Transcript RNA
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 tRNA ?
?
gRNA Ribozyme Antisense RNA SRP-RNA snRNA Telomerase RNA snoRNA microRNA pRNA
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
RNA non-coding RNA (20-20000nt) small non-coding RNA (sncRNA) (20-500nt) long non-coding RNA (lncRNA) (>500-20000nt) 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 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
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 Functional Analysis by Experimental Method Structural Prediction Functional Prediction
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
(a) Box C/D snoRNA (b) Box C/D snoRNAs direct rRNA methylation
(a) Box H/ACA snoRNA (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
(2) (3) (4) (1) Diversity of genomic organization of ncRNAs
snoRNA gnene organization
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 snR90 SPAC1B3.05
Exon 2 Intron Exon 1 Transcription snR80 snR90 Polycistronic precursor Exon 2 Intron Exon 1 Splicing Intron lariat Nucleases snR80 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 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 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 Craig Mello published their discovery ： and
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 Developmental regulation by MicroRNA
~22nt siRNAs
processing target recognition
mRNA
degradation
~22nt
lin-4
processing
~22nt
let-7 lin-14
mRNA
lin-41
mRNA
target recognition 3’UTR 3’UTR 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
Exon 1
U1 U1
ATP
U2 A
Exon 2
E
(Commitment Complex)
A
(Pre-spliceosome)
U1 U6 U4 U2 U5 B
(spliceosome)
U4 U6 U2 U5 C
(Activated Spliceosome)
U6 U2 U5
Exon 1 mRNA Exon 2
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 GA U U G U A U A CCU GGU AGG UGU AAU 蛋白质顺序
DNA
正链 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 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.
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 (AATCCC) n A G G G T T 3’ 5’ Protein C A A U C C C A A U C ’3 RNA 5’
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG) n (AATCCC) n A G G G T T A G G G T T 3’ 5’ C A A U C C C A A U C ’3 RNA 5’
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG) n (AATCCC) n A G G G T T A G G G T T 3’ 5’ C A A U C C C A A U C RNA ’3 5’
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG) n (AATCCC) n A G G G T T A G G G T T 3’ 5’ C A A U C C C A A U C RNA ’3 5’
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG) n (AATCCC) n A G G G T T A G G G T T A G G G T T 3’ 5’ C A A U C C C A A U C RNA ’3 5’
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG) n (AATCCC) n A G G G T T A G G G T T A G G G T T 3’ 5’ RNA C A A U C C C A A U C ’3 5’
Telomerase
a reverse transcriptase to elongate telomeric DNA
(TTAGGG) n (AATCCC) n A G G G T T A G G G T T A G G G T T 3’ 5’
primer DNA polymerase
A myriad of RNAs and functional diversity
mRNA, tRNA, rRNA: protein biosynthesis gRNA: snRNA: mRNA editing snoRNA: RNA P: mRNA processing (splicing and maturation) rRNA processing( cleavage and modification) tRNA processing Telomerase RNA: SRP-RNA: miRNA: DNA replication and life transport regulation of gene expression in transcription and post-transcription levels siRNA: gene silence Xist and Tsix: …… X chromosome inactivation