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Presented by,
Kavitha Premkumar
NE/604/565
CONTENTS……
Overview of replication
Players in replication initiation
Small non coding RNAs
RNAs involved in replication
Tetrahymena 26T RNA
EBV G rich RNA
Vertebrate Y RNA
Concluding remarks
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Bidrectional DNA synthesis
Replication forks will
merge
DNA Replication Initiation
1. Recognition: label the
origin with the ORC
2. Initiative assembly or
licensing: load the DNA
helicase to form the pre-RC
3. Unwinding: activate the
DNA helicase
4. Elongative assembly:
load the replisome including
DNA polymerase (POL)
holoenyzmes and SSB
(single-stranded DNA
binding protein).
ORC-origin interaction – key to choosing the start
site?
ORC- 6 subunit
Cdc6
G1
Cdt1
MCM
Dpb11, GINS complex,
MCM10, Cdc45, S and DNA polymerase
Missing link-the mechanism
that recruits ORC to
chromatin
ORCA/LRWD1
HBO1
14-3-3
Cdt1-Geminin associated
proteins
HOX etc...
Non-coding RNAs
NON CODING RNAs
DNA replication
26T
RNA
Y
RNAG rich
RNA
ROLE OF NON CODING RNA IN REPLICATION INITIATION
A review on
INTEGRAL PART OF
TETRAHYMENA ORC-
26T RNA
Tetrahymena thermophila
• Unicellular ciliate
• Model organism
• Nuclear dimorphism -germline micronuclie, vegetative macronuclie.
• Macronuclie- 5 monocentric chromosome-exceptional case rDNA chromosome-highly amplified.
• cis-acting replication determinants, type I elements that associate with sequence-specific, single-stranded binding factors, TIF1 through TIF4.
• TIF4- tetrahymena ORC. (Mohammad et. Al, 2003,2007).
• Binds to T rich strand.
• RNase treatment eliminates binding, its an RNP (Mohammad et. al,2009)
• ORC binds to rDNA origin throughout the cell cycle. HOW?
Why rDNA ORC is stable?
26T RNA
Binding of TtORC to rDNA ori is regulated by a noncoding RNA.
(Mohammad et al.,2007)
26T RNA
• Isolated and sequenced from TIF4
• Corresponds to 282 nts of 3’ terminus of
mature 23S rRNA. (Mohammad et al., 2007)
• Hence the name
• Sequence specifically interact with rDNA
origin.
• Confirmed by pull down assay and
complementary mutations in 26TRNA.
• Molecular mechanism- specific base pairing.
Proposed sequence of 26T RNA
Characterization of 26T
RNA
Sequence alignment with rDNA ori
Mohammad et al., 2007
• ChIP studies shows that ORC binds
specifically to rDNA origin, but not other sites
on the rDNA macronuclear chromosome.
• Recently, another ori-ARS 1 was identified,
to which ORC binds independent of
26TRNA.( Donti et al., 2009)
• But, the binding is on random sites.
• Concluding, 26T RNA needed for differential
binding of ORC to rDNA origin.
• In conclusion, the key role of 26T RNA in the
control of rDNA origin is linked to the efficient
and site specific recruitment of ORC to this
region, which is highly amplified during
development.
• Give selective advantage under competitive
demand for limited initiation factors.
• A new role for ribosomal RNA in
chromosome biology
Unanswered Question
• ORC-26T RNA does not bind to the perfectly
complementary rDNA region
• Does not bind to any other type 1 Element
• Something beyond the specific base
pairing????????
Viral encoded G rich RNA
recruits human ORC to
EBV genome.
Epstein- Barr Virus
• Oncogenic human gamma herpes virus.
• EBV origin of plasmid replication (OriP)
provides an attractive model to study ORC
recruitment in human cells.
• EBNA 1 , a virus encoded protein recruits
host ORC to the particular element of Ori P,
the dyad symmetry element. (Lindner and
Sugden, 2007)
EBV episomal
minichromoso
me
Ori P
EBNA 1ORC
ORC binding to the viral origin
Norseen et al., 2009
• Human ORC does not have any sequence specificity, then how it is targeted to a particular sequence in EBV OriP?
• A structured non coding RNA plays key role in this sequence specific interaction. (Norseen et al., 2008)
• EBNA1 have amino terminal linking regions LR1 and LR2 binding to ORC.
• Further study revealed LR contains RGG motif.
• RGG motif is known for RNA binding.
Mutation analysis confirmed this motif is essential for ORC binding.
• EBNA 1 binds to RNA through RGG
motif.(Snudden et al., 1994, Lu et al., 2004)
suggesting that ORC binding via these motif
may influenced by RNA.
• Confirmed by treatment of immuno
precipitants by RNase treatment- disrupted
the interaction between EBNA and ORC.
(Norseen et al., 2008)
• EBNA1 binds to its own mRNA.
• This RNAs are heterogeneous in size,
EBNA1 shows preference to G rich RNA with
particular secondary structures.(Lu et al., 2004,
Norseen et al., 2009))
• G rich RNA can form G quadruplexes,
suggesting G quadruplex mediate interaction
of ORC and EBNA.
• EBNA1 mRNA have G rich motif.
Model of G rich RNA mediating ORC
recruitment
Norseen et al., 2008
What’s next?
• Exact mechanism unknown.
• FMRP and HMGA1a proteins also contains
RGG motifs
• MNase treatment of nuclear extracts
released 40% ORC, Rnase treatment, 20%.
(Norseen et al., 2008)
• Involved in cellular replication also???
• G-quadruplex-interacting compounds may be
useful for the pharmacological inhibition of
EBNA1-dependent replication.
Vertebrate Y RNA is
essential for DNA
replication
Y RNA
• First identified in 80’s from the patients with
autoimmune diseases. (Lerner et al,
• 1981)
• Component of Ro ribonucleoprotein.
• Found in all vertebrates, C.elegans and
D.radiodurans.
• In humans, Located in chromosome
7,transcribed by RNA polymerase III , 85-112
nucleotides.
• most abundant in heart and brain tissue
Y RNAs are highly conserved among vertebrates
Teunissen et al. Nuc Acids Res. 2000
Specific secondary structure is
responsible for function
Cellular functions
• Effect on Ro 60 well understood
• UV resistance in mammalian cells and
bacteria
• Localization of Ro 60
• Inhibit chaperone activities of La, hn RNP I
and K
• Essential role in DNA replication
• Required for the functional reconstitution of mammalian chromosomal replication in cell free system. (Christov et al.,2006)
• Isolated from cellular extract of S phase cell.
• Interacts with two proteins, RPA and PCNA.
• All vertebrate YRNAs are functionally redundant with each other.
• Non vertebrate Y RNAs could not replace function.
Cytosolic extract
(RPA)QA QB(PCNA+X)
ArFT(Protein) ArE (NA)
DNase I RNase A
Digestion
RNA
•Depletion of hY RNA inhibits
chromosomal DNA replication
in late G1 template nuclei.
•Other hY RNAs could
reconstitute the function
•Y RNA is not priming the
replication
Christov et al.,2006
Functional substitution of hY1 RNA with
other Y RNAs
• Upper stem contains
conserved motif for
function in replication
• Non vertebrate Y RNAs
do not have this motif.
• Only this motif is needed.
• two mutant Y RNAs with
shuffled nucleotide
sequences.SH and SH:US
SH SH:US
Replication
Gardiner et al. 2009
What’s the Y doing?
Y
?
?
Replication of chromosomal DNA in isolated G1- and S-phase nuclei.
Krude T et al. J Cell Sci 2009;122:2836-2845
©2009 by The Company of Biologists Ltd
Y RNA is not needed in extension
DNA combing assay
• aligns DNA fibres on glass cover
slips in a parallel fashion
• individual tracks of replicated
DNA can be visualised by
fluorescent microscopy
• two distinct labels allows
detection of replication origin
activation and quantification of
individual replication fork
progression rates
• digoxigenin-dUTP at the
beginning of an in vitro
replication reaction, and biotin-
dUTP after 60 minutes
Nascent DNA assay
• Nascent DNA was released and
analyzed on alkaline agarose gel
• For elongation studies S phase
cells pre-labelled in vitro, then
incubated in unlabelled medium.
DNA COMBING ASSAY
Krude T et al. J Cell Sci 2009;122:2836-2845
©2009 by The Company of Biologists Ltd
Nascent DNA analysis
Krude et al., 2009
The catch and Release model
• Y RNAs bind to euchromatin dynamically
• Y RNAs interact with initiation proteins (ORC,
Cdt1, Cdc6, DUE-B and Ku)
• First associate with unreplicated G1 nuclei
• Trigger initiation proteins
• After initiation, leaves the region
• Discriminates replicated and unreplicated
regions,
• Thus licensing “once and only once
replication”
Krude et al., 2011
Role in human diseases
• YRNAs are highly expressed in cancers. (Christov et al
2008)
• It was hypothesized that Y RNAs may have a critical
role in maintaining the neoplastic phenotype of
human tumors.
urinary bladder cervix colon
Kidney Lung Postrate
Potential as a tool
• could serve as a potential biomarker for
identification of proper therapeutic
intervention
• could be used as a tool to manipulate cell
cycle regulation and cell proliferation in
culture.
Unanswered questions
• The functional replacement of hY1 and hY3
with other Ys. Is it just a numbers issue?
• Involvement of Ro 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?
• HOW DOES IT WORK?
CONCLUDING REMARKS…
• The emergence of non-coding RNAs from these studies implies the existence of additional factors assisting ORC for origin targeting and replication licensing.
• Upon RNase A treatment, a fraction of ORC is released from chromatin, indicating that the ORC association with chromatin can be partially stabilized by RNA ,
• It is highly possible that some structured RNAs mediate ORC recruitment to certain origins. These findings reinforce the important role of non-coding RNAs in the regulation of replication initiation.
REFERNCES
• Timothy J. Gardiner, Christo P. Christov, Alexander R. Langley, et al.
(2009). A conserved motif of vertebrate Y RNAs essential for
chromosomal DNA replication. RNA 15: 1375-1385.
• Alice Tianbu Zhang, Alexander R. Langley, Christo P. Christov,
Eyemen Kheir, Thomas Shafee, Timothy J. Gardiner and Torsten
Krude. (2011)Dynamic interaction of Y RNAs with chromatin and
initiation proteins during human DNA replication. Journal of Cell
Science. 124, 2058-2069.
• Christo P. Christov, Timothy J. Gardiner, David Szuts, and Torsten
Krude. (2006). Functional Requirement of Noncoding Y RNAs for
Human Chromosomal DNA Replication. Molecular and Cellular
Biology.26, 6993–7004.
• Julie Norseen, F. Brad Johnson, and Paul M. Lieberman. (2009). Role
for G-Quadruplex RNA Binding by Epstein-Barr Virus Nuclear Antigen
1 in DNA Replication and Metaphase Chromosome Attachment.
Journal of Virology. 83(20),10336–10346.
•De Pamphilis, M. L., Blow, J. J., Ghosh, S., Saha, T., Noguchi, K., and Vassilev, A.
(2006) Regulating the licensing of DNA replication origins in metazoa. Curr Opin Cell
Biol, 18, 231–239.
•Donti, T. R., Datta, S., Sandoval, P. Y., and Kapler, G. M. (2009) Differential targeting
of Tetrahymena ORC to ribosomal DNA and non-rDNA replication origins. EMBO J,
28, 223–233.
•Krude, T. (2006) Initiation of chromosomal DNA replication in mammalian cell-free
systems. Cell Cycle, 5, 2115–2122.
•Mohammad, M. M., Donti, T. R., Sebastian Yakisich, J., Smith, A. G., and Kapler, G.
M. (2007) Tetrahymena ORC contains a ribosomal RNA fragment that participates in
rDNA origin recognition. EMBO J, 26, 5048–5060.
•Norseen, J., Thomae, A., Sridharan, V., Aiyar, A., Schepers, A., and Lieberman, P. M.
(2008) RNA-dependent recruitment of the origin recognition complex. Embo J, 27,
3024–3035.
THANK YOU…….