Noncoding Y RNAs: A Functional Role in DNA

Replication

Mahta NiliBMCB 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 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

Methods III: Fractionation

Figure 1A

HeLa Cell Extracts from 4C Biotech

Cleared by ultracentrifugation

Pre-equilibrated in buffer containing 200mM KCl

Purification of RNA Necessary for Replication

Figure 1

Propidium Iodide Fab

Identification of RNAs

Figure 2

Out of 19 cDNA clones: 4 – 5S rRNA 8 – U2 snRNA 2 – hY4 5 – hY5

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

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

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 semi-conservative 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?

http://users.ox.ac.uk/~kearsey/

Y?

?

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?