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Noncoding Y RNAs: A Functional Role in DNA Replication

Noncoding Y RNAs: A Functional Role in DNA Replication. Mahta Nili BMCB 625 June 13, 2007. 60 kDa Ro Autoantigen Y RNA Sequence and Structure DNA Replication Review “Functional Requirement of Noncoding Y RNAs for Human Chromosomal DNA Replication”

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Noncoding Y RNAs: A Functional Role in DNA Replication

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  1. Noncoding Y RNAs: A Functional Role in DNA Replication Mahta Nili BMCB 625 June 13, 2007

  2. 60 kDa Ro Autoantigen • Y RNA Sequence and Structure • DNA Replication Review • “Functional Requirement of Noncoding Y RNAs for Human Chromosomal DNA Replication” • Conclusions / Remaining Questions

  3. Ro 60kD Autoantigen • Major target of the immune response in rheumatic disease • Lupus (anti-Ro antibodies 25-60% of patients) • Photosensitive skin lesions • Neonatal – congenital heart block • Sjogren’s Syndrome (anti-Ro antibodies 50-90% of patients) • Mice lacking Ro develop autoimmune syndrome similar to lupus

  4. Possible Roles for Ro / Ro-Y RNA Complex • RNA stability • QC - Ro binds incorrectly folded small RNAs • Facilitates cell survival after exposure to UV • Prevention of autoimmune disease

  5. Ro 60kD Autoantigen RNP Stein et al. Cell. 2005

  6. Noncoding Y RNAs • 4 human Y RNAs (hY1, hY3, hY4, hY5) • Little primary sequence conservation • Highly conserved structural elements • Found in all vertebrates and highly conserved across species • Not in yeast, plants, or insects • Approximately 100 nucleotides long

  7. Noncoding Y RNAs • Transcribed by PolIII • Y RNAs most abundant in heart and brain tissue • Ro / Y RNA complex may contain additional proteins • La – protein which binds new RNA PolIII transcripts

  8. Y Genes • All hY genes on chromosome 7 • hY genes all have class III promoters • Numerous hY-homologous pseudogenes

  9. Noncoding Y RNAs Van Gelder et al. Nuc Acids Res. 1994

  10. Noncoding Y RNAs Teunissen et al. Nuc Acids Res. 2000

  11. Noncoding Y RNAs Possible Secondary Structures Chen and Wolin. J Mol Med. 2005

  12. DNA Replication Initiation http://users.ox.ac.uk/~kearsey/

  13. Methods I: Cell Synchronization • HeLa, EJ30, and NIH3T3 cells • Cells arrested in G1 with mimosine • Iron/Zinc chelator • Causes DNA strand breaks • Impairs DNA replication initiation or elongation? • Synchronization verified by flow cytometry

  14. Methods II: Preparation of Nuclei Nuclei from HeLa, EJ30, and NIH3T3 cells swollen in hypotonic buffer mitotic cells lost interphase cells Homogenized, pelleted, washed in PBS and re-pelleted Permeabilization in triton x-100, sucrose, spermidine concentration - hemocytometer

  15. Methods III: Fractionation • HeLa Cell Extracts from 4C Biotech • Cleared by ultracentrifugation • Pre-equilibrated in buffer containing 200mM KCl Figure 1A

  16. Purification of RNA Necessary for Replication Propidium IodideFab Figure 1

  17. Identification of RNAs Out of 19 cDNA clones: 4 – 5S rRNA 8 – U2 snRNA 2 – hY4 5 – hY5 Figure 2

  18. Human Y RNA Required for Replication Figure 4

  19. Table 1

  20. Secondary Structures Revisited Figure 3

  21. Degradation of hY RNAs Figure 5A

  22. Inhibition of Chromosomal DNA Replication Figure 5

  23. Mouse NIH3T3 template nuclei from cells synchronized in late G1 phase naturally by the release of contact-inhibited quiescent cells through sub-cultivation Table 2

  24. Human Y RNAs Required for Semi-Conservative Replication Figure 6

  25. Are Y RNAs Acting as Primers? • Y RNAs have ss 3’ polyU tail and unmodified 3’ OH end • Used radioactive hY RNAs to initiate DNA replication • Not able to detect extensions by in vitro reactions • Also capped 3’ OH end of hY1 with 3’ deoxyuridine • Capped hY1 RNA also initiated DNA replication

  26. Are Y RNAs Acting as Primers? • Y RNAs have ss 3’ polyU tail and unmodified 3’ OH end • Used radioactive hY RNAs to initiate DNA replication • Not able to detect extensions by in vitro reactions • Also capped 3’ OH end of hY1 with 3’ deoxyuridine • Capped hY1 RNA also initiated DNA replication Y RNAs not acting as primers

  27. Is Binding to Ro Necessary for DNA Replication? Figure 7

  28. Ro Binding Not Essential for Replication Figure 7

  29. KD of hY1 by RNAi Inhibits DNA Replication Figure 8

  30. Conclusions • Deletion of hY RNAs inhibits DNA replication in late G1 phase nuclei • Y RNAs required for reconstitution of semi-conservative DNA replication • Y RNAs not primers • Y RNA role in replication is Ro independent • Y RNAs required for replication in vivo

  31. What’s the Y doing? ? Y ? http://users.ox.ac.uk/~kearsey/

  32. Remaining Questions • This paper makes no mention of Ro (other than knocking out the binding site on Y RNAs) 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? • What other effects are mimosine having on the nuclei? • The functional replacement of hY1 and hY3 with other Ys. Is it just a numbers issue? • Mechanisms by which Y RNAs regulate replication?

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