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