non-coding rna. what is noncoding rna? non-coding rna (ncrna) is a rna molecule that functions...
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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 ?
mRNA
rRNA
tRNA
Protein
snRNA
snoRNA
gRNA
Antisense RNA
SRP-RNA
microRNApRNA
Ribozyme
Telomerase RNA
??
Functional diversity of ncRNAs
Scheme for the function of different sncRNAs by targeting bacterial or eukaryal mRNAs or pre-mRNAs leading to regulation of gene expression
The function of diffenent small non-coding RNAs by targeting mRNAs or pre-mRNAs
non-coding RNA (20-20000nt)
RNA
mRNA
small non-coding RNA (sncRNA) (20-500nt)
long non-coding RNA (lncRNA) (>500-20000nt)
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.
sncRNAs are very small
sncRNAs contain no specific features at their
5’ and 3’ ends
How to identify the ncRNA genes in genomic studies?
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 Computational Screen for Methylation Guide snoRNAs
SCIENCE, 283: 1168-1171, FEBRUARY 19, 1999
A example of computational approach for screening box C/D snoRNAs
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 im
perative
orthologous ( a1 in species I, a1 in species II)
paralogous ( a1 and a2 in species I )
Orthologous and paralogous
Repeat Sequence
repeat sequence
Inverted repeat, palindrome sequence
mirror repeat
(Inverted repeat)
G A A T T C
C T T A A 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
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 !
Tertiary stuctures are much less well understood
L-shaped tRNA molecule
methods for finding novel non-coding RNA genes
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
new method
by non-coding RNA libraries and deep sequencing
Experimental RNomics
Deep sequencing
Functional analysis
Combination of bioinformatical methods and experimental methods in ncRNA functional analysis
structure and functional analysis
Computational Analysis
Functional Prediction
Structural
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)
Box C/D and box H/ACA guide snoRNAs and the core associated proteins
snoRNA
methyl groups or pseudouridine groups
methylation and pseudouridylation guided by snoRNAs
RNA processing and modification
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
(1)
(2)
(3)
(4)
Diversity of genomic organization of ncRNAs
Trends Plant Science, 8(1): 42-49, 2003
snoRNA gnene organization
Diversity of genomic organization of ncRNAs
microRNA gnene organization
Diversity of genomic organization of ncRNAs
snoRNA and microRNA gene cluster
Polycistronic and intronic pre-snoRNA transcripts are processed by either a splicing or a non-splicing pathway
Procession of polycistronic and intronic pre-snoRNA transcripts
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 snR90 SPAC1B3.05
Exon 2 Intron Exon 1
snR90 precursor
snR80
Nucleases
Intron lariat
Splicing
Exonuclease trimming
snR90
snR80 snR90
Exon 2 Intron Exon 1
Polycistronic precursor
Transcription
microRNA
The discovery of miRNAs
• miRNA was first discovered in 1993 by Victor Ambros at miRNA was first discovered in 1993 by Victor Ambros at Harvard (Harvard (lin-4lin-4))• The second miRNA The second miRNA Let-7Let-7 was discovered in 2000 by Frank was discovered in 2000 by Frank Slack as a postdoc at HarvardSlack as a postdoc at Harvard (Ruvkun lab)(Ruvkun lab)
Victor AmbrosVictor Ambros Gary Ruvkun
The first discovered miRNA The first discovered miRNA lin-4 in 1993lin-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
Bartel, Cell 116: 281-297, 2004
The foundingmembers
Animals Plants
microRNAs had been neglected for so many years because of their small size.
The underlying reason is: The underlying reason is: people never dream that people never dream that small RNAs will have imsmall RNAs will have important biological roles.portant biological roles.
miRNA biogenesismiRNA biogenesis
Pri-miRNA( 原初 miRNA)
DroshaDrosha(1)(1)
pre-miRNA( 前体 miRNA)
DicerDicer(2)(2)
成熟 miRNA
Exportin 5 (Exp5) transports pre-miRNA 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 an
notated to show their similar structure with added lower case lette
r 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 annot
ation 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
Andrew Z. FireAndrew Z. FireCraig C. MelloCraig C. MelloAndrew Z. FireAndrew Z. FireCraig C. MelloCraig C. Mello
The Nobel Prize in Physiology or Medicine 2006
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
processing~22nt
siRNAs
target
recognition
mRNA
degradation
Developmental regulationby MicroRNA
~22ntlin-4
processing
target
recognitionlin-14mRNA
lin-41mRNA
3’UTR
3’UTR
~22ntlet-7
Translational repression
Base Pairing Differences between miRNAs and siRNAs
Transcriptional Gene Silencing by Transcriptional Gene Silencing by Directing Chromatin Modification 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
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.
Expression of hairpin RNA (shRNA) usinExpression of hairpin RNA (shRNA) using a Pol III promoterg a Pol III promoter
Vector-based SiRNAplasmid 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
– 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
snRNA snRNA (( small nuclear RNAsmall nuclear RNA ))
是细胞内稳定表达的一类是细胞内稳定表达的一类 RNARNA ,转录后需与多种蛋白子结,转录后需与多种蛋白子结合形成合形成 snRNP(small nuclear ribonucleoprotein particles)snRNP(small nuclear ribonucleoprotein particles)
The Spliceosome Assembly Pathway
U1
U1 U2A
U6
U5U4
U2
U1
U4U6
U5
U2
ATP
E(Commitment Complex)
A(Pre-spliceosome)
B(spliceosome)
C(Activated Spliceosome)
U6
U5
U2mRNA
Exon 1
Exon 1
Exon 2
Exon 2
gRNA RNA editing
in RNA editing, the coding sequence of an mRNA
molecule is altered after transcription, and so t
he protein has an amino acid sequence that dif
fers from that encoded by the gene.
observed in mRNAs, tRNAs, and rRNAs from a wi
de range of organisms;
include the insertion and the deletion of nucleotid
es 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
UUA GGU AUA AAA GUA GAU UGU AUA CCU GGU AGG UGU AAU
L G I K V D C I P G R C N
T TA GGT ATA AAA GTA GA G A A CCT GGT AGG TGT AAT
mRNA 顺序
蛋白质顺序
DNA 正链
480 490 500 510
480 490 500 510
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.
The Xist RNA is a large non-coding RNA which has been shown to necessary for developmentally regulated chromosomal silencing in females.
Xist RNA
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)
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
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
RNA
Protein
’3 5’
C A A U C C C A A U C
G G G T T
(AATCCC)n
A
5’3’
(TTAGGG)n
dATPdTTP
dG
TP
RNA
’3 5’
C A A U C C C A A U C
G G G T T
(AATCCC)n
A
5’
(TTAGGG)n
A G G G T T3’
Telomerase a reverse transcriptase to elongate telomeric DNA
RNA
’3 5’
C A A U C C C A A U C
G G G T T
(AATCCC)n
A
5’
(TTAGGG)n
A G G G T T3’
Telomerase a reverse transcriptase to elongate telomeric DNA
RNA
’3 5’
C A A U C C C A A U C
G G G T T
(AATCCC)n
A
5’
(TTAGGG)n
A G G G T T3’
Telomerase a reverse transcriptase to elongate telomeric DNA
RNA
’3 5’
C A A U C C C A A U C
G G G T T
(AATCCC)n
A
5’
(TTAGGG)n
A G G G T T A G G G T T3’
Telomerase a reverse transcriptase to elongate telomeric DNA
RNA
’3 5’
C A A U C C C A A U C
G G G T T
(AATCCC)n
A
5’
(TTAGGG)n
A G G G T T A G G G T T3’
Telomerase a reverse transcriptase to elongate telomeric DNA
DNA polymerase
G G G T T
(AATCCC)n
A
5’
(TTAGGG)n
A G G G T T A G G G T T
primer3’
Telomerase a reverse transcriptase to elongate telomeric DNA
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