Scuola di specializzazione in Ostetricia e Ginecologia Facolt

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Scuola di specializzazione in Ostetricia e
Ginecologia Facoltà di Medicina e Chirurgia,
Università di PadovaLezioni di Biologia
ApplicataStefania Bortoluzzi

• Long non-coding RNAs in disease and cancer

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PREVASIVE GENOME TRANSCRIPTION

• C-value paradox genome size does not correlate
  with organismal complexity
• Gene-value paradox relationship between
  morphological complexity and the number of
  protein-coding genes within a genome
• simplistic expectation contradictory data
  paradox

• The number of human genes is about the same as
  the number required to specify a nematode worm

• The secret of evolution lies in gene regulation
  complexity?

• FACTS
• Less than 2 of the human genome encodes
  proteins
• Recent evidence from genomic tiling arrays and
  transcriptome deep sequencing showed that gt50 of
  the human genome is transcribed
• The bulk of transcriptional products consists of
  small and long RNAs with very reduced coding
  potential
• ? most eukaryotic transcribed DNA is non-coding

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PREVASIVE GENOME TRANSCRIPTION

• Main observations
• Intronic transcripts
• Non-polyadenylated RNAs
• Antisense and overlapping transcription
• Concept of transcribed dark matter, i.e.
  transcripts with unknown function and meaning
• Tests of function that depend on gene knockout
  or overexpression only work for a fraction even
  of known protein-coding genes. Need to establish
  non coding transcripts function

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NON-coding RNA

• RNA molecules both encode sequence information
  and possess great structural plasticity
• RNA can directly interact with DNA and with
  other RNAs by base pairing
• Highly structured RNA can also provide docking
  sites for binding proteins
• RNA has a compact size and significant sequence
  specificity
• non-coding RNA known from long time
• rRNA and tRNA in translation
• snRNA and snoRNA in mRNA processing
• ribozymes
• remind the RNA world hypothesis

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sMALL AND LONG NON-coding RNA

• Genome-wide surveys have revealed that
  eukaryotic genomes are extensively transcribed
  into thousands of long and short ncRNAs
• Important small ncRNA with regulatory roles
• miRNAs
• siRNAs
• piRNAs (Piwi-interacting RNA, transposon
  silencing in spermatogenesis)
• Long ncRNAs, lncRNAs gt200 nt
• Many lncRNAs show spatial- and temporal-specific
  patterns of expression, indicating that lncRNA
  expression is strongly regulated
• lncRNAs have specific biological functions
• if they are by-products of other regulatory
  events, they can be convenient biomarkers of
  ongoing regulation

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lncRNAs molecular function

• lncRNA Mechanisms can be described according to
  four, not mutually exclusive, archetypes
• As signals
• As decoys
• As guides
• As scaffolds

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lncRNAs molecular function
lncRNAs as signals

• lncRNAs can serve as molecular signals, because
  transcription of individual lncRNAs occurs at a
  very specific time and place to integrate
  developmental cues, interpret cellular context,
  or respond to diverse stimuli.
• Some lncRNAs in this archetype possess
  regulatory functions, while others are merely
  by-products of transcriptionit is the act of
  initiation, elongation, or termination that is
  regulatory.
• The advantage of using RNA as a medium suggests
  that potential regulatory functions can be
  performed quickly without protein translation.

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lncRNAs molecular function
lncRNAs as signals

• Allele specificity Xist ion X chromosome
  inactivation, Air expressed only in paternal
  chromosome and repress inmprineted genes
• Anatomic specific expression HOTAIR and HOTTIP
  from Hox loci
• Induction by DNA damage LinkRNA-p21 acts as
  transcriptional repressor of P53 pathway and
  triggers apoptosis
• Induction by cold RNAs expressed after
  vernalization controls flowering
• Coordinated activity eRNAs (enhancer RNAs)

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lncRNAs molecular function
lncRNAs as decoys

• as decoys, lncRNAs can titrate transcription
  factors and other proteins away from chromatin or
  titrate the protein factors into nuclear
  subdomains
• as decoys, lncRNAs can compete with mRNAs for
  miRNA target sites

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lncRNAs molecular function
lncRNAs as decoys (molecular sinks)

• TERRA telomeric repeat-containing RNA
  sequestrates the telomerase. It is large
  non-coding RNA in animals and fungi, which forms
  an integral component of telomeric
  heterochromatin. The accumulation of TERRA at
  telomeres interferes with telomere replication,
  leading to a sudden loss of telomere tracts.
• ncRNA compete for miRNA binding. The 3' UTR of
  PTENP1 (tumor suppressor pseudogene) RNA was
  found to bind the same set of regulatory miRNA
  sequences that target the tumor-suppressor gene
  PTEN, reducing the downregulation of PTEN mRNA
  and allowing its translation into the
  tumor-suppressor protein PTEN.

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lncRNAs molecular function
lncRNAs as guides

• The third archetype of lncRNA is the guideRNA
  binds protein(s), then directs the localization
  of ribonucleoprotein complex to specific targets.
  
• lncRNAs can guide changes in gene expression
  either in cis (on neighboring genes) or in trans
  (distantly located genes) in a manner that is not
  easily predicted based on lncRNA sequence.

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lncRNAs molecular function
lncRNAs as guides

• The gene regulatory components brought on by the
  lncRNAs include both repressive (e.g., polycomb)
  and activating (MLL) complexes, as well as
  transcription factors (TFIIB). However, no matter
  the distance or mechanism (either cis or trans),
  the principle remains the same to convey
  regulatory information across an intervening
  stretch of DNA to control target gene expression,
  bringing about changes in the epigenome.
• Expression of the Hox lncRNA HOTAIR has recently
  been associated with cancer metastasis and was
  observed in primary and metastatic breast cancer.
  
• Depletion of HOTAIR from cancer cells leads to a
  reduced invasiveness of cells that express a high
  level of polycomb proteins (PRC2). These findings
  suggest that ncRNA-mediated targeting of polycomb
  complexes is a crucial event in breast
  tumorigenesis.
• lncRNAs such as HOTAIR are able to alter and
  regulate epigenetic states in cells through their
  targeting of chromatin-modifying complex
  occupancy/localization/enzymatic activity in
  trans.

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lncRNAs molecular function
lncRNAs as scaffolds

• Traditionally, proteins were thought to be the
  major players in various scaffolding complexes

• As scaffolds, lncRNAs can bring together
  multiple proteins to form ribonucleoprotein
  complexes.
• The lncRNA-RNP may act on chromatin as
  illustrated to affect histone modifications.
• In other instances, the lncRNA scaffold is
  structural and stabilizes nuclear structures or
  signaling complexes.The lncRNA scaffold is
  structural and stabilizes nuclear structures or
  signaling complexes.

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lncRNAs molecular function
lncRNAs as scaffolds

• LncRNAs can serve as central platforms upon
  which relevant molecular components are
  assembled.
• Telomerase catalytic activity requires the
  association of two universal telomerase subunits
  an integral RNA subunit, the telomerase RNA
  (TERC) that provides the template for repeat
  synthesis, and a catalytic protein subunit, the
  TERT, as well as several species-specific
  accessory proteins. The TERC in particular also
  possesses structures that contribute to TERT
  binding and catalytic activity, in addition to
  those that play major roles in stability of the
  complex.
• Thus, the primary functional role for TERC is to
  be a scaffold

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lncRNAs and disease

• Alterations in the primary structure, secondary
  structure, and expression levels of lncRNAs as
  well as their cognate RNA-binding proteins
  underlie diseases ranging from neurodegeneration
  to cancer.
• Recent progress suggests that the involvement of
  lncRNAs in diverse human disease.
• Evidence highlight fundamental concepts in
  lncRNA biology that still need to be clarified to
  provide a robust framework for lncRNA genetics.

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lncRNAs and disease

• LncRNAs involved in epigenetic silencing
• lncRNA ANRIL
• The INK4b/ARF/INK4a locus encodes three tumor
  suppressor genes that have been linked to various
  types of cancers.
• A recent study has characterized the mechanism by
  which the lncRNA ANRIL mediates INK4a
  transcriptional repression in cis.
• ANRIL was shown to interact with the Pc/Chromobox
  7 (CBX7) protein, a member of the polycomb
  repressive complex 1 (PRC1).
• Altered ANRIL activity might result in
  dysregulated silencing of the INK4b/ARF/INK4a
  locus, contributing to cancer initiation.
• Genome-wide association studies have shown that
  the intergenic region encompassing ANRIL is
  significantly associated with increased
  susceptibility to coronary disease, intracranial
  aneurysm, type 2 diabetes, as well as several
  types of cancers

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lncRNAs and disease

• Splicing regulation by lncRNAs
• lncRNA MALAT-1
• MALAT-1 (metastasis-associated in lung
  adenocarcinoma transcript) was identified in an
  attempt to characterize transcripts associated
  with early-stage non-small-cell lung cancer
  (NSCLC).
• MALAT-1 is an abundant 6.5 kb lncRNA
  transcribed from chromosome 11q13 and primarily
  localized in nuclear speckles.
• MALAT-1 regulates alternative splicing through
  its interaction with the serine/arginine-rich
  (SR) family of nuclear phosphoproteins which are
  involved in the splicing machinery.
• In NSCLC metastasizing tumors, MALAT-1
  expression is three-fold higher than in
  non-metastasizing tumors.
• In patients with stage I disease, MALAT-1
  expression is closely correlated with poor
  prognosis.
• lcnRNAs as prognostic marker for metastasis and
  survival?

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lncRNAs and disease
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Human genetics of lncRNAs

• The bulk of sequence mutations in the genome
  occur in non-coding and intergenic regions.
• A substantial portion of the genome is
  transcribed
• Mutations are transmitted to the transcriptome,
  potentially affecting a large number of lncRNAs.
• How small mutations in lncRNAs contribute to
  disease?
• How primary sequence translates into lncRNA
  function?