Terra (Telomeric repeat-containing RNA)

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lncRNALong non-protein-coding RNAs

functional surprises from the RNA world

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LONG NON-PROTEIN-CODING RNAS (LNCRNAS) largest transcript class in the mouse and

human transcriptomes large amount of noncoding RNAs :

• microRNAs • long noncoding RNAs(lncRNAs)

regulation of cellular processes :cell growth and apoptosis, as well as cancer progression and metastasis

CRITICAL ROLE

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LONG NON-PROTEIN-CODING RNAS (LNCRNAS) biggest class of ncRNAs approximately 10,000 lncRNA genes

annotated in humans. The FANTOM3 project, by using physical

cDNA clone analysis alone, identified more than 23,000 lncRNAs

Transcribed:• RNA polymerase II• RNA polymerase III

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FUNCTION

The function of the majority of lncRNAs is still unknown.

negatively or positively regulating gene expression differentiation human disease regulate protein-coding (pc) gene expression:

posttranscriptional transcriptional

Paucity of Introns (nuclear localization) Low GC content (low expression level) Predicted ORFs have poor start codon and contexts Significant similarity between lncRNA and 3’-UTR of

mRNA (structural feature + sequence composition)

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LOCATION mRNAs cytoplasm

lncRNAs nucleus cytoplasm

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HUMAN DISEASES:

Cancers (breast cancer, colorectal cancer, prostate cancer, hepatocellular carcinoma, leukemia, melanoma

Alzheimer Psoriasis Heart disease Transient neonatal diabetes mellitus (lncRNA HYMAI) Pseudohypoparathyroidism (lncRNA NESP-AS) Atheromatosis and atherosclerosis Beckwith–Wiedeman syndrome

(lncRNAs H19 and KCNQ1OT1) Silver–Russell syndrome (lncRNA H19( McCune–Albright syndrome (lncRNA NESP-AS)

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LOCATION IN GENOME LncRNAs can be categorized according to

their proximity to protein coding genes in the genome, using this criteria lncRNAs are generally placed into five categories:

sense antisense bidirectional intronic intergenic

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Sense - The lncRNA sequence overlaps with the sense strand of a protein coding gene.Antisense - The lncRNA sequence overlaps with the antisense strand of a protein coding gene.Bidirectional - The lncRNA sequence is located on the opposite strand from a protein coding gene whose transcription is initiated less than 1000 base pairs away.Intronic - The lncRNA sequence is derived entirely from within an intron of another transcript. This may be either a true independent transcript or a product of pre-mRNA processingIntergenic - The lncRNA sequence is not located near any other protein coding loci

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HUMAN CANCER-ASSOCIATED LNCRNAS

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TERRA

telomeric repeat-containing RNA

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TELOMERES Identified in the 1930s nucleoprotein structures that protect the

ends of Chromosomes From DNA double-strand breaks (DSBs) and

from degradation At the cellular level for genome stability At the organismal level, they act as tumour

suppressors and may contribute to ageing Human telomeres extend from 9–15 Kb, but can be as long as 100 Kb in rodents

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Human and mouse telomeres are composed: long tracts of double-stranded G rich TTAGGG

repeats complex, shelterin The shelterin complex consists of six protein

subunits: 1. Telomeric repeat binding factor-1 (TRF1)2. TRF2, 3. Repressor activator protein-1 (RAP1)4. TRF1-interacting protein-2 (TIN2)5. TINT1/PIP1/PTOP 1 (TPP1) 6. protection of telomeres-1 (POT1) regulated the maintenance of telomere length

and protects natural chromosome ends from being recognized as damaged

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Single-stranded repeat structures created by 5′ to 3′ exonucleases

The invasion of the 3′ telomeric overhang into the duplex telomeric array forms a T-loop TRF2.

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THE FOLLOWING ROLES OFTELOMERES ARE WELL ESTABLISHED Regulated the lifespan of cells (Harley et al, 1990;

Allsopp et al, 1992; Levy et al, 1992; Bodnar et al, 1998)

• Telomere shortening occurs at the distal ends• Telomerase is regulated at individual chromosome

ends• in humans, telomerase is expressed in most tissues

only during the first weeks of embryogenesis (Ulaner and Giudice, 1997).

• Repression of telomerase in somatic cells is thought to result in a powerful tumour-suppressive function.

Telomeres protect natural chromosome ends from unwanted DNA repair activities

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TELOMERIC REPEAT-CONTAINING RNA (REFERRED TO AS TERRA) A noncoding RNA molecule, has recently been found

in mammalian cells Forms an integral component of telomeric

heterochromatin TERRA transcription occurs at most or all chromosome

ends TERRA regulated by RNA surveillance factors and in

response to changes in telomere length Roles in the regulation of telomerase and in

orchestrating chromatin remodelling throughout development and cellular differentiation

transcribed by RNA polymerase II (RNAPII). RNAPI and RNAPIII)

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inhibition of RNAPII by a-amanitin reduces the abundance of TERRA in both human and mouse cells

Actinomycin D treatment resulted in a 50%reduction in TERRA levels within 2.5–3 h

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Approximately 7% of human TERRA molecules are polyadenylated

most or all yeast TERRA molecules carry a poly(A) tail

all human and most yeast TERRA 5′ ends contain a 7 -methylguanosine (m7G) cap structure

increase the stabilityof TERRA molecules

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TERRA forms stable G-quadruplexes in vitro and in vivo

there is direct evidence that TERRA binds to the telomeric protein TRF2 by forming an intramolecular G-quadruplex structure

TERRA participates in multiple regulatory functions:

telomerase activity Heterochromatinization of telomeres cellular differentiation

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REGULATION OF TELOMERE LENGTH VIA SEVERAL PATHWAYS TERRA can inhibit telomerase activity to

promote telomere shortening. TERRA can promote Exo1-dependent

resection at chromosome ends to initiate telomere shortening.

the loss of TRF1 increases the recruitment of telomerase

Rap1 binds to the telomeres via TRF2 and plays a negative regulatory role on telomerase recruitment.

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HOW DOES TERRA REGULATE TELOMERE LENGTH DURING THIS PROCESS? The shelterin components TRF1 and TRF2

directly associate with TERRA in multiple cell types in vivo

It has also been reported that the T-loops can be deleted by homologous recombination (HR), which results in the rapid shortening of telomeres.

TRF2 is proposed to suppress T-loop HR The amino-terminal define (GAR) domain of

TRF2 has a high affinity for RNA Binding of TERRA to TRF2 could alleviate

protection of the t-loop and induce t-loop HDR

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TERRA AT TELOMERES TERRA is exclusively found in nuclear RNA

fraction TERRA can be detected using RNA

fluorescence in situ hybridization (RNAFISH) at a subset of telomeres in interphase cells

present on human and mouse chromosome ends in the metaphase of the cell cyclens from human and mouse cells

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TERRA EXPRESSION LEVELS DO NOT CORRELATE WITH TELOMERE LENGTH AND RADIATION SENSITIVITY IN HUMAN CANCER CELL LINES In human cells, CpG-island promoters drive

TERRA transcription and are regulated by methylation

suggestion:the amount of TERRA may be related to telomere length.

five human cell lines: HeLa (cervical carcinoma) BRC-230 (breast cancer) AKG (gastric cancers) GK2 (gastric cancers) GM847 (SV40 immortalized skin fibroblasts)

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in cancer cells telomere length is maintained, usually through telomerase activation

in about 15% of human cancers, an alternative telomere lengthening (ALT) mechanism operates which does not require telomerase activation.

In ALT cells, telomere maintenance is due toa recombination based mechanism causinggreat intracellular variability of telomere length TERRA is regulated during the cell cycle being lowest in late S phase peaking in early G1

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TERRA

telomerase

3’

3’

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regulation of telomerase by TERRA and hnRNPA1

multifunctional RNA-binding protein

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The Reference Database For Functional Long Noncoding RNAs

http://www.lncrnadb.org/

the LncRNADisease databasehttp://www.cuilab.cn/lncrnadisease

the latest version of this long non-coding RNA database contains 113,513 human annotated lncRNAs

http://www.lncipedia.org/

http://deepbase.sysu.edu.cn/chipbase/lncrna.php

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THE ENDThanks

Sogand VahidiEmail: [email protected]