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…….