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Mariusz Nowacki
University of Bern Institute of Cell Biology Switzerland
RNA-mediated genome remodeling in ciliates
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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
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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.
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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
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scrambled non-scrambled
human
yeast
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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
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MAC 50 Mb
MIC 1 Gb
Deleted 95%
Maintained 5%
Gene Unscrambling in Oxytricha
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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
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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)
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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
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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
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Conclusion
In Oxytricha, matrnal RNA play an essential role during development by: - Providing templates for DNA unscrambling - Regulating chromosome amount in the new macronucleus
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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
Precise excision of short Internal Eliminated Sequences (IES)
MIC
MAC
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"#$%&'&()!$'*$+$(,'!,-!$'.&/'01!&1$2$'0.&3!4&56&')&!78"9:!&;)$4$,'<!
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"#$%&'&()!$'*$+$(,'!,-!$'.&/'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!-,/!.*&$/!&;)$4$,'!
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!"#"$%&'($!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!
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N*&!4)0'!DEF!2,3&1!-,/!#/,%/022&3!%&',2&!/&0//0'%&2&'.4<!
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N*&!4)0'!DEF!2,3&1!-,/!#/,%/022&3!%&',2&!/&0//0'%&2&'.4<!
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New class of small RNAs in Paramecium?
25 nt scnRNA 23 nt siRNA
???
!"
#!!!"
$!!!"
%!!!"
&!!!"
"'()"
"*(+"
,-.)" /('0" /('#" /('1"
DCL3 *
DCL2 *
DCL5 *
DCL4
DCL1veg
development
0
2
4
6
8DCL Gene Expression
relat
ive e
xpre
ssio
n
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DCL Gene Expression!
relat
ive e
xpre
ssio
n!
Dcl5!
Dcl1/2!
Dcl3/4!
97.5"
Dcl3 *!
Dcl2 *!
Dcl5 *!
Dcl4!
Dcl1!
Tetrahymena Dcl1"
Oxytricha Dcl2"
Oxytricha Dcl3"
Dcr1!
Dcr3"Dcr2"
Oxytricha Dcr1"
Oxytricha Dcl1"
Tetrahymena Dcr2"
P. biaurelia Dcl4"
P. sexaurelia Dcl3"
P. sexaurelia Dcl4"
P. multimicronucleatum Dcl3"
P. caudatum Dcl3"
P. caudatum Dcl5"
P. sexaurelia Dcl1-2"
P. multimicronucleatum Dcl1-2"
P. caudatum Dcl1-2"
P. biaurelia Dcl1-2B"
P. biaurelia Dcl1-2A"
P. biaurelia Dcl5"
P. sexaurelia Dcl5"
0.2"
100"
100"
100"
100"
99.9"
100"
51.5"
99.2"
52.2"
100"
99.8"
100"
100"
100" 100"
86.6"
100"
99.7"
69.0"
100"89.5"
90.9"
Human Dicer"
Ichthyophthirius Dcl1"
P. biaurelia Dcl3"
100"100"
73.3"
84.6"
100"
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97.5"
Dcl3 *!
Dcl2 *!
Dcl5 *!
Dcl4!
Dcl1!
Tetrahymena Dcl1"
Oxytricha Dcl2"
Oxytricha Dcl3"
Dcr1!
Dcr3"Dcr2"
Oxytricha Dcr1"
Oxytricha Dcl1"
Tetrahymena Dcr2"
P. biaurelia Dcl4"
P. sexaurelia Dcl3"
P. sexaurelia Dcl4"
P. multimicronucleatum Dcl3"
P. caudatum Dcl3"
P. caudatum Dcl5"
P. sexaurelia Dcl1-2"
P. multimicronucleatum Dcl1-2"
P. caudatum Dcl1-2"
P. biaurelia Dcl1-2B"
P. biaurelia Dcl1-2A"
P. biaurelia Dcl5"
P. sexaurelia Dcl5"
0.2"
100"
100"
100"
100"
99.9"
100"
51.5"
99.2"
52.2"
100"
99.8"
100"
100"
100" 100"
86.6"
100"
99.7"
69.0"
100"89.5"
90.9"
Human Dicer"
Ichthyophthirius Dcl1"
P. biaurelia Dcl3"
100"100"
73.3"
84.6"
100"
DCL Gene Expression!
relat
ive e
xpre
ssio
n!
???
Objective:!!
Characterize products of different Dcl proteins!- Do Dcl2 and Dcl3 produce the same sRNA molecules?!- Is Dcl5 responsible for the late 26-29nt sRNAs?!- Possible role for the 26-29nt sRNAs?!
!
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?)1@AB!0'3!?)1I!0/&!/&56$/&3!-,/!8"9!&;)$4$,'!
Swart et al. 2014 Nucleic Acids Res, in press"
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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!
![Page 24: RNA-mediated genome remodeling in ciliates Nowacki.pdf · Nowacki_Salzburg2014.ppt Author: Mariusz Nowacki Created Date: 8/31/2014 4:35:32 PM](https://reader033.vdocument.in/reader033/viewer/2022060711/6076fe66b042f90e210649bb/html5/thumbnails/24.jpg)
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
![Page 25: RNA-mediated genome remodeling in ciliates Nowacki.pdf · Nowacki_Salzburg2014.ppt Author: Mariusz Nowacki Created Date: 8/31/2014 4:35:32 PM](https://reader033.vdocument.in/reader033/viewer/2022060711/6076fe66b042f90e210649bb/html5/thumbnails/25.jpg)
iesRNAs sometimes map across IES/IES junctions!
IES! IES! IES!MAC!scnRNAs!26-29 nt
iesRNAs!
!!!!!
Conclusion:!!
iesRNAs are produced from excised IESs!!
!!!!!
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Part 2:!!!Analysis of Dcl proteins cleavage products!
DCL Gene Expression!
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."
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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
Sandoval, Swart et al. 2014, Dev Cell."
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$&4DEF4!0/&!0!'&R!)1044!,-!3&S&1,#2&'.01!4DEF4!
Early development!
Late development!
IES length (nt)"
Frac
tion
of to
tal r
eads"
5!"! 3!"!23!25!27!
Control!
Sandoval, Swart et al. 2014, Dev Cell."
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F!),2+$'&3!)*+,!0'3!)*+-!T',)T3,R'!4&S&/&1>!3&.&4!4)'DEF4!
5!"! 3!"!
DCL2
+3-K
D!Ct
rl!
Sandoval, Swart et al. 2014, Dev Cell."
25!
25!
25!
25!
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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!
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W',)T3,R'!,-!)*+.!4&S&/&1>!3&.&4!$&4DEF4!DC
L5-K
D!
5!"! 3!"!
Ctrl!
Sandoval, Swart et al. 2014, Dev Cell."
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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
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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
![Page 34: RNA-mediated genome remodeling in ciliates Nowacki.pdf · Nowacki_Salzburg2014.ppt Author: Mariusz Nowacki Created Date: 8/31/2014 4:35:32 PM](https://reader033.vdocument.in/reader033/viewer/2022060711/6076fe66b042f90e210649bb/html5/thumbnails/34.jpg)
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
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Sandoval, Swart et al. 2014, Dev Cell."
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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: