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




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

 


  BLAST, Sequence Alignment Genes that code for proteins are conserved across genomes (e.g. low rate of mutation)


  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)


Triple helix

ncRNA: Sequence vs Structure

The specificity of



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


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 (


miRNA) Drosha (1) pre-miRNA (


miRNA) Dicer (2)


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







let-7 lin-14




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












small nuclear RNA

) 是细胞内稳定表达的一类


,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


U2 A

Exon 2


(Commitment Complex)



U1 U6 U4 U2 U5 B


U4 U6 U2 U5 C

(Activated Spliceosome)

U6 U2 U5

Exon 1 mRNA Exon 2


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 (


)gCYB gRNA 68nt






480 490 500 510




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)


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) 。


人 端粒

RNA( 451nt)



是真核细胞染色体的生理性末端 ,由高含



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


和蛋白质 是端粒




聚合酶,而是专一的逆转录酶, 能以自身的




, 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


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’


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’


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’


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’


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’


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’


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

a hidden “RNA world” within modern DNA world