Brief introduction to non- protein-coding RNAs Mihaela Zavolan - - PowerPoint PPT Presentation

▶
brief introduction to non protein coding rnas
SMART_READER_LITE
LIVE PREVIEW

Brief introduction to non- protein-coding RNAs Mihaela Zavolan - - PowerPoint PPT Presentation

Jul 27, 2023 363 likes •681 views

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

slide-1
SLIDE 1

Brief introduction to non- protein-coding RNAs

Mihaela Zavolan Biozentrum, Basel Swiss Institute of Bioinformatics

slide-2
SLIDE 2

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
slide-3
SLIDE 3

From genes to proteins

pre-mRNA Transcription Splicing, capping, polyadenylation mature mRNA cDNA genome Start Stop Capture, cDNA synthesis Translation protein

) ) ) ( ( (

m3G A A A A A

Exon 1 Exon 2 Exon 3 Exon 4

5’ splice site ) 3’ splice site (

) ) )

Start

) )) ) ( ( (

Stop

slide-4
SLIDE 4

snRNA

slide-5
SLIDE 5

tRNA

slide-6
SLIDE 6

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

slide-7
SLIDE 7

rRNA

RNA - gray Peptide - gold backbone

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

slide-8
SLIDE 8

snoRNA

Weinstein & Steitz, 1999

slide-9
SLIDE 9

snoRNA

Cavaille et al. 2002 Cavaille et al. 2000

slide-10
SLIDE 10

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
slide-11
SLIDE 11

Short history of RNAi

slide-12
SLIDE 12

Short history of RNAi

  • 1990 - multiple copies of a pigment transgene result in

colorless petunias (Napoli et al.)

slide-13
SLIDE 13

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

slide-14
SLIDE 14

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
slide-15
SLIDE 15

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

slide-16
SLIDE 16

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

slide-17
SLIDE 17

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

slide-18
SLIDE 18

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

  • 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.

slide-19
SLIDE 19

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

AAAA

3’-UTR miRNA

RISC-like miRNP

RNA interference pathway MicroRNA pathway

Me Me Me Me

Chromatin silencing Translational repression

RITS

rasiRNA

Drosha

slide-20
SLIDE 20

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)
slide-21
SLIDE 21

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

slide-22
SLIDE 22

Main problems with RNAi technology

siRNAs

RISC mRNA

Cleavage

AAAA

3’-UTR

RISC-like miRNP

Off-target effects Delivery of siRNAs to the appropriate tissue Sustainability of the inhibitory effect

slide-23
SLIDE 23

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

slide-24
SLIDE 24

Applications of RNAi

High-throughput RNAi screens for elucidating gene function

slide-25
SLIDE 25

Applications of RNAi

Development of RNA- based anti-viral (HIV1, Influenza, Coxsackie, HCV) and anti-cancer (targets: angiogenesis, DNA repair, STAT3, bcl-2) therapeutics

slide-26
SLIDE 26

Functions of miRNAs

Let-7 miRNA regulates developmental timing in C.elegans. Reinhart et al. 2000

slide-27
SLIDE 27

Functions of miRNAs

Ras is regulated by let-7 miRNA. Johnson et al. 2005

slide-28
SLIDE 28

Functions of miRNAs

Pancreatic-specific miR-375 inhibits insulin secretion. Poy et al. 2004

slide-29
SLIDE 29

Functions of miRNAs

miRNAs regulate brain morphogenesis in zebrafish. Giraldez et al. 2005

slide-30
SLIDE 30

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.

slide-31
SLIDE 31

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/