RNA

Report
Non-coding RNA
What is noncoding RNA?
Non-coding RNA (ncRNA) is a RNA
molecule that functions without
being translated into a protein
How many RNAs in cells ?
Protein
mRNA
?
?
rRNA
gRNA
Ribozyme
tRNA
Antisense RNA
SRP-RNA
Telomerase RNA
snRNA
snoRNA
pRNA
microRNA
Functional diversity of ncRNAs
The function of diffenent small non-coding
RNAs by targeting mRNAs or pre-mRNAs
Scheme for the function of different sncRNAs by targeting bacterial or eukaryal
mRNAs or pre-mRNAs leading to regulation of gene expression
non-coding RNA
(20-20000nt)
small non-coding RNA (sncRNA)
(20-500nt)
long non-coding RNA (lncRNA)
(>500-20000nt)
RNA
mRNA
Non-coding RNA:
Versatility in form and function
 Noncoding
RNA genes are
surprisingly numerous.
 Noncoding
RNA have a very
different functions.
Time for RNomics
Cell, 89: 669–672, May 30, 1997
PNAS, 97(26): 14035-14037, Dec 19, 2000
Understanding RNomics from
an expending snoRNA world
Couzin J.
Breakthrough of the year. Small RNAs make big splash.
Science. 2002 Dec 20;298(5602):2296-7.
DNA
RNA
Protein
Study non-coding RNAs on the genomic scale
Study the identification, expression, biogenesis,
structure, regulation of expression, targets, and
biological functions of noncoding RNAs on the
genomic scale.
How to identify the ncRNA genes in genomic
studies?
 sncRNAs are very small
 sncRNAs contain no specific features at their
5’ and 3’ ends
methods for
finding novel non-coding RNA genes
Computational RNomics
 Searching conserved intronic sequences by comparative analysis of
introns
 Searching conserved intergenic sequences
 Searching well-defined sequence elements or characteristics (boxC/D,
functional regions, complementary and other conserved sequence etc.)
 Novel algorithm taking the folding parameters in RNA molecule into
account
All predictions of novel ncRNA genes need to be
confirmed by direct detection of these transcripts !!!
A example of computational approach for
screening box C/D snoRNAs
A Computational Screen for Methylation Guide snoRNAs
SCIENCE, 283: 1168-1171, FEBRUARY 19, 1999
Similarity Searching

Proteins



BLAST, Sequence Alignment
Genes that code for proteins are conserved
across genomes (e.g. low rate of mutation)
ncRNA


Secondary structure usually conserved
Alignment scoring based on structure is
imperative
Orthologous and paralogous
orthologous(a1 in species I, a1 in species II)
paralogous(a1 and a2 in species I)
Repeat Sequence
 repeat sequence
 Inverted repeat, palindrome sequence
 mirror repeat
(Inverted repeat)
G AAT T C
C T TAA G
Triple helix
ncRNA: Sequence vs Structure
The specificity of RNA search
ncRNA is defined by
primary and secondary structure
RNA structure
Base-pairing defines a secondary structure
Tertiary stuctures are much less well understood
RNA is extremely difficult to crystallize:
RNA is enzymatically unstable molecule (RNAses are
everywhere!)
RNA is conformationally flexible molecule.
Thus Bioinformatic approach –
RNA structure prediction is very important !
L-shaped tRNA molecule
methods for
finding novel non-coding RNA genes
Experimental RNomics
Traditional methods
 by PAGE separation of non-coding RNAs and sequencing
 by immunoprecipitation of specific RNPs
by non-coding RNA enriched cDNA libraries and sequencing
 by microarray analysis
new method
 by non-coding RNA libraries and deep sequencing
New
Deep sequencing
Functional analysis
Combination of bioinformatical methods and
experimental methods in ncRNA functional analysis
structure and functional analysis
Computational Analysis
Structural
Prediction
Functional
Prediction
Functional Analysis by
Experimental Method
Nomenclature of non-coding RNA

Bacterial RNAs --- Small RNA(sRNA)
 Eukaryotic RNA --- Non-coding RNA (ncRNA),
functional RNA (fRNA), small nonmessenger
RNAs (snmRNA)
 Based on subcellular localization --Small nucleolar RNAs (snoRNA)
 Based on size --- micro RNA (miRNA),small
interfering RNAs (siRNA), long non-coding
RNA(lnRNA)
snoRNA
Box C/D and box H/ACA guide snoRNAs and the core associated proteins
RNA processing and modification
methylation and pseudouridylation
guided by snoRNAs
methyl groups or pseudouridine groups
Box C/D snoRNA
(a)
(b)
Box C/D snoRNAs direct rRNA methylation
Box H/ACA snoRNA
(a)
(b)
Box H/ACA snoRNAs direct rRNA pseudouridylation
Box C/D-H/ACA snoRNA (scaRNA)
snoRNA target
snoRNA --------------------------------------rRNA, U6
scaRNA---------------------------------------snRNA
imprinted snoRNA------ -------------------mRNA
Homologs of snoRNAs in Archaea-----rRNA and tRNA
Orphan guide snoRNAs-------------------No target
Diversity of genomic organization of ncRNAs
snoRNA gnene organization
(1)
(2)
(3)
(4)
Trends Plant Science, 8(1): 42-49, 2003
Diversity of genomic organization of ncRNAs
microRNA gnene organization
Diversity of genomic organization of ncRNAs
snoRNA and microRNA gene cluster
Procession of polycistronic and intronic pre-snoRNA transcripts
Polycistronic and intronic pre-snoRNA transcripts are processed
by either a splicing or a non-splicing pathway
Non-coding RNA host gene
 Protein Coding Gene------Most intronic snoRNA genes of
vertebrates and yeast are nested in genes encoding
proteins involved in ribosome biogenesis.
 Non-coding RNA gene------These ‘‘host’’ genes harbour
snoRNAs in multiple introns but their exon does not code
for proteins
SPAC1B3.05
snR80
Exon 2
Intron
snR90
SPAC1B3.05
Exon 1
Transcription
snR80
snR90
Polycistronic precursor
Exon 2
Intron
Exon 1
Intron lariat
snR80
Splicing
Nucleases
snR90 precursor
Exonuclease trimming
snR90
microRNA
The discovery of miRNAs
Victor Ambros
Gary Ruvkun
• miRNA was first discovered in 1993 by Victor Ambros at
Harvard (lin-4)
• The second miRNA Let-7 was discovered in 2000 by Frank
Slack as a postdoc at Harvard (Ruvkun lab)
The first discovered miRNA lin-4 in 1993
Ruvkun G, Wightman B, Ha I. The 20 years it took to recognize the importance of tiny RNAs. Cell. 2004 Jan
23;116 (2 Suppl):S93-6.
Lee R, Feinbaum R, Ambros V. A short history of a short RNA. Cell. 2004 Jan 23;116 (2 Suppl):S89-92
Thought to be an oddity not a general phenomenon
Breakthrough with BlastN of the second
miRNA (stRNA) let-7
Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, Hayward DC,
Ball EE, Degnan B, Muller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine
M, Leahy P,Davidson E, Ruvkun G. Conservation of the sequence and temporal
expression of let-7 heterochronic regulatory RNA. Nature. 2000 Nov 2;408(6808):86-9.
MicroRNAs: 22-25 nt Noncoding RNAs
The founding
members
Animals
Plants
Bartel, Cell 116: 281-297, 2004
microRNAs had been neglected for
so many years because of their
small size.
The underlying reason is:
people never dream that
small RNAs will have
important biological roles.
miRNA biogenesis
Pri-miRNA
(原初miRNA)
Drosha
(1)
pre-miRNA
(前体miRNA)
Dicer
(2)
成熟miRNA
Exportin 5 (Exp5)
transports premiRNA to the
cytoplasm
Cell 125,
887–901, 2006
Another View
Microprocessor
Complex
Differences in miRNA Mode of
Action
microRNA nomenclature
Experimentally confirmed microRNAs are given a number that is
attached to the prefix mir followed by a dash eg mir-123.
 miRNAs with similar structures bar at 1 or 2 nucleotides are
annotated to show their similar structure with added lower case
letter eg miR-1a and miR-1b.
 miRNAs at different loci to produce the same miRNA and these
are show with additional number eg miR-1-1 and miR-1-2
 microRNA nomenclature should also be preceded by the
annotation for the species they are observed in eg homo sapiens
= hsa-miR-xxx.
Discovery of siRNA
In 1998, the American scientists Andrew Fire and
Craig Mello published their discovery:
RNA interference
The Nobel Prize in Physiology or Medicine 2006
Andrew Z. Fire
Craig C. Mello
siRNA-Mediated Gene Silencing
What is the Difference between
miRNA and siRNA?
 siRNA originates with dsRNA;
miRNA originates with ssRNA that forms a hairpin
secondary structure.
 siRNA is often 100% complementary to the target;
miRNA is often not 100% complementary to the target.
A comparison between
miRNA and siRNA
RNAi by siRNAs
~22nt
siRNAs
Developmental regulation
by MicroRNA
processing
processing
~22nt
lin-4
target
recognition
mRNA
lin-14
mRNA
lin-41
mRNA
~22nt
let-7
target
recognition
3’UTR
3’UTR
degradation
Translational repression
Base Pairing Differences between miRNAs
and siRNAs
Transcriptional Gene Silencing by
Directing Chromatin Modification
RNA silencing in different organisms
RNA-Mediated Gene Silencing
Post-transcriptional Gene Silencing (PTGS)
or RNA Interference (RNAi)
Transcriptional Gene Silencing (TGS)
(RNA-dependent DNA Methylation)
Gene Silencing By MicroRNAs
Expression of hairpin RNA (shRNA)
using a Pol III promoter
 Transcription from RNAP III promoters of U6 and H1
are well characterized.
 RNAP III transcription uses a well-defined termination
signal (TTTTT) and the products have no extra sequence.
 Transcription from these promoters is very efficient in
various tissues.
Vector-based SiRNA
plasmid and viral vectors
establishing long-term RNAi:
let the cell make the siRNA for you!
Example of Expression Vector
lentiviral construct for siRNAs
siRNA Delivery & Processing
21世纪初RNA研究正在兴起
2000年世界十大科技突破的第二条
2001年世界十大科技突破的第二条
2002年世界科技十大突破的第一条
2004年世界科技十大突破均来自RNA
snRNA (small nuclear RNA)
是细胞内稳定表达的一类RNA,转录后需与多种蛋白子结
合形成snRNP(small nuclear ribonucleoprotein particles)
–种类:主要有5种 U1、U2、U4、U5、U6;其它如:
U11、U12等
–功能:
• 识别剪接点并与之结合
• 形成剪接体的三维结构,助于反应进行
• 可能有催化转酯反应的作用
The Spliceosome Assembly Pathway
U1
Exon 1
Exon 2
(Commitment Complex)
ATP
U1
E
U2
A
A
(Pre-spliceosome)
U1
U6
B
U5
U4
(spliceosome)
U2
U4
C
U6
(Activated Spliceosome)
U5
U2
U6
U5
U2
Exon 1
Exon 2
mRNA
gRNA
RNA editing
in RNA editing, the coding sequence of an mRNA
molecule is altered after transcription, and so
the protein has an amino acid sequence that
differs from that encoded by the gene.
observed in mRNAs, tRNAs, and rRNAs from a
wide range of organisms;
include the insertion and the deletion of
nucleotides and the conversion of one base
into another
T. brucei (布氏锥虫)gCYB
gRNA 68nt
导致RNA编辑中U的加入与去除
480
490
500
510
mRNA顺序 UUA GGU AUA AAA GUA GAU UGU AUA CCU GGU AGG UGU AAU
蛋白质顺序
DNA正链
L
G
I
K
V
D
T TA GGT ATA AAA GTA GA
480
490
C
I
P
G
R
C
N
G A A CCT GGT AGG TGT AAT
500
锥虫COII基因片段及其表达产物的序列比较
核酸序列的数字是以起始密码子AUG(ATG)的A开始编码.
510
Xist RNA
The Xist RNA is a large non-coding
RNA which has been shown to
necessary for developmentally regulated
chromosomal silencing in females.
Human XistRNA 16,500nt X
有丝分裂中失活X染色体(蓝色)上的Xist RNA(红色)
Cell, 93, 309-312, (1998)
pRNA
在双链DNA病毒增殖和成熟的过程中, 需要将
相当长的子代DNA装入一个空间极为有限的新
生病毒衣壳中。早在1987年, Guo P X等在对
噬菌体ф29 DNA的转运进行研究时发现了一
种具有转运功能的RNA分子, 该RNA分子在噬
菌体ф29的DNA包装中有着重要的作用, 这种
RNA分子被称为pRNA(packaging RNA)。
pRNA
人 端粒RNA( 451nt)
端粒(telomere)是真核细胞染色体的生理性末端
,由高含G的DNA序列和相应的蛋白组成。
端粒的维持需端粒酶(telomerase)的激活。端粒酶
是一种核糖-核蛋白复合体,其中RNA和蛋白质
是端粒DNA合成所必须的。它不同于经典的
DNA聚合酶,而是专一的逆转录酶,能以自身的
RNA为模板,逆转录合成端粒DNA,以补偿细
胞分裂时染色体末端缩短.
Telomerase RNA
• Component of telomerase
• Provides template for
telomere synthesis
• Role in Cancer and Aging
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG)n
A G G G T T
5’
(AATCCC)n
3’
Protein
C A A U C C C A AUC
RNA
’3
5’
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG)n
A G G G T T A G G G T T
5’
(AATCCC)n
3’
C A A U C C C A AUC
RNA
’3
5’
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG)n
A G G G T T A G G G T T
3’
5’
(AATCCC)n
C A A U C C C A A U C RNA
’3
5’
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG)n
A G G G T T A G G G T T
5’
3’
C A A U C C C A AUC
(AATCCC)n
RNA
’3
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG)n
A G G G T T A G G G T T A G G G T T
5’
3’
C A A U C C C A AUC
(AATCCC)n
RNA
’3
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG)n
A G G G T T A G G G T T A G G G T T
3’
5’
(AATCCC)n
RNA
C A A U C C C A AUC
’3
5’
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG)n
A G G G T T A G G G T T A G G G T T
5’
primer
(AATCCC)n
DNA polymerase
3’
A myriad of RNAs and functional diversity
 mRNA, tRNA, rRNA: protein biosynthesis
 gRNA: mRNA editing
 snRNA: mRNA processing (splicing and maturation)
 snoRNA: rRNA processing( cleavage and modification)
 RNA P: tRNA processing
 Telomerase RNA: DNA replication and life
 SRP-RNA: transport
 miRNA: regulation of gene expression in transcription and
post-transcription levels
 siRNA: gene silence
 Xist and Tsix: X chromosome inactivation
……
a hidden “RNA world”
within modern DNA world

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