Noncoding Y RNAs: A Functional Role in DNA Replication

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Noncoding Y RNAs
A Functional Role in DNA Replication
Mahta Nili BMCB 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
• Sjogrens 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 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
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Methods III Fractionation

• HeLa Cell Extracts from 4C Biotech
• Cleared by ultracentrifugation
• Pre-equilibrated in buffer containing 200mM KCl

Figure 1A
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Purification of RNA Necessary for Replication
Propidium Iodide Fab
Figure 1
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Identification of RNAs
Out of 19 cDNA clones 4 5S rRNA
8 U2 snRNA 2 hY4
5 hY5
Figure 2
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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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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
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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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Whats the Y doing?
?
Y
?
http//users.ox.ac.uk/kearsey/
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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?