Brief introduction to non- protein-coding RNAs · 2013-06-28 · miRNA , siRNA , rasiRNA biogenesis dsRNA DICER Exogenous dsRNA ¥ Viral infection ¥ Transfection Endogenous dsRNA

Brief introduction to non-

protein-coding RNAs

Mihaela Zavolan

Biozentrum, Basel

Swiss Institute of Bioinformatics

Classes of functional RNAs

• rRNA - protein synthesis

• tRNA - transport of amino acids

• snRNA - spliceosome

• snoRNA - rRNA methylation and pseudouridylation

• RNase P - removal of 5’ sequence from tRNAs

• SRP RNA - protein secretion pathway

• miRNA,siRNA - translation inhibition / mRNA degradation

• antisense RNAs (Xist) - X chromosome inactivation

• bacterial noncoding RNAs

From genes to proteins

pre-mRNA

Transcription

Splicing, capping, polyadenylation

mature mRNA

cDNA

genome

Start Stop

Capture, cDNA synthesis Translation

protein

) ) )( ( (

m3G A AA

AA

Exon 1 Exon 2 Exon 3 Exon 4

5’ splice site )3’ splice site (

) )

)

Start

) )) )( ( (

Stop

snRNA

tRNA

rRNA

70 S

2.5x106 Da

50 S

1.6x106 Da 30 S

0.9x106 Da

5 S rRNA

120 nc

23 S rRNA

2900 nc

16 S rRNA

1540 nc

34 proteins 21 proteins

80 S

4.2x106 Da

60 S

2.8x106 Da 40 S

1.4x106 Da

5 S rRNA

120 nc

28 S rRNA

4700 nc

18 S rRNA

1900 nc

~49 proteins

~33 proteins

5.8 S rRNA

160 nc

Prokaryotic Eukaryotic

rRNA

RNA - gray

Peptide - gold backbone

Large ribosomal subunit of Haloarcula mortui - Ban et al. Science 289:905-920 (2000)

snoRNA

Weinstein & Steitz, 1999

snoRNA

Cavaille et al. 2002

Cavaille et al. 2000

RNAi: why so much excitement?

• Unsuspected, intricate regulatory pathways

• Involving many new enzymes and RNA-binding proteins

• Regulating a large fraction of the genes

• Extremely versatile tool to knock down gene expression

Short history of RNAi

Short history of RNAi• 1990 - multiple copies of a pigment transgene result in

colorless petunias (Napoli et al.)



• 1993 - The group of V. Ambros discovers the first

microRNA (lin-4) in the worm





• 1995 - Guo & Kemphues discover RNA interference






• 2000 - The group of G. Ruvkun discovers the first

evolutionarily conserved microRNA (let-7) in the worm








• 2001 - The group of T. Tuschl shows that RNAi

is mediated by small (21-22 nc) RNAs










• 2002 - Hutvagner & Zamore show that miRNAs and

siRNAs share components of the effector pathway










• 2002 - Hutvagner & Zamore show that miRNAs and

siRNAs share components of the effector pathway

• 2004 - RITS complex purified by the group of D. Moazed

• 2005 - hundreds on miRNA genes in human (321),

mouse (245), rat (194), chicken (122), fly (78),

worm (114); hundreds more predicted.

miRNA, siRNA, rasiRNA biogenesis

dsRNA

DICER

Exogenous dsRNA

• Viral infection

• Transfection

Endogenous dsRNA

• Repetitive elements

• Antisense transcripts

RISC

mRNA

mRNA cleavage

siRNA

MicroRNA precursors

encoded in the genome

DICER

AAAA3’-UTR

miRNA

RISC-like

miRNP

RNA interference pathway MicroRNA pathway

Me Me Me Me

Chromatin silencing Translational repression

RITS

rasiRNA

Drosha

Biological functions of RNAi/miRNAs

• Defense mechanisms against foreign nucleic acids

- Viral infection

- Active transposons

• Regulation of gene expression

- globally (formation of heterochromatin, centromers)

- locally (stellate locus silencing)

- translational regulation by miRNAs

- transcriptional regulation by miRNAs (plants)

• Regulation of genomic rearrangement (Tetrahymena, Paramecium)

Proteins involved in RNAi function

Components of RISC and miRNPs

Paz (siRNA-) Piwi (Dicer-binding) domain family (Ago, Hiwi)

Fragile X Mental Retardation Protein (FMRP) and related proteins

VIG, Gemins, Tudor-SN, R2D2, Dicer

Other proteins RNA-dependent RNA polymerases (RdRPs)

RNA helicases

Proteins modulating RNAi (eri-1)

Viral supressors of RNAi

Various genes of unknown function identified by screening

Main problems with RNAi technology

siRNAs

RISC

mRNACleavage

AAAA

3’-UTR

RISC-like

miRNP

Off-target effects

Delivery of siRNAs to the appropriate tissue

Sustainability of the inhibitory effect

Specificity of RNAi/miRNAs

5’ end of the miRNA is critical for miRNA

function. Doench & Sharp 2004.

RISC

mRNA

mRNA cleavage

Ago2

Ago2 is a cleaving endonuclease.

Joshua-Tor & Hannon 2004

Applications of RNAi

High-throughput RNAi screens for elucidating gene function

Applications of RNAi

Development of RNA-

based anti-viral

(HIV1, Influenza,

Coxsackie, HCV)

and anti-cancer

(targets:

angiogenesis, DNA

repair, STAT3, bcl-2)

therapeutics

Functions of miRNAs

Let-7 miRNA regulates

developmental timing in

C.elegans. Reinhart et al. 2000

Functions of miRNAs

Ras is regulated by let-7 miRNA.

Johnson et al. 2005

Functions of miRNAs

Pancreatic-specific miR-375

inhibits insulin secretion.

Poy et al. 2004

Functions of miRNAs

miRNAs regulate brain

morphogenesis in zebrafish.

Giraldez et al. 2005

rasiRNAs in epigenetic regulation

Heterochromatic RITS-associated siRNAs in fission

yeast originate from diverse DNA elements.

Cam et al. 2005.

Not demonstrated

yet in mammals.

Some ncRNA databases

• general: ncRNA database

http://noncode.bioinfo.org.cn/

• microRNA repository

http://microrna.sanger.ac.uk/sequences/

• tRNA databases http://lowelab.ucsc.edu/GtRNAdb/

http://medstat.med.utah.edu/RNAmods/trnabase/

http://www.staff.uni-bayreuth.de/~btc914/search/

• rRNA database http://www.psb.ugent.be/rRNA/

Documents

Brief introduction to non- protein-coding RNAs · 2013-06-28 · miRNA , siRNA , rasiRNA biogenesis dsRNA DICER Exogenous dsRNA ¥ Viral infection ¥ Transfection Endogenous dsRNA