Download Noncoding Y RNAs

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?