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/

15. Methods I: Cell Synchronization  HeLa, EJ30, and NIH3T3 cells  Cells arrested in G 1 with mimosine  Iron/Zinc chelator  Causes DNA strand breaks  Impairs DNA replication initiation or elongation?  Synchronization verified by flow cytometry

16. 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 concentration - hemocytometer Permeabilization in triton x-100, sucrose, spermidine

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

18. Purification of RNA Necessary for Replication Figure 1 Propidium Iodide Fab

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

20. Human Y RNA Required for Replication Figure 4

21. Table 1

22. Secondary Structures Revisited Figure 3

23. Degradation of hY RNAs Figure 5A

24. Inhibition of Chromosomal DNA Replication Figure 5

25. Table 2 Mouse NIH3T3 template nuclei from cells synchronized in late G 1 phase naturally by the release of contact-inhibited quiescent cells through sub-cultivation

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

27. 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

28. 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

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

30. Ro Binding Not Essential for Replication Figure 7

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

32. 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

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

34. 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?