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lncRNALong non-protein-coding RNAs

functional surprises from the RNA world

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DISCOVERY Many initial lncRNAs, such as XIST and H19,

were discovered in the 1980s and 1990s by searching cDNA libraries for clones of interest

Early 2000s: development of large scale cDNA sequencing leads to discovery of surprising number of lncRNA transcripts.

Mid 2000s: the number of detected lncRNA transcripts increases exponentially.

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Long non-protein-coding RNAs (lncRNAs) areproposed to be the largest transcript class in themouse and human transcriptomes The transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA,

and other non-coding RNA transcribed in one cell or a population of cells In addition to protein-coding genes, the human

genome makes a large amount of noncoding RNAs, including microRNAs and long noncoding RNAs (lncRNAs). Both microRNAs and lncRNAs have been shown to have a critical role in the regulation of cellular processes such as cell growth and apoptosis, as well as cancer progression and metastasis

 Such RNAs were previously disregarded as useless, but recent functional studies have revealed that they have multiple regulatory functions.

 In addition to mRNA and microRNA, lncRNA is a major hotspot in functional genomics research.

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LNCRNAS - A NEW LAYER OF GENOME REGULATORYINFORMATION It is now well appreciated that less than two percent of the

human genome codes for proteins and the majority of the genome gives rise to non-protein-coding RNAs (ncRNAs)

The focus of this review is long ncRNAs (known as lncRNAs), which constitute the biggest class of ncRNAs with approximately 10,000 lncRNA genes so far annotated in humans.

.Based on large-scale sequencing and prediction from chromatin-state maps of full length cDNA libraries in FANTOM2 and 3 as well as human transcriptomes, more than 4,600 LncRNAs in mouse and over 3,300 LncRNAs in human have been identified with a total of approximately 23,000 LncRNAs in a mammalian genome.

http://fantom3.gsc.riken.jp/ The number of lncRNAs may still rise if next-generation

sequencing studies focus on cell types that are not yet completely characterized.

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 lncRNAs are RNA polymerase II (RNAPII) transcripts that lack an open reading frame and are longer than 200 nucleotides.

Majority of long ncRNAs have been shown to be transcribed through RNA polymerase II, although some long ncRNAs are generated by RNA polymerase III

This size cut-off distinguishes lncRNAs from small RNAs such as microRNAs, piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs) and small interfering RNAs (siRNAs) and arises from RNA preparation methods that capture RNA molecules above this size.

?Two important questions are whether all lncRNAs are functional and how they could exert a function. Several lncRNAs have been shown to function through

their product, but this is not the only possible mode of action.

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FUNCTION The function of the majority of lncRNAs is still

unknown. the number of characterized lncRNAs is growing and

they play roles in negatively or positively regulating gene expression in development, differentiation and human disease

lncRNAs may regulate protein-coding (pc) gene expression at both the posttranscriptional and transcriptional level.

Posttranscriptional regulation could occur by lncRNAs acting as competing endogenous RNAs to regulate microRNA levels as well as by modulating mRNA stability and translation by homologous base pairing, or as in the example of NEAT1 that is involved in nuclear retention of mRNAs .

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FUNCTION Long ncRNAs that regulate transcription are

divergent molecules. Paucity of Introns (nuclear localization) Low GC content (low expression level) Predicted ORFs have poor start codon and

contexts (activation of nonsense-mediated decay pathway)

Significant similarity between lncRNA and 3’-UTR of mRNA (structural feature + sequence composition)

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The most mRNAs, which ultimately localized to the cytoplasm after processing, most lncRNAs are permanently localized in the nucleus. There are also some lncRNAs selectively localized in the cytoplasm

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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 processing

Intergenic - The lncRNA sequence is not located near any other protein coding loci

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MODES OF TRANSCRIPTIONAL REGULATION BY LNCRNAS Regulation of transcription is considered:

to be an interplay of tissue and developmental specific transcription factors (TFs)

chromatin modifying factors acting on enhancer promoter sequences to facilitate the assembly of

the transcription machinery at gene promoters. Transcriptional regulation by lncRNAs could work

either in cis or in trans, and could negatively or positively control pc gene expression.

lncRNAs work in cis when their effects are restricted to the chromosome from which they are transcribed, and work in trans when they affect genes on other chromosomes.

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LncRNAs fall into one of three categories. (A) Long intervening non-coding RNAs (lincRNAs) are transcribed from regions far away from protein-coding genes. (B) Natural Antisense Transcripts (NATs) are transcribed from the opposite strand of a protein-coding gene. (C) Intronic lncRNAs (shown in green) are transcribed from within introns of protein-coding genes.

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TRANSCRIPTION OF LONG NCRNAS Many of long ncRNAs represent tissue-specific pattern of

expression the expression of these long ncRNAs should: 1.these long ncRNAs contain trimethyl marks of histone

H3-lysine (K) 4 at their promoter regions and trimethyl marks of histone H3-K36 along the length of the transcribed region, which are observed in usual transcripts by RNA polymerase II.

Trimethyl marks are designated as “chromatin signature (a K4-K36 domain)

2. long ncRNAs generally possess the 50CAP (7-methylguanosine cap) structure at the 50 edge and also poly (A) tail at their 30 end as well

3. the long ncRNAs have well-defined transcription factor binding sites like NF-kB in their promoter regions

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Transcriptional Regulation Through Targeting Basic Transcription Factors and RNA

Polymerase II by Long ncRNAs***************

1. Alu RNA SINE retrotransposon elements including Alu repeats

generate numerous species of long ncRNAs Alu RNAs and SINE B2 RNAs exert transcriptional repression

under the heat-shock condition SINE B2 and Alu RNA directly target RNA polymerase II. SINE B2 turns out to have similar repressive effect on the

transcription as well repetitive sequence that occupies the half of the human

genome could be transcribed, and their transcripts, the long ncRNAs, exert transcriptional repression

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2. Dehydrofolate Reductase ncRNA

In mammalian cells, expression of dehydrofolate reductase (DHFR) is repressed

a transcript of a minor promoter located upstream of a major promoter is involved in the repression of DHFR

The alternative promoters within the same gene have been observed in various loci.

It could be a general mechanism that the transcripts from the alternative promoters have a regulatory role in transcription of the promoter

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Transcriptional repression of dehydrofolate reductase (DHFR) gene by the ncRNA transcribed from the minor promoter of the DHFR gene. The DHFR ncRNA represses the DHFR gene expression by blocking the preinitiation complex through targeting TFIIB and RNA polymerase II

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REGULATION IN TRANS

Some significant examples of lncRNAs that act in trans are those that can influence the general transcriptional output of a cell by directly affecting RNAPII activity 

EXAMPLE: The 331 nucleotide 7SK lncRNA, which represses

transcription elongation by preventing the PTEFβ transcription factor from phosphorylating the RNAPII carboxy-terminal domain

THE 178 nucleotide B2 lncRNA, a general repressor of RNAPII activity upon heat shock.

**  The B2 lncRNA acts by binding RNAPII and inhibiting phosphorylation of its CTD by TFIIH, thus disturbing the ability of RNAPII to bind DNA 

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REGULATION IN TRANS

Regulation in trans can also act locus-specifically. While the ability of lncRNAs to act locus-specifically to

regulate a set of genes was first demonstrated for imprinted genes where lncRNA expression was shown to silence from one to ten flanking genes in cis, lncRNAs that lie outside imprinted gene clusters, such as the HOTAIR lncRNA, were later found also to have locus-specific action.

 HOTAIR is expressed from the HOXC cluster and was shown to repress transcription in trans across 40 kb of the HOXD cluster 

 HOTAIR interacts with Polycomb repressive complex 2 (PRC2) and is required for repressive histone H3 lysine-27 trimethylation (H3K27me3) of the HOXD cluster.

 

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REGULATION IN CIS

In contrast to trans-acting lncRNAs, which act via their RNA product, cis-acting lncRNAs have the possibility to act in two fundamentally different modes.

1. depends on a lncRNA productEXAMPLE:  general cis-regulation is induction of X inactivation by the Xist lncRNA in female mammals. Xist is expressed from one of the two X chromosomes and induces silencing of the whole chromosome

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It was also proposed to act in plants. In Arabidopsis thaliana, the COLDAIR lncRNA is

initiated from an intron of the FLC pc gene and silences it by targeting repressive chromatin marks to the locus to control flowering time

2. regulation by lncRNAs involves the process of transcription itself, which is a priori cis-acting Several lines of evidence suggest that the mere

process of lncRNA transcription can affect gene expression if RNAPII traverses a regulatory element or changes general chromatin organization of the locus.

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MECHANISMS BY WHICH LNCRNA TRANSCRIPTION SILENCES GENE EXPRESSION Transcription-mediated silencing, also referred to as

‘transcriptional interference’ (TI) TI has been reported in unicellular and multicellular

organisms Mechanistic details are still largely unclear, but TI

could theoretically act at several stages in transcription: by influencing enhancer or promoter activity or by blocking RNAPII elongation, splicing or polyadenylation.

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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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THE FUNCTIONAL ROLE OF LONG NON-CODING RNA INHUMAN CARCINOMAS

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Type Subclasses Symbol

Small ncRNA (18 to 200 nt in size)

Transfer RNAs tRNAsMicroRNAs miRNAsRibosomal 5S and 5.8S RNAs rRNAsPiwi interacting RNAs piRNAsTiny transcription initiation RNAs tiRNAsSmall interfering RNAs siRNAPromoter-associated short RNAs PASRsTermini-associated short RNAs TASRsAntisense termini associated short RNAs

aTASRs

Small nucleolar RNAs snoRNAsTranscription start site antisense RNAs TSSa-RNAsSmall nuclear RNAs snRNAsRetrotransposon-derived RNAs RE-RNAs3’UTR-derived RNAs uaRNAsx-ncRNA x-ncRNAHuman Y RNA hY RNAUnusually small RNAs usRNAsSmall NF90-associated RNAs snaRsVault RNAs vtRNAs

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Type Subclasses Symbol

Long ncRNA (lncRNAs, 200 nt to >100 kb in size)

Ribosomal 18S and 28S RNAs rRNAs

Long or large intergenic ncRNAs lincRNAs

Transcribed ultraconserved regions T-UCRs

Pseudogenes None

GAA-repeat containing RNAs GRC-RNAs

Long intronic ncRNAs None

Antisense RNAs aRNAs

Promoter-associated long RNAs PALRs

Promoter upstream transcripts PROMPTs

Stable excised intron RNAs None

Long stress-induced non-coding transcripts

LSINCTs

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accumulating reports of misregulated lncRNA expression across numerous cancer types suggest that aberrant lncRNA expression may be a major contributor to tumorigenesis

This surge in publications reflects the increasing attention to this subject and a number of useful lncRNA databases have been created

transcribed ultraconserved regions (T-UCRs), within human carcinomas.

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With advancements in cancer transcriptome profiling and accumulating evidence supporting lncRNA function, a number of differentially expressed lncRNAs have been associated with cancer

LncRNAs have been implicated to regulate a range of biological functions and the disruption of some of these functions, such as genomic imprinting and transcriptional regulation, plays a critical role in cancer development.

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MPRINTED LNCRNA GENES Imprinting is a process whereby the copy of a gene

inherited from one parent is epigenetically silenced imprinted regions often include multiple maternal and

paternally expressed genes with a high frequency of ncRNA genes

The imprinted ncRNA genes are implicated in the imprinting of the region by a variety of mechanisms including enhancer competition and chromatin remodeling

A key feature of cancer is the loss of this imprinting resulting in altered gene expression

Two of the best known imprinted genes are in fact lncRNAs.

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The H19 gene encodes a 2.3 kb lncRNA that is expressed exclusively from the maternal allele

H19 and its reciprocally imprinted protein-coding neighbor the Insulin-Like Growth Factor 2 or IGF2 gene at 11p15.5.

The expression of H19 is high during vertebrate embryo development, but is down regulated in most tissues shortly after birth with the exception of skeletal tissue and cartilage

loss of imprinting at the H19 locus resulted in high H19 expression in cancers of the esophagus, colon, liver, bladder and with hepatic metastases

H19 has been implicated as having both oncogenic and tumor suppression properties in cancer

H19 is upregulated in a number of human cancers, including hepatocellular, bladder and breast carcinomas, suggesting an oncogenic function for this lncRNA

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In colon cancer H19 was shown to be directly activated by the oncogenic transcription factor c-Myc

H19 transcripts also serve as a precursor for miR-675, a miRNA involved in the regulation of developmental genes

miR-675 is processed from the first exon of H19 and functionally down regulates the tumor suppressor gene retinoblastoma (RB1) in human colorectal cancer, further implying an oncogenic role for H19

There is evidence suggesting H19 may also play a role in tumor suppression

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Using a mouse model for colorectal cancer, it was shown that mice lacking H19 manifested an increased polyp count compared to wildtype

a mouse teratocarcinoma model demonstrated larger tumor growth when the embryo lacked H19

in a hepatocarcinoma model, mice developed cancer much earlier when H19 was absent

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XIST - X-INACTIVE-SPECIFIC TRANSCRIPT XIST is arguably an archetype for the study of

functional lncRNAs in mammalian cells, having been studied for nearly two decades

In female cells, the XIST transcript plays a critical role in X-chromosome inactivation by physically coating one of the two X-chromosomes, and is necessary for the cis-inactivation of the over one thousand X-linked genes

Like the lncRNAs HOTAIR and ANRIL, XIST associates with polycombrepressor proteins, suggesting a common pathway of inducing silencing utilized by diverse lncRNAs

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In mice, X inactivation in the extra embryonic tissues is nonrandom , and the initial expression of Xist is always paternal in origin, followed later by random X inactivation in the epiblast associated with random mono-allelic expression

It is unclear, however, how much of this regulation is conserved in humans, who do not show imprinted X inactivation

While XIST expression levels are correlated with outcome in some cancers, such as the therapeutic response in ovarian cancer, the actual role that XIST may play in human carcinomas, if any, is not entirely clear.

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For tumors in which two active X chromosomes are observed, as has been frequently observed in breast cancer, the most common mechanism involves loss of the inactive X and duplication of the active X, often resulting in heterogeneous XIST expression in these tumors

many cancers do show different onsets and progressions in males and females

XIST expression will increase with the number of inactive X chromosomes.

It has been shown that approximately 15% of human X linked genes continue to be expressed from the inactive X chromosome

X CHROMOSOME INACTIVATION IN MAMMALS

X X X Y

X

Dosage compensation

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Many of the described lncRNAs are expressed in a variety of cancers, however a select few thus far have been associated with a single cancer type:

HOTAIR, has only been described in breast cancer

three lncRNAs PCGEM1, DD3 and PCNCR1 have been associated solely with prostate cancer

the liver associated lncRNA HULC is highly expressed in primary liver tumors, and in colorectal carcinomas that metastasized to the liver, but not in the primary colon tumors or in non-liver metastases

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The HOTAIR lncRNA is transcribed from the HOXC locus binding of the PRC2 and LSD1 complex to the HOXD locus the HOTAIR-PRC2-LSD1 complex is redirected to the HOXD locus

on chromosome 2 where genes involved in metastasis suppression are silenced through H3K27 methylation and H3K4 demethylation.

This drives breast cancer cells to develop gene expression patterns that more closely resemble embryonic fibroblasts than epithelial cells.

mechanism of HOTAIR

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(1)The kinase PRKACB functions as an activator of CREB.

(2) Phosphorylated (activated) CREB forms part of the RNA pol II transcriptional machinery to activate HULC expression.

(3) Abundant HULC RNA acts as a molecular sponge to sequester and inactivate the repressive function miR-372.

(4) PRKACB levels increase, as transcripts are normally translationally repressed by high miR-372 levels

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The long ncRNAs involving in transcriptional regulation through chromosomal modification

(a) Evf2 activates transcription by removing the methylase MeCP2 on CpG regions and releasing HDAC activity from the target gene.

(b) HOTAIR activates transcription by binding PRC2 and histone methylation of HOXD locus

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HUMAN CANCER-ASSOCIATED LNCRNAS

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RNA POLYMERASE III TRANSCRIPTION OF LNCRNA The lncRNAs described thus far are products of RNA

pol II transcription, yet many ncRNAs are transcribed by RNA polymerase III (RNA pol III)

RNA pol III is frequently deregulated in cancer cells resulting in increased activity

Aberrant RNA pol III function may have consequences to the expression of lncRNAs transcribed by this polymerase.

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EXAMPLE the lncRNA BC200 is a small cytoplasmic lncRNA in the

neurons of primate nervous systems and human cancers, but not in non-neuronal organs

Unlike the majority of lncRNAs described thus far, BC200 is transcribed by RNA pol III and shares unique homology with human Alu elements

The BC200 RNA has been characterized as a negative regulator of eIF4A-dependent translation initiation

Due to the fact that many whole transcriptome sequencing methods were developed to enrich for poly(A) purified transcripts, RNA pol III transcripts may have been excluded from analysis

This suggests that other, yet unidentified RNA pol III lncRNAs over-expressed in cancer may be participating in tumorigenesis

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LONG INTERGENIC NON-CODING RNAS, ONE OF LIFE’S BARE ESSENTIALS (2013) Researchers from the Rinn Lab set out to investigate

the functional relevance of lincRNAs under a number of physiological conditions, given that they are a key player in the regulation of gene expression.

The scientists focused on a subgroup of  lncRNAs called long intergenic noncoding RNAs (lincRNAs)

Here’s what they discovered: Knockout of 3 of the lincRNAs of interest produces a lethal

phenotype, a true TKO (at least in the boxing world). Several other knockouts also produced severe growth and

homeotic defects. Further detailed characterization was also done on a

particular lincRNA knockout (linc-Brn1b), which led developmental defects in the brain.

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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 VAHIDI sogand.v4@gmail.com

http://www.powershow.com/view.php?id=7268935&t=lncRNA