lncRNA

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• lncRNA
• Long 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) are
• proposed to be the largest transcript class in
  the
• mouse 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 PTEFß
  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 product
• EXAMPLE  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
• BeckwithWiedeman syndrome (lncRNAs H19 and KCNQ1O
  T1)
• SilverRussell syndrome (lncRNA H19(
• McCuneAlbright 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 tRNAs
Small ncRNA (18 to 200 nt in size) MicroRNAs miRNAs
Small ncRNA (18 to 200 nt in size) Ribosomal 5S and 5.8S RNAs rRNAs
Small ncRNA (18 to 200 nt in size) Piwi interacting RNAs piRNAs
Small ncRNA (18 to 200 nt in size) Tiny transcription initiation RNAs tiRNAs
Small ncRNA (18 to 200 nt in size) Small interfering RNAs siRNA
Small ncRNA (18 to 200 nt in size) Promoter-associated short RNAs PASRs
Small ncRNA (18 to 200 nt in size) Termini-associated short RNAs TASRs
Small ncRNA (18 to 200 nt in size) Antisense termini associated short RNAs aTASRs
Small ncRNA (18 to 200 nt in size) Small nucleolar RNAs snoRNAs
Small ncRNA (18 to 200 nt in size) Transcription start site antisense RNAs TSSa-RNAs
Small ncRNA (18 to 200 nt in size) Small nuclear RNAs snRNAs
Small ncRNA (18 to 200 nt in size) Retrotransposon-derived RNAs RE-RNAs
Small ncRNA (18 to 200 nt in size) 3UTR-derived RNAs uaRNAs
Small ncRNA (18 to 200 nt in size) x-ncRNA x-ncRNA
Small ncRNA (18 to 200 nt in size) Human Y RNA hY RNA
Small ncRNA (18 to 200 nt in size) Unusually small RNAs usRNAs
Small ncRNA (18 to 200 nt in size) Small NF90-associated RNAs snaRs
Small ncRNA (18 to 200 nt in size) Vault RNAs vtRNAs
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Type Subclasses Symbol
Long ncRNA (lncRNAs, 200 nt to gt100 kb in size) Ribosomal 18S and 28S RNAs rRNAs
Long ncRNA (lncRNAs, 200 nt to gt100 kb in size) Long or large intergenic ncRNAs lincRNAs
Long ncRNA (lncRNAs, 200 nt to gt100 kb in size) Transcribed ultraconserved regions T-UCRs
Long ncRNA (lncRNAs, 200 nt to gt100 kb in size) Pseudogenes None
Long ncRNA (lncRNAs, 200 nt to gt100 kb in size) GAA-repeat containing RNAs GRC-RNAs
Long ncRNA (lncRNAs, 200 nt to gt100 kb in size) Long intronic ncRNAs None
Long ncRNA (lncRNAs, 200 nt to gt100 kb in size) Antisense RNAs aRNAs
Long ncRNA (lncRNAs, 200 nt to gt100 kb in size) Promoter-associated long RNAs PALRs
Long ncRNA (lncRNAs, 200 nt to gt100 kb in size) Promoter upstream transcripts PROMPTs
Long ncRNA (lncRNAs, 200 nt to gt100 kb in size) Stable excised intron RNAs None
Long ncRNA (lncRNAs, 200 nt to gt100 kb in size) 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

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X chromosome inactivation in mammals
X
X
X
Y
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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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mechanism of HOTAIR

• 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.

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• 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 Lifes
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)
• Heres 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 database
• http//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 end

• Thanks
• SOGAND VAHIDI
• sogand.v4_at_gmail.com