incubated in unlabelled medium. • two distinct labels allows detection of replication origin activation and quantification of individual replication fork progression rates • digoxigenin-dUTP at the beginning of an in vitro replication reaction, and biotin- dUTP after 60 minutes
- 36. DNA COMBING ASSAY Krude T et al. J Cell Sci 2009;122:2836-2845 ©2009 by The Company of Biologists Ltd
- 37. Nascent DNA analysis Krude et al., 2009
- 38. The catch and Release model • Y RNAs bind to euchromatin dynamically • Y RNAs interact with initiation proteins (ORC, Cdt1, Cdc6, DUE-B and Ku) • First associate with unreplicated G1 nuclei • Trigger initiation proteins • After initiation, leaves the region • Discriminates replicated and unreplicated regions, • Thus licensing “once and only once replication” Krude et al., 2011
- 39. Role in human diseases • YRNAs are highly expressed in cancers. (Christov et al 2008) • It was hypothesized that Y RNAs may have a critical role in maintaining the neoplastic phenotype of human tumors. urinary bladder cervix colon Kidney Lung Postrate
- 40. Potential as a tool • could serve as a potential biomarker for identification of proper therapeutic intervention • could be used as a tool to manipulate cell cycle regulation and cell proliferation in culture.
- 41. Unanswered questions • The functional replacement of hY1 and hY3 with other Ys. Is it just a numbers issue? • Involvement of Ro in terms of functional role in replication – how stable are the Y RNAs without this association? • Are they associated with another factor in the nucleus that stabilizes them? • HOW DOES IT WORK?
- 42. CONCLUDING REMARKS… • The emergence of non-coding RNAs from these studies implies the existence of additional factors assisting ORC for origin targeting and replication licensing. • Upon RNase A treatment, a fraction of ORC is released from chromatin, indicating that the ORC association with chromatin can be partially stabilized by RNA , • It is highly possible that some structured RNAs mediate ORC recruitment to certain origins. These findings reinforce the important role of non-coding RNAs in the regulation of replication initiation.
- 43. REFERNCES • Timothy J. Gardiner, Christo P. Christov, Alexander R. Langley, et al. (2009). A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication. RNA 15: 1375-1385. • Alice Tianbu Zhang, Alexander R. Langley, Christo P. Christov, Eyemen Kheir, Thomas Shafee, Timothy J. Gardiner and Torsten Krude. (2011)Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication. Journal of Cell Science. 124, 2058-2069. • Christo P. Christov, Timothy J. Gardiner, David Szuts, and Torsten Krude. (2006). Functional Requirement of Noncoding Y RNAs for Human Chromosomal DNA Replication. Molecular and Cellular Biology.26, 6993–7004. • Julie Norseen, F. Brad Johnson, and Paul M. Lieberman. (2009). Role for G-Quadruplex RNA Binding by Epstein-Barr Virus Nuclear Antigen 1 in DNA Replication and Metaphase Chromosome Attachment. Journal of Virology. 83(20),10336–10346.
- 44. •De Pamphilis, M. L., Blow, J. J., Ghosh, S., Saha, T., Noguchi, K., and Vassilev, A. (2006) Regulating the licensing of DNA replication origins in metazoa. Curr Opin Cell Biol, 18, 231–239. •Donti, T. R., Datta, S., Sandoval, P. Y., and Kapler, G. M. (2009) Differential targeting of Tetrahymena ORC to ribosomal DNA and non-rDNA replication origins. EMBO J, 28, 223–233. •Krude, T. (2006) Initiation of chromosomal DNA replication in mammalian cell-free systems. Cell Cycle, 5, 2115–2122. •Mohammad, M. M., Donti, T. R., Sebastian Yakisich, J., Smith, A. G., and Kapler, G. M. (2007) Tetrahymena ORC contains a ribosomal RNA fragment that participates in rDNA origin recognition. EMBO J, 26, 5048–5060. •Norseen, J., Thomae, A., Sridharan, V., Aiyar, A., Schepers, A., and Lieberman, P. M. (2008) RNA-dependent recruitment of the origin recognition complex. Embo J, 27, 3024–3035.