Transcript Stefania Bortoluzzi Lezioni di Biologia Applicata

Scuola di specializzazione in Ostetricia e Ginecologia Facoltà di
Medicina e Chirurgia, Università di Padova
Lezioni di Biologia Applicata
Stefania Bortoluzzi
 Long non-coding RNAs in disease and
cancer
PREVASIVE GENOME TRANSCRIPTION
• 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 >50% 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
• C-value paradox
genome size does
not correlate with
organismal
complexity
• Gene-value
paradox;
relationship between
morphological
complexity and the
number of proteincoding genes within
a genome
• simplistic
expectation +
contradictory data
= “paradox”
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
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
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 >200 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
lncRNAs MOLECULAR FUNCTION
lncRNA Mechanisms can be described according to four, not
mutually exclusive, archetypes:
1. As signals
2. As decoys
3. As guides
4. As scaffolds
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 transcription—it 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.
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)
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
lncRNAs MOLECULAR FUNCTION
lncRNAs as decoys (molecular sinks)
TERRA: telomeric repeat-containing RNA
sequestrates the telomerase. It is large noncoding 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.
lncRNAs MOLECULAR FUNCTION
lncRNAs as guides
• The third archetype of lncRNA is the guide—RNA 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.
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.
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.
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
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.
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
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?
lncRNAs AND DISEASE
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?