rna-mediated genome remodeling in ciliates nowacki.pdf · nowacki_salzburg2014.ppt author: mariusz...

Mariusz Nowacki

University of Bern Institute of Cell Biology Switzerland

RNA-mediated genome remodeling in ciliates

Nuclear dimorphism in ciliates

Landweber

MICRONUCLEUS: •  germline •  transcriptionally silent •  diploid •  contains transposons and other

germline-limited DNA

MACRONUCLEUS: •  somatic •  transcriptionally active •  highly polyploid •  short chromosomes •  replaced at each sexual process Paramecium cell

Genome amplification MIC 2n MAC up to 10!000n !!!

Precise excision of short Internal Eliminated Sequences (IES)

MIC

MAC

Genome reduction up to 98% !!! Chromosome fragmentation Down to a 1 gene per chr. !!!

MIC

MAC

Macronuclear development A.

B.

C.

D.

E.

Paramecium, Tetrahymena

Two distinct types of genome rearrangement in ciliates

Stichotrichs Oligohymenophorea Oxytricha, Stylonychia

MAC development: -  Long chromosomes -  DNA excision and repair

MAC development: -  Short chromosomes

(nanochromosomes) -  DNA excision and repair -  DNA unscrambling

scrambled non-scrambled

human

yeast

MAC 50 Mb

MIC 1 Gb

Deleted 95%

Maintained 5%

Gene Unscrambling in Oxytricha

D.M.Prescott, 2000

Detangling of scrambled actinI gene

3 4 6 5 7 9 2 1 8

3 4 6 5 7 9 2 1 8

MDS n MDS n+1

Pointer (2-14 bp)

Pointers are too short to guide unambiguously their own alignment

Not enough information

to assure correct unscrambling

New MAC development

Conjugation

! ! 4 5 3

Microinject RNA template

Microinjection of alternative RNA template

!

Progeny MAC DNA:

inj ctrl inj ctrl inj ctrl ! ! 5 4 3

! ! 4 5 3

inj

F1 Generation F2 Generation

Nowacki M., et al. (2008), Nature, 451(7175)

parental MAC DNA

RNA template

new mature MAC DNA

zygotic MAC DNA

1 2 3 4

C

B

A

5 6

1 2 3 4 5 6

1 2 3 4 5 6

1 2 3 4

5

6

1 2 3 4 5 6

RNA template-guided DNA unscrambling

RNA template model in Oxytricha

Transposons involved in excision Nowacki et al. (2009), Science

Nowacki et al. (2008), Nature

piRNAs protect MAC DNA against loss Fang et al. (2012), Cell

Epigenetic inheritance of gene copy number in Oxytricha

ActinI probe

non-inj. F1 F2 F1 F2

ActinI RNA inj

Loading control

2 4 6 8 10 12

Pol-! probe

Loading control

0.5

1.0

1 2 3 4

5.0 kb

RNA injection: RNAi:

Nowacki M., et al. (2010), PNAS

Conclusion

In Oxytricha, matrnal RNA play an essential role during development by: - Providing templates for DNA unscrambling - Regulating chromosome amount in the new macronucleus







Precise excision of short Internal Eliminated Sequences (IES)

MIC

MAC

"#$%&'&()!$'*$+$(,'!,-!$'.&/'01!&1$2$'0.&3!4&56&')&!78"9:!&;)$4$,'<!

"#$%&'&()!$'*$+$(,'!,-!$'.&/'01!&1$2$'0.&3!4&56&')&!78"9:!&;)$4$,'<!

Works for nearly half of IESs (maternally controlled IESs)

=0.&/'011>!),'./,11&3!8"94!/&56$/&!?)1@ABC#/,36)&3!4)'DEF4!-,/!&;)$4$,'!!E,'C=0.&/'011>!),'./,11&3!8"94!3,!',.!'&&3!4)'DEF4!-,/!.*&$/!&;)$4$,'!

!"#"$%&'($!8'.&/'01!"1$2$'0.&3!9&56&')&4!78"9:!

•  G!HI!JJJ!#&/!*0#1,$3!%&',2&!

•  #/&4&'.!$'!%&'$)!0'3!$'.&/%&'$)!/&%$,'4!

•  9*,/.!7@K!+#!1&'%.*!2,3&:L!4$'%1&C),#>L!','C),3$'%!4&56&')&4!

•  IMCNFOFPOEDCBM!),'4&'464!4&56&')&!&;.&'3$'%!$'.,!8"9!/&4&2+1&4!!.*0.!,-!N)QA=0/$'&/!./0'4#,4,'4!

N*&!4)0'!DEF!2,3&1!-,/!#/,%/022&3!%&',2&!/&0//0'%&2&'.4<!

New class of small RNAs in Paramecium?

25 nt scnRNA 23 nt siRNA

???

!"

#!!!"

$!!!"

%!!!"

&!!!"

"'()"

"*(+"

,-.)" /('0" /('#" /('1"

DCL3 *

DCL2 *

DCL5 *

DCL4

DCL1veg

development

0

2

4

6

8DCL Gene Expression

relat

ive e

xpre

ssio

n

DCL Gene Expression!

relat

ive e

xpre

ssio

n!

Dcl5!

Dcl1/2!

Dcl3/4!

97.5"

Dcl3 *!

Dcl2 *!

Dcl5 *!

Dcl4!

Dcl1!

Tetrahymena Dcl1"

Oxytricha Dcl2"

Oxytricha Dcl3"

Dcr1!

Dcr3"Dcr2"

Oxytricha Dcr1"

Oxytricha Dcl1"

Tetrahymena Dcr2"

P. biaurelia Dcl4"

P. sexaurelia Dcl3"

P. sexaurelia Dcl4"

P. multimicronucleatum Dcl3"

P. caudatum Dcl3"

P. caudatum Dcl5"

P. sexaurelia Dcl1-2"

P. multimicronucleatum Dcl1-2"

P. caudatum Dcl1-2"

P. biaurelia Dcl1-2B"

P. biaurelia Dcl1-2A"

P. biaurelia Dcl5"

P. sexaurelia Dcl5"

0.2"

100"

100"

100"

100"

99.9"

100"

51.5"

99.2"

52.2"

100"

99.8"

100"

100"

100" 100"

86.6"

100"

99.7"

69.0"

100"89.5"

90.9"

Human Dicer"

Ichthyophthirius Dcl1"

P. biaurelia Dcl3"

100"100"

73.3"

84.6"

100"

97.5"

Dcl3 *!

Dcl2 *!

Dcl5 *!

Dcl4!

Dcl1!

Tetrahymena Dcl1"

Oxytricha Dcl2"

Oxytricha Dcl3"

Dcr1!

Dcr3"Dcr2"

Oxytricha Dcr1"

Oxytricha Dcl1"

Tetrahymena Dcr2"

P. biaurelia Dcl4"

P. sexaurelia Dcl3"

P. sexaurelia Dcl4"

P. multimicronucleatum Dcl3"

P. caudatum Dcl3"

P. caudatum Dcl5"

P. sexaurelia Dcl1-2"

P. multimicronucleatum Dcl1-2"

P. caudatum Dcl1-2"

P. biaurelia Dcl1-2B"

P. biaurelia Dcl1-2A"

P. biaurelia Dcl5"

P. sexaurelia Dcl5"

0.2"

100"

100"

100"

100"

99.9"

100"

51.5"

99.2"

52.2"

100"

99.8"

100"

100"

100" 100"

86.6"

100"

99.7"

69.0"

100"89.5"

90.9"

Human Dicer"

Ichthyophthirius Dcl1"

P. biaurelia Dcl3"

100"100"

73.3"

84.6"

100"


relat

ive e

xpre

ssio

n!

???

Objective:!!

Characterize products of different Dcl proteins!- Do Dcl2 and Dcl3 produce the same sRNA molecules?!- Is Dcl5 responsible for the late 26-29nt sRNAs?!- Possible role for the 26-29nt sRNAs?!

!

?)1@AB!0'3!?)1I!0/&!/&56$/&3!-,/!8"9!&;)$4$,'!

Swart et al. 2014 Nucleic Acids Res, in press"

sRNA size distributions and source

Early development

Ctr

l

Late development

Vector

MAC

IES

Total0.56

25nt scnRNA

22-31ntIES-matching

sRNAs

Frac

tion

of to

tal r

eads

=! iesRNA!

sRN

A co

vera

ge

5’ s

RN

A st

arts

3’ s

RN

A en

ds

Ctrl early; 25 nt sRNAs Ctrl late; 27 nt sRNAs; 26-36 bp IESsCtrl late; 25 nt sRNAs

TAMAC IES

Early development

Ctr

l

Late development

Vector

MAC

IES

Total0.56

Frac

tion

of to

tal r

eads

- scnRNAs overlap MAC/IES junctions and concentrate at IES ends- iesRNAs map exclusively to IESs and concentrate at IES ends

iesRNAs

iesRNAs sometimes map across IES/IES junctions!

IES! IES! IES!MAC!scnRNAs!26-29 nt

iesRNAs!

!!!!!

Conclusion:!!

iesRNAs are produced from excised IESs!!

!!!!!

Part 2:!!!Analysis of Dcl proteins cleavage products!


relat

ive e

xpre

ssio

n!

Silencing of Dcl2, Dcl3 and Dcl5 followed by sRNA sequencing and analysis!

Sandoval, Swart et al. 2014, Dev Cell."

Knockdown of Dcls causes dramatic changes in developmental sRNAs

E L E L E L E L E L

Contro

l

DCL5-K

D

DCL2-K

D

DCL3-K

D

DCL2/3-

KD

≥ 26 nt

25 nt

23 nt

5S rRNA


$&4DEF4!0/&!0!'&R!)1044!,-!3&S&1,#2&'.01!4DEF4!

Early development!

Late development!

IES length (nt)"

Frac

tion

of to

tal r

eads"

5!"! 3!"!23!25!27!

Control!


F!),2+$'&3!)*+,!0'3!)*+-!T',)T3,R'!4&S&/&1>!3&#1&.&4!4)'DEF4!

5!"! 3!"!

DCL2

+3-K

D!Ct

rl!


25!

25!

25!

25!

8'3&#&'3&'.!)*+,!0'3!)*+-!T',)T3,R'4!/&S&01!3$U&/&'.!4DEF!4$V&!0'3!1&'%.*!#/&-&/&')&4!

DCL2

-KD!

DCL3

-KD!

5!"! 3!"!

Ctrl!

W',)T3,R'!,-!)*+.!4&S&/&1>!3&#1&.&4!$&4DEF4!DC

L5-K

D!

5!"! 3!"!

Ctrl!


Dcl cleavage preferences!Dcl2

0.0

0.5

1.0

G

CAUC

U

G

A

C

U

G

A

20

U

A

U

C

G

A

C

G

U

A

G

AUC

25

G

A

C

U

Dcl3

size 25 nt size 22-32 nt

0.0

0.5

1.0

C

A

UACUAGC

U

G

A

20

A

C

U

G

A

C

U

G

A

G

AUC

25

G

A

C

U

size 25 ntscnRNA

0.0

0.5

1.0

C

G

AUA

C

UAGG

U

A

20

G

U

C

A

G

C

U

AC

A

U

25

C

A

U

responsible for the size sequ

ence

pre

fere

nce

Dcl cleavage preferences!

Dcl5

size 22-31 nt

0.0

0.5

1.0

G

AC

UC

G

UAUCAGA

U

AGAUC

G

C

A

U

25

C

UGAG

ACU

WebLogo 3.2

0.0

0.5

1.0

bits

C

G

AUG

U

ACUAG

C

G

UA

5

C

G

U

A

G

U

A

G

U

A

G

U

A

U

A

10

U

A

15 20

U

U

A

A

G

CAU

A

U

25

C

G

UAG

ACU

Internal, non-terminalIES positions for ≥ 60 bp IESs

0.0

0.5

1.0

C

A

UACUAGC

U

G

A

20

A

C

U

G

A

C

U

G

A

G

AUC

25

G

A

C

U

scnRNA

0.0

0.5

1.0

G

AC

UC

G

UAUCAGA

U

AGAUC

G

C

A

U

25

C

UGAG

ACU

iesRNA

IES end

WebLogo 3.2

0.0

1.0

2.0

bits

TAG

A

CT

T

G

A5

C

TAG

A

TA

TG

T

AG

T

A

10

T

A A

Paramecium sRNAs concentrate at IES boundaries

iesRNAs locate 10 fold more frequently at the terminal TNG than at internal TNGs,suggesting that iesRNAs production is biased towards the ends of IESs

F)T',R1&3%2&'.4!

Nowacki Lab: Pamela Sandoval Estienne Swart Patricia Romero Miroslav Arambasic Michael Ignarski Dominique Furrer Aditi Singh Tina Hoehener Matthias Kraft Nasikhat Stahlberger

Collaborators: Linda Sperling, CNRS, Gif-sur-Yvette, France Mireille Betermier, CNRS, Gif-sur-Yvette, France Hans-Joachim Lipps, Uni-Witten-Herdecke, Germany

Funding:

rna-mediated genome remodeling in ciliates nowacki.pdf · nowacki_salzburg2014.ppt author: mariusz...

Documents