Transcript Non-coding RNAs

Gene
Molecular definition:
Entire nucleic acid sequence necessary for the
synthesis of a functional polypeptide (protein
chain) or functional RNA
Genes in the genome:
• Protein-coding genes (mRNA): around 20500
(as of 10/2012)
• Non-coding RNAs
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Small nuclear RNA (SnRNA)
Small nucleolar RNA (SnoRNA)
microRNA (miRNA)
Other non-coding RNAs (Xist, 7SK, etc.)
• Pseudogenes
Ratio of non-coding to protein coding DNA
Raises as a function of developmental complexity
The genetic basis of human complexity and variation
• ~ 98% of the transcriptional output in humans is noncoding RNA
- 95-97% of the primary transcript of protein-coding genes is intronic
- there are enormous numbers of noncoding RNA genes in the
mammalian genome, which are only now beginning to be recognized,
and which appear to account for between 1/2 and 3/4 of all transcripts
• The majority of the human genome is transcribed
Known or
predicted
transcribed
Noncoding
percent of
transcription
60%
59%
70%
71%
56%
- 1.9% exonic (1.2% protein-coding) x 20 indicates
that 30-40%
of the
58%
98%
Human
genome is transcribed, just to account for protein-coding
genes
55%
98%
Mouse
Fruitfly
- if equal number of noncoding RNA transcripts,
then >60% is transcribed
Worm
- a direct summation of ’known genes', mRNAs,Yeast
and spliced ESTs 0.6%
7Mb
130Mb
2.6Gb
from the
UCSC100Mb
database
shows
that2.9Gb
(a minimum of) 58% of the
human genome is transcribed, 24% from both strands (total 2.3 Gb).
• Either the genome is replete with useless transcription or these nonprotein-coding RNAs are fulfilling some unexpected function
Non Coding RNAs: ‘RiboRegulators’
(~97% of RNAs Present in Human Cells are Non-Coding)
rRNA
tRNA
Vault
Y RNAs
7SK, 7SL
Xist, H19
snRNAs
snoRNAs
Guide RNA
Introns
5’ UTR
3’ UTR
Antisense RNAs
Catalytic:
Ribozymes
Telomerase
MicroRNAs
Viral RNAs
Retrotransposons
Many pseudogenes
Processes affected by ncRNAs.
Process
Example
Function
Transcription
184-nt E. coli 6S
Modulates promoter use
331-nt human 7SK
Inhibits transcription
factor P-TEFb
Steroid receptor
875-nt human SRA
Gene silencing
16,500-nt human Xist
Required for Xchromosome Inactivation
100,000-nt human Air
Required for autosomal
gene imprinting
Replication
451-nt human telomerase
RNA Core of
telomerase
and telomere template
RNA processing
377-nt E. coli RNase P
186-nt human U2 snRNA
Translation… 28S+18S RNAs
Catalytic core of RNase
Core of spliceosome
Ribosomes
Non Coding RNAs: ‘RiboRegulators’
(~97% of RNAs Present in Human Cells are Non-Coding)
rRNA
tRNA
Vault
Y RNAs
7SK, 7SL
Xist, H19
snRNAs
snoRNAs
Guide RNA
Introns
5’ UTR
3’ UTR
Antisense RNAs
Catalytic:
Ribozymes
Telomerase
MicroRNAs
Viral RNAs
Retrotransposons
Many pseudogenes
RNA-Mediated Gene Silencing
Post-transcriptional Gene Silencing (PTGS) or RNA
Interference (RNAi)
Gene Silencing By MicroRNAs
OTHER GENE SILENCING MECHANISMS
MEDIATED BY NON-CODING RNAs
RNA Silencing: The Genome’s
Immune System
Ronald H. A. Plasterk Science vol. 296 2002
Genomes are databases sensitive to invasion by viruses
(foreign nucleic acids). In recent years, a defense
mechanism has been discovered, which turns out to be
conserved among eukaryotes. The system can be
compared to the immune system in several ways: It has
specificity against foreign elements and the ability to
amplify and raise a massive response against an
invading nucleic acid. The latter property is beginning to
be understood at the molecular level.
RNA-Mediated Gene Silencing
Science 2002 296:1263-1265
Remarkable Properties of RNAi
• dsRNA (not ssRNA) is interfering agent
• Sequence-specific loss of mRNA and protein
• Effective against exons not introns
• Potent (few dsRNA molecules/cell effective)
• Persistent (affects next generation)
• Effects can cross cell barriers (feed, soak)
dsRNA
EXÓGENO O
ENDÓGENO
MicroRNAs:
Expanding Family of ‘RiboRegulators’
•
•
•
•
lin-4 and let-7 RNAs (from worm) were first examples
Also known as stRNAs (small temporal RNAs)
Regulate expression of proteins and developmental timing
Tip of the iceberg………..MicroRNAs are everywhere!
Science Vol. 297 Sept. 13, 2002
RNAi and Heterochromatin – a Hushed-Up Affair
R. Allshire 297:1818-1819
Regulation of Heterochromatic Silencing and Histone H3
Lysine-9 Methylation by RNAi
Volpe et al 297:1833-1837
Small RNAs Correspond to Centromere Heterochromatic
Repeats
Reinhart & Bartel 297:1831
siRNA and Silent Chromatin
- Model
RNA homologous to
centromeric repeats
are processed –
siRNAs
siRNAs may recruit Clr4
histone H3 methylase
result in meth. of H3
Lys9
Swi6 binds chromatin
Gene silencing
OTHER SILENCING SMALL NON-CODING RNAs…
(piRNAs, rasiRNAs…)
Piwi RNAs
Long non-coding RNAs
Paradigms for how long ncRNAs function.
Wilusz J E et al. Genes Dev. 2009;23:1494-1504
©2009 by Cold Spring Harbor Laboratory Press
Other cell processes…
Genomic organization of the transcription of short and large ncRNAs. A. Small non-coding RNAs (sRNAs) are transcribed from 5′nucleosome depleted region (5′-NDRs) i.e. PASR (Promoter-Associated Small RNAs, brownish-red arrows), tiRNA (transcription initiationassociated RNAs, orange arrows), TSSa-RNAs (Transcription Start Site-associated RNAs, red arrows), unstable PROMPTs (PROMoter
upstream Transcripts; black, dotted arrows) and from 3′-NDR i.e. TASR (Terminator-Associated Short RNAs, in blue). These ncRNAs are
transcribed in both senses. During gene looping RNAPII can possibly swap genomic regions (depicted with doted double-sense arrow) and
in consequence transcribe different coding or non-coding regions. B. Large non-coding RNAs (lncRNAs) depicted here are: lincRNAs (long
intervening ncRNAs, green arrows), PALRs (Promoter-Associated Long ncRNAs, magenta), lancRNAs (long antisense non-coding RNAs/NAT,
blue) and eRNAs (enhancer-associated ncRNAs, yellow). lncRNAs are transcribed in both senses from promoters, enhancers or inter-genic
regions.
Mammalian X inactivation
In female somatic cells, one X chromosome becomes
inactive and is cytologically detected as a Barr body.
The inactive X chromosome in female cells is more
heavily methylated and later replicating than the
active X chromosome
Consequence: one allele is expressed in
some areas of the body and the other allele
is expressed in other areas of the body (cells
are hemizygotic and all females are
mosaics)
There are pseudoautosomal regions of the X
chromosome that are transcriptionally active on both
active and inactive X chromosomes.
XIST: the first discovered long ncRNA
(X-Inactive Specific Transcript)
-- Identified in 1991(Willard, Brockdorff)
-- 17 kb spliced, noncoding RNA
-- Stable expression only from inactive X
-- “Paints” inactive X chromosome
-- Required to initiate silencing
The Xist RNA has an important role in X chromosome inactivation
XIST: the first discovered long ncRNA
Xist – X inactive-specific transcript
Avner and Heard, Nat. Rev. Genetics 2001 2(1):59-67
Random parental X inactivation in somatic cells
Mammalian X inactivation
In female somatic cells, one X chromosome becomes
inactive and is cytologically detected as a Barr body.
The inactive X chromosome in female cells is more
heavily methylated and later replicating than the
active X chromosome
Consequence: one allele is expressed in
some areas of the body and the other allele
is expressed in other areas of the body (cells
are hemizygotic and all females are
mosaics)
There are pseudoautosomal regions of the X
chromosome that are transcriptionally active on both
active and inactive X chromosomes.
X inactivation
maternal
chromosome
paternal
chromosome
embryo
dermal displasia
Duchenne Muscular Dystrophy (DMD)
• X-L recessive
-progressive weakness and loss of muscle
-symptoms by 5 yrs, in wheelchair by 11 yrs, death in
or early 20s
• 5-10% of carrier females have muscle weakness, a few
severe disease
• very large gene (>2.5 Mb)
-protein is called dystrophin
-most mutations are deletions
-Becker Muscular Dystrophy is allelic
late teens
have
X-linked diseases: female intermediate phenotypes
(f.e. color blindness)
XIST: the first discovered long ncRNA
Mammalian X inactivation
In female somatic cells, one X chromosome becomes
inactive and is cytologically detected as a Barr body.
The inactive X chromosome in female cells is more
heavily methylated and later replicating than the
active X chromosome
Consequence: one allele is expressed in
some areas of the body and the other allele
is expressed in other areas of the body (cells
are hemizygotic and all females are
mosaics)
There are pseudoautosomal regions of the X
chromosome that are transcriptionally active on both
active and inactive X chromosomes.
Pseudogenes
• Two types: processed and non-processed
• 70% processed vs 30% non-processed
• ~ 20,000
Torrents et al. Genome Res. 2003 13: 2559-67.
Human pseudogenes
Non-processed
pseudogenes
Contain introns;
Arise by duplications;
Frequency of transfer depend on
chromosomal context
(pericentromeral fragment are
transferred more often)
Processed
pseudogenes
Do not contain introns;
Arise by retrotransposition;
Frequency of transfer depends
on initial level of gene
expression
(Highly expressed genes are
transferred more often)
Complete
Partial
Both types of pseudogenes are raw material for evolution
NF1 gene and its pseudogenes on different
chromosomes
All NF1 pseudogenes are partial;
11 of them are found in the genome
Mechanism of processed pseudogene transfer
into new location
Could be very prolific: there are 95 functional ribosomal genes
and 2090 pseudogenes
R e tro p se u d o g e n e s
• R e ve r se -tr a n sc r ib e d g e n e s a r e (1)
w id e ly e x p r e sse d , (2 ) h ig h ly
c o n se r ve d , (3 ) sh o r t, a n d (4 ) G C -p o o r .
• T h e h u m a n g e n o m e is e stim a te d to
c o n ta in 2 3 ,0 0 0 t o 3 3 ,0 0 0
r e tr o p se u d o g e n e s.
PROMOTOR ??
ncRNAs and transcription…
Other cell processes…
Triple helix formation