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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” Conclusions / Remaining Questions 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 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 Ro 60kD Autoantigen RNP Stein et al. Cell. 2005 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 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 Y Genes All hY genes on chromosome 7 hY genes all have class III promoters Numerous hY-homologous pseudogenes Noncoding Y RNAs Van Gelder et al. Nuc Acids Res. 1994 Noncoding Y RNAs Teunissen et al. Nuc Acids Res. 2000 Noncoding Y RNAs Possible Secondary Structures Chen and Wolin. J Mol Med. 2005 DNA Replication Initiation http://users.ox.ac.uk/~kearsey/ 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 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 Methods III: Fractionation HeLa Cell Extracts from 4C Biotech Cleared by ultracentrifugation Pre-equilibrated in buffer containing 200mM KCl Figure 1A Purification of RNA Necessary for Replication Propidium Iodide Figure 1 Fab Identification of RNAs Out of 19 cDNA clones: 4 – 5S rRNA 8 – U2 snRNA 2 – hY4 5 – hY5 Figure 2 Human Y RNA Required for Replication Figure 4 Table 1 Secondary Structures Revisited Figure 3 Degradation of hY RNAs Figure 5A Inhibition of Chromosomal DNA Replication Figure 5 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 Human Y RNAs Required for Semi-Conservative Replication Figure 6 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 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 Is Binding to Ro Necessary for DNA Replication? Figure 7 Ro Binding Not Essential for Replication Figure 7 KD of hY1 by RNAi Inhibits DNA Replication Figure 8 Conclusions Deletion of hY RNAs inhibits DNA replication in late G1 phase nuclei Y RNAs required for reconstitution of semiconservative DNA replication Y RNAs not primers Y RNA role in replication is Ro independent Y RNAs required for replication in vivo What’s the Y doing? ? Y ? http://users.ox.ac.uk/~kearsey/ 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?