Non-coding RNA
26.04.2024
MDSD
If protein-coding portions of the human
genome make up only 1.5% what is the rest
doing?
Noncoding RNA:
Housekeeping noncoding RNAs (usually expressed constitutively)
•Transfer RNA (tRNA)
•Ribosomal RNA (rRNA)
•Splicing
•small nuclear RNAs
Regulatory noncoding RNAs
Short
•microRNAs
•small interfering RNAs
•Piwi-associated RNAs
Long >200bp
•lncRNAs
Noncoding RNA:
Proportion of the various classes of RNA in mammalian somatic cells by total mass
(A) and by absolute number of molecules (B).
Non-coding RNA: what is functional and what is junk? Front. Genet., 2015
Discovery of small RNAs Rosalind Lee
• The first small RNA:
• In 1993 Rosalind Lee (Victor Ambros lab) was studying
a non-coding gene in C. elegans, lin-4, that was
involved in silencing of another gene, lin-14, at the
appropriate time in the development of the worm C.
elegans.
• Two small transcripts of lin-4 (22nt and 61nt) were
found to be complementary to a sequence in the 3'
UTR of lin-14.
• Because lin-4 encoded no protein, she deduced that it
must be these transcripts that are causing the
silencing by RNA-RNA interactions.
• The second small RNA wasn't discovered until 2000!
Biogenesis and functional mechanisms of miRNAs, piRNAs and snoRNAs
Esteller, M. Nature Reviews Genetics 12, 861-874 (2011)
miRNAs
•miRNAs are small non-coding RNAs, usually consisting
of 20-22 nt for animals and 20-24 for plants
•All miRNA precursors have a well-predicted stem loop
hairpin structure
•Evolutionarily conserved
•Regulate gene expression by binding complementary
regions at 3’ regions of target mRNAs
•Act as negative regulators of gene expression (not
always)
•miRNA expression profile is tissue specific
miRNAs
• Currently - 38589 known miRNA entries in 271
species (miRBase release 22.1)
• Mechanism is far reaching and complex
– each miRNA may control many genes
• Operate by one of two hypothesized mechanisms:
–Complete pairing
mRNA is degraded - predominant in plants
–Imperfect pairing
Translation is repressed but mRNA remains intact - predominant
in animals
Genomic location of miRNAs
Kim et al., Biogenesis of small RNAs in animals, 2009.
miRNA biogenesis
Beezhold et al., Microprocessor of microRNAs: regulation and potential for therapeutic intervention, 2010.
Non-canonical pathways of miRNA biogenesis
Summary of Players
• Drosha and Pasha are part of the “Microprocessor”
protein complex (~600-650kDa)
• Drosha and Dicer are RNase III enzymes
• Pasha is a dsRNA binding protein
• Exportin 5 is a member of the karyopherin
nucleocytoplasmic transport factors that requires
Ran and GTP
• Argonautes are RNase H enzymes
Differences in miRNA Mode of Action
Post-Transcriptional Regulation
• Event • Outcome
• miRNA – target – Target mRNA
binding at 3’ UTR
degradation
of mRNA, due to
approximate – Translation is blocked
complementation by miRNA binding
of miRNA to mRNA – Target protein is not
produced
… no protein… no function
miRNA Target Binding
Bulges
The MRE is known as
Hairpin is more stable the “miRNA recognition
than a simple bulge element.” This is simply
the sequence in the
target that an miRNA
binds to
Processing
bodies are
sites of
storage
and/or
degradation
of mRNA
Diversity of miRNA functions
• Development
• Timing
• Cell proliferation
• Stem cell
• Cell death
• DNA methylation and chromatin formation
• Diseases
• Cancer
• Diabetes
• Cholesterol biosynthesis
• Nervous system disorders
• Autoimmune diseases
• Metabolic diseases
miRNA and Cancer
• Majority of miRNA genes are located
in cancer associated regions and
chromosome fragile sites
• Indicate a differential expression
pattern in normal and tumor tissues
• miRNA display tissue and cell lineage
specificity
• Altered miRNA expression pattern can
serve as markers for
• Tumor diagnosis
• Disease prognosis
• Therapeutic response prediction
• Cancer of unknown primary
The microcosmos of cancer, Nature review, 2012
microRNAs: Oncogene / Tumor Suppressor
Importance
• Translational regulators
[Link]
miRNAs in plants
miRNA Biogenesis Pathway
a) Animal b) Plant miRNA
biogenesis.
Mature miRNAs are
indicated in red and
miRNA* strands are in
blue.
Volume 17, Issue 3, p123–125, March 2012
Front. Plant Sci., 10 December 2015
Viral miRNAs
many viruses encode RNA silencing
suppressors (RSS) which are
employed to moderate the
potency of the cell’s miRNA
selection against viral replication
Harwig et al., Retroviral microRNAs, 2014.
siRNAs
• Small interfering RNA (siRNA), sometimes known as
short interfering RNA or silencing RNA, is a class of
double-stranded RNA molecules, 20-25 base pairs
in length.
• siRNA plays many roles, but it is most notable in the
RNA interference (RNAi) pathway, where it
interferes with the expression of specific genes
with complementary nucleotide sequences.
• These siRNAs are generated from long double
strand RNAs through various biological processes.
siRNA Pathway à
miRNA pathway
What is the Difference between
miRNA and siRNA?
• Function of both species is regulation of gene expression
• Difference is in where they originate
• siRNA originates with dsRNA
• siRNA is most commonly a response to foreign RNA (usually
viral) and is often 100% complementary to the target
• miRNA originates with ssRNA that forms a hairpin secondary
structure
• miRNA regulates post-transcriptional gene expression and is
often not 100% complementary to the target
Generation and action of siRNAs and miRNAs
• [Link]
RNA-induced transcriptional silencing (RITS)
Molecular Biology of RNA, Second Edition, Oxford University Press, 2015
RNA-induced transcriptional silencing (RITS)
Molecular Biology of RNA, Second Edition, Oxford University Press, 2015
piRNAs
• piRNAs are slightly longer than miRNAs (24–31 nt in
length).
• piRNAs derive from long single-stranded RNAs
transcribed from specific regions within the genome.
• In fact, piRNAs are generated from regions harboring
transposons, and they were firstly described as a
mechanism to protect the cells against the internal
attacks of transposon.
• piRNAs are able to interact with a specialized family of
argonaute proteins called PIWI that will guide them to
their targets and silence the transposon transcripts by
their slicing activity.
ncRNAs in transposons
Molecular Biology of RNA, Second Edition, Oxford University Press, 2015
ncRNAs in transposons
Molecular Biology of RNA, Second Edition, Oxford University Press, 2015
ncRNAs in transposons
Molecular Biology of RNA, Second Edition, Oxford University Press, 2015
Ping-pong
loop I
PIWI- interacting RNAs: small RNAs with big functions, Nature Reviews, 2019.
Ping-pong
loop II
PIWI- interacting RNAs: small RNAs with big functions, Nature Reviews, 2019.
Functions of piRNA
PIWI- interacting RNAs: small RNAs with big functions, Nature Reviews, 2019.
Molecular Biology of RNA, Second Edition, Oxford University Press, 2015
Long noncoding RNAs (lncRNA)
lncRNAs allow the cell to fine-tune gene expression in response to its environment or
to up-regulate, down-regulate, or completely silence a gene to ensure proper tissue
patterning during programmed embryonic development. This includes:
• control of cell division, development, cell metabolism, human diseases
(including cancer progression and metastasis)
• regulation of transcription (can act in either cis or trans)
• regulation of RNA splicing, RNA transport, translation, mRNA processing and
stability
• chromatin remodeling (can act in either cis or trans) and 3D architecture of
the genome, nuclear organization
• scaffolding for assembly of RNA-protein complexes, ligands for proteins
involved in gene regulation and in base-pairing interactions capable of guiding
lncRNA-containing ribonucleoprotein complexes to specific target sites on
DNA or RNA, decoys for sequestering of regulatory proteins from their target
DNA sequences, localization and/or activity of proteins
Many lncRNAs are differentially expressed during individual stages of differentiation
and their expression and activity often exhibit tissue specificity.
Categories of lncRNA action
Sana, J. et al. Journal of Translational Medicine 10, 103-124 (2012)
X chromosome inactivation
In mammals females carry two X chromosomes whereas males carry only a single X
giving a double dosage of X-linked genes in XX females relative to XY males.
To balance X-linked gene expression levels in males and females, female cells invoke a
mechanism (X chromosome inactivation, XCI) to randomly silence one of the X
chromosomes (Xm) during early embryogenesis.
Prior to cell differentiation: both X chromosomes are active (Xa)
During cell differentiation:
• each cell counts its number of X chromosomes
• one X chromosome is randomly selected for inactivation
• the lncRNA, Xist, is up-regulated on the future inactive X (Xi)
• a gradual chromosome-wide silencing process is then initiated on Xi
Once established, this silent state is stably transmitted through each round of cell
division in a heritable manner.
X chromosome inactivation
During development females undergo two forms of XCI - imprinted and random.
imprinted XCI – occurs during early embryogenesis - the paternal X chromosome (Xp) is
preferentially silenced (maintained in extra-embryonic tissues throughout
development)
random XCI – all imprinted epigenetic marks on Xp are erased in cells of epiblast lineage (these
form the future embryo) - a second round of XCI is then initiated where either Xp
or the maternal X chromosome (Xm) is randomly silenced (this randomness in
chromosome selection is directly observable in the coloration pattern of female
calico cats)
X chromosome inactivation
XIC is initiated from the X inactivation center (Xic) which encodes five ncRNAs of known function:
Xist, Tsix, Xite, RepA, and Jpx.
Xist (X-inactive specific transcript) is up-regulated only after transient pairing of the two X
chromosomes (pairing is the way in which the cell counts the number of X chromosomes).
Depletion of any of the factors involved in pairing (e.g., CTCF and OCT4) blocks pairing Þ cells
with an aberrant number of active (2 Xa) or inactive (2 Xi) X chromosomes.
Xist expression is regulated by Tsix ( – ), Xite ( – ), Jpx (+), and RepA (+)
Regulation of X-chromosome inactivation by the X-inactivation centre, Nature Reviews Genetics, 2011
XCI in mammals
Molecular Biology of RNA, Second Edition, Oxford University Press, 2015
XCI in mammals
Curious Case of Calico Cats
Curious Case of Calico Cats
• The areas of the white coat color are the result of an autosomal gene that affects the migration of
pigment-producing cells known as melanocytes.
• The amount of white fur a particular calico cat has depends on how slow the migration of
melanocytes is, so a very slow migration results in larger areas of white fur.
Are Calico Cats always Female?
• Yes, calico cats need to have two X
chromosomes, so they are generally
female (XX) not male (XY). But there
are “X’ ceptions !
• Male calico cats can have an extra X
chromosome (aneuploidy – XXY),
similar to Klinefelter syndrome in
humans. Such individuals are often
infertile.
• Male calico cats can also be a
chimera – a fusion of two different
individuals early in the pregnancy (as
an embryo). If a black and an orange
male embryo fuse then you will get a
male calico cat that will be fertile,
and pass on orange or black.
video
• lncRNA
[Link]
Circular RNAs (circRNAs)
• a large class of non-coding RNAs
produced by a non-canonical splicing event called backsplicing;
- a downstream splice-donor site is covalently linked to an upstream
splice-acceptor site
• most circRNAs are expressed from known protein-coding genes
and consist of a single exon or of multiple exons
• internal intron retention may lead to the production of circRNAs
that contain sequences derived from both exons and introns
(known as exon–intron circRNAs)
• a failure in the debranching of intronic lariats during canonical
splicing can lead to the production of so-called circular intronic
RNAs (ciRNAs)
• circRNAs generally localize to the cytoplasm
• exon–intron circRNAs and ciRNAs reside in the nucleus
The biogenesis
of circRNAs I
BSJ: backsplice junction
circRNA: circular RNA
ciRNA: circular intronic RNA
EIcircRNA: exon–intron circRNAs
The biogenesis, biology and characterization of circular RNAs, Nature Reviews, 2019
The biogenesis
of circRNAs II
The biogenesis, biology and characterization of circular RNAs, Nature Reviews, 2019
circRNA functions
The biogenesis, biology and characterization of circular RNAs, Nature Reviews, 2019