noncoding y rnas: a functional role in dna replication mahta nili bmcb 625 june 13, 2007
TRANSCRIPT
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Noncoding Y RNAs: A Functional Role in DNA
Replication
Mahta NiliBMCB 625
June 13, 2007
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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
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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
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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
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Ro 60kD Autoantigen RNP
Stein et al. Cell. 2005
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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
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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
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Y Genes
All hY genes on chromosome 7
hY genes all have class III promoters
Numerous hY-homologous pseudogenes
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Noncoding Y RNAs
Van Gelder et al. Nuc Acids Res. 1994
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Noncoding Y RNAs
Teunissen et al. Nuc Acids Res. 2000
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Noncoding Y RNAs Possible Secondary Structures
Chen and Wolin. J Mol Med. 2005
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DNA Replication Initiation
http://users.ox.ac.uk/~kearsey/
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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
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Methods II: Preparation of NucleiNuclei 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
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Methods III: Fractionation
Figure 1A
HeLa Cell Extracts from 4C Biotech
Cleared by ultracentrifugation
Pre-equilibrated in buffer containing 200mM KCl
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Purification of RNA Necessary for Replication
Figure 1
Propidium Iodide Fab
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Identification of RNAs
Figure 2
Out of 19 cDNA clones: 4 – 5S rRNA 8 – U2 snRNA 2 – hY4 5 – hY5
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Human Y RNA Required for Replication
Figure 4
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Table 1
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Secondary Structures Revisited
Figure 3
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Degradation of hY RNAs
Figure 5A
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Inhibition of Chromosomal DNA Replication
Figure 5
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Table 2
Mouse NIH3T3 template nuclei from cells synchronized in late G1 phase naturally by the release of contact-inhibited quiescent cells through sub-cultivation
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Human Y RNAs Required for Semi-Conservative Replication
Figure 6
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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
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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
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Is Binding to Ro Necessary for DNA Replication?
Figure 7
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Ro Binding Not Essential for Replication
Figure 7
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KD of hY1 by RNAi Inhibits DNA Replication
Figure 8
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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
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What’s the Y doing?
http://users.ox.ac.uk/~kearsey/
Y?
?
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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?