Download Non-coding RNA

Lecture 3
GCATCCATCTTGGGGCGTCCCAATTGCTGAGTAACAAATGAGACGC
TGTGGCCAAACTCAGTCATAACTAATGACATTTCTAGACAAAGTGAC
TTCAGATTTTCAAAGCGTACCCTGTTTACATCATTTTGCCAATTTCG
CGTACTGCAACCGGCGGGCCACGCCCCCGTGAAAAGAAGGTTGTT
TTCTCCACATTTCGGGGTTCTGGACGTTTCCCGGCTGCGGGGCGG
GGGGAGTCTCCGGCGCACGCGGCCCCTTGGCCCCGCCCCCAGTC
ATTCCCGGCCACTCGCGACCCGAGGCTGCCGCAGGGGGCGGGCT
GAGCGCGTGCGAGGCGATTGGTTTGGGGCCAGAGTGGGCGAGGC
GCGGAGGTCTGGCCTATAAAGTAGTCGCGGAGACGGGGTGCTGGT
TTGCGTCGTAGTCTCCTGCAGCGTCTGGGGTTTCCGTTGCAGTCCT
CGGAACCAGGACCTCGGCGTGGCCTAGCGAGTTATGGCGACGAAG
GCCGTGTGCGTGCTGAAGGGCGACGGCCCAGTGCAGGGCATCAT
CAATTTCGAGCAGAAGGCAAGGGCTGGGACGGAGGCTTGTTTGCG
AGGCCGCTCCCACCCGCTCGTCCCCCCGCGCACCTTTGCTAGGAG
CGGGTCGCCCGCCAGGCCTCGGGGCCGCCCTGGTCCAGCGCCCG
GTCCCGGCCCGTGCCGCCCGGTCGGTGCCTTCGCCCCCAGCGGT
GCGGTGCCCAAGTGCTGAGTCACCGGGCGGGCCCGGGCGCGGG
GCGTGGGACCGAGGCCGCCGCGGGGCTGGGCCTGCGCGTGGCG
GGAGCGCGGGGAGGGATTGCCGCGGGCCGGGGAGGGGCGGGGG
CGGGCGTGCTGCCCTCTGTGGTCCTTGGGCCGCCGCCGCGGGTC
TGTCGTGGTGCCTGGAGCGGCTGTGCTCGTCCCTTGCTTGGCCGT
GTTCTC
Much of the genome remains to be annotated
Non-coding
•
Non-protein
coding Genes
•
cis-regulatory
elements
•
Other functional
sequences
Protein Coding
Human genome
repeats
The first non-coding RNA!
The first non-coding RNA to be characterized was an alanine tRNA in
baker’s yeast (Holley et al, Science, 1965). The “cloverleaf” secondary
structure was revealed using X-ray crystallography in 1974.
Robert Holly
Ribosomal RNAs
George
Palade
Abundant (80% of total RNA)!
Humans : many copies of the rRNA
genes in tandem repeats
300-400 rDNA repeats are localized on
chromosomes 13,14,15,21 and 22
Euraryotes: Ribosomes 80S: 2 subunits
Large (5S, 5.8S, 28S) 60S; Small (18S)
40S
28S, 18S and 5.8S rRNAs are made
from a single transcript (45S)
Venkatraman Ramakrishnan, Thomas A. Steitz, Ada E. Yonath
http://www.nobelprize.org
snoRNAs (small nucleolar RNAs)
150 species, 70-250 nt in
length
*Ribosomal RNA processing
**rRNA modification (2'-Oribose methylation, or
pseudouridylation)
The majority of vertebrate
snoRNA genes are encoded
in the introns of proteins
involved in ribosome
synthesis or translation, and
are synthesized by RNA
polymerase II
Small nuclear
RNAs

assembly
U RNAs: U1, U2, U4, U5,
U6 small nuclear RNA
(for splicing mRNAs)
first transesterification
second
transesterification
Lariat intron released,
debranched, degraded
Other snRNPs released
Other ncRNAs
Enhancer-associated RNA (eRNA)
Long non-coding RNAs

Have intron/exon structures like protein-coding
genes, but lack protein-coding potential.

First several were discovered in early 90s’

H19, only transcribed from the maternal allele, is
linked to Beckwith-Wiedemann syndrome (BWS)
and cancer

Xist, involved in X inactivation
Beckwith-Wiedemann syndrome
Long non-coding RNAs

Tiling microarray experiments led to the finding that a large
fraction of the mammalian genome is transcribed (Bertone,
Science 306, 2242–2246 (2004).18. Kapranov. Science 316, 1484–
1488 (2007).19. Rinn,Genes Dev. 17, 529–540 (2003).20. Kapranov,
Science 296, 916–919 (2002).)
Bertone,
Science 2004
Long non-coding RNAs
Large scale full length cDNA
cloning and sequencing efforts,
such as the RIKEN’s FANTOM
project, led to the finding that
62% of the mouse genome is
transcribed, and the identification
of thousands of long non-coding
RNAs in early 2000s (Okazaki et
al., Nature 2002, 420 (6915) pp.
563-73; Carninci et al., Science
2005, 309 (5740) pp. 1559-63).
 Non-coding RNA genes exhibit
similar levels of conservation as
the protein-coding genes at
promoters.
 Many lncRNAs are dynamically
expressed

Carninci et al., Science 2005
Long non-coding RNAs identified by
chromatin signatures
H3K4m3 is a mark for
promoters
 H3K36me3 is a mark
for gene body of
actively transcribed
genes
 Genomic regions with
H3K4me3 and
H3K36me3 are likely
genes

Chromatin profiles can be obtained
using ChIP-seq
Long non-coding RNAs identified by
chromatin signatures
H3K4m3 is a mark for
promoters
 H3K36me3 is a mark
for gene body of
actively transcribed
genes
 Genomic regions with
H3K4me3 and
H3K36me3 are likely
genes

Guttman et al., Nature 2009, 458 (7235) pp. 223-7
Long non-coding RNAs identified by
chromatin signatures
LncRNAs have low
protein coding potential
 LncRNAs are
evolutionarily
conserved
 LncRNA promoters are
evolutionarily
conserved

Guttman et al., Nature 2009, 458 (7235) pp. 223-7
Protein coding potential of
lncRNA
Guttman & Rinn, Nature 2012, 482 (7385) pp. 339-46
Long non-coding RNAs
LncRNA gene structures
can be reconstructed from
RNA-seq data using
Scripture (Guttman et al.,
Nature Bioltech. 2010)
 On average the
reconstructed lncRNAs
have 3.7 exons, compared
to 7 for protein coding
genes

Guttman et al. Nature Bioltech. 2010 vol.
28 (5) pp. 503-10
Long non-coding RNAs
Cabili et al., Genes & Dev. 2011, 25 (18) pp. 1915-27
Properties of lncRNAs


Tissue specific
expression
Lower
abundance
Functions of lncRNAs
Guttman & Rinn, Nature 2012, 482 (7385) pp. 339-46
Many LncRNAs affect transcription either
in cis- or. trans
Xist
HOTAIR
Guttman & Rinn, Nature 2012, 482 (7385) pp. 339-46
Finding chromatin
binding sites of lncRNA
48 complementary DNA oligonucleotides
that were 20-mer each and tiled the
entire length of HOTAIR
 ‘‘split-probe’’ strategy helps remove off
target sequences.
 Glutaraldehyde crosslinking

Chu et al., Molecular Cell 44, 667–678, 2011
Mode of action of lncRNAs
CCND1 ncRNA
GAS5
Guttman & Rinn, Nature 2012, 482 (7385) pp. 339-46
Proposed models of lncRNA
functions
Proposed models of lncRNA
functions – modular principles
Promoter associated small RNAs
Enhancer associated RNA (eRNA)





Bi-directional
Short (few hundred bp to 1000bp)
Lacks 5’ cap and 3’ polyA
Transient and levels correlate with enhancer activities
Functions unknown
Kim et al., Nature 465,182–187(2010).
Summary

A large number long ncRNAs have now been found in
the mammalian genome

However, their functions are generally unknown, with
the exception of a few.

It is proposed that lncRNAs could function in a
modular fashion to affect gene regulation, and other
cellular processes.

Clearly, one of the most exciting development in the
future is to characterize their function.
Small RNAs are regulatory!!
~20-30 nt long RNAs
 First small RNA, lin-4 microRNA (genetic screens in
nematode worms)
 The number of small RNAs have exploded, and are
implicated from heterochromatin formation to mRNA
destabilization and translational control

Diversity of small RNAs
• siRNA
Cellular gene silencing
Generated by RNAi pathway 21-23nt
Cleave target mRNA
Target RNA from virus and transposons
Generated by miRNA pathway 21-23nt
•miRNA
Cleave target mRNA inhibit translation
initiation
Differentiated
cells
NPC
glia
Function in many processes and cells
neuron
IGF/serum
miR-34a
•piRNA
Dicer-independent 24-30 nt
Fly, fish and mouse ovary and testis
Derived from transposons and other
repeated sequence elements
ovary
Self-renewing division
MicroRNAs





~21-22 nt long non-coding RNAs
More than 60% of protein-coding genes are predicted to be
controlled by microRNAs
Almost every cellular process investigated to date have a
microRNA component
microRNAs function in the form of ribonucleoprotein
complexes (miRISCs: miRNA-induced silencing complexes).
Ago, GW182 are components of miRISC
Biogenesis and function of microRNAs
~160
miRNAs
~150
miRNAs
~1000
miRNAs
Potential functions of long non-coding RNAs