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Characterization of

Deadenylases in Malaria

Transmission

Kevin Hart Lindner Lab

5/21/15

Outline for Today

Why we care about CCR4

Background

CCR4 Deadenylases

Caf-1 Previous work

Summary: Deadenylases play an important role in the proper

development of Malaria parasites

Plasmodium has a Complex Life Cycle

Plasmodium has a Complex Life Cycle Original Image By: Maria Mota

Original Image By: Maria Mota

PyPuf2-KO results in a loss of spz infectivity

with prolonged salivary gland residence

of spz infectivity with prolonged salivary gland residence Down: 129 Up: 513 Lindner et al. Cellular
Down: 129 Up: 513
Down: 129
Up: 513

Lindner et al. Cellular Microbiology. 2013

The CCR4-Not Proteins have different functions

NOT 4

500

159

221

0

WT

Puf2 KO

CAF40 110 99 200 0 Ubiquitination (-/-) H3K4me3 reduction CCR4 -1 WT Puf2 KO 7.22
CAF40
110
99
200
0
Ubiquitination
(-/-) H3K4me3 reduction
CCR4 -1
WT
Puf2 KO
7.22
10
0.56
CCR4
NOT 2
CAF40
0
25.74
50
0.65
WT
Puf2 KO
Not4
0
Deadenylation
WT
Puf2 KO
Not1
Not2
CAF 1
50
24
24
159
151
200
CAF1
Not5
0
0
WT
Puf2 KO
WT
Puf2 KO
100
Not7
57
NOT 5
CAF16
4
0
CAF16
WT
Puf2 KO
FPKM

100

0

52

2

57 NOT 5 CAF16 4 0 CAF16 WT Puf2 KO FPKM 100 0 52 2 WT

WT

Puf2 KO

4

3

2

1

0

CCR4 -2

3.37

0.02
0.02

WT

Puf2 KO

Modified from Collart, M., & Panasenko, O. (2012). The Ccr4Not complex. Gene, 492(1), 42-53.

Caf1 is the only Not complex protein in

Plasmodium to be characterized

is the only Not complex protein in Plasmodium to be characterized • Forward genetic screen •

Forward genetic

screen

Piggybac transposon

is the only Not complex protein in Plasmodium to be characterized • Forward genetic screen •

Caf1 disruption in Pf results in a decreased

parasitemia due to a defect in egress

results in a decreased parasitemia due to a defect in egress Conclusions: • KO results in

Conclusions:

KO results in a decrease in proliferation efficiency

KO results in a severe disruption of merozoite egress

Premature merozoites bursting RBC

membrane

Attempts to KO in P. berghei unsuccessful

bursting RBC membrane • Attempts to KO in P. berghei unsuccessful Balu et al. (John Adams)

Balu et al. (John Adams) Eukaryotic Cell 2011

Lindner Lab

Scott Lindner Laura Bowman

Steve Griffin Brooke Kanaskie Mark Kennedy Allen Minns Elyse Munoz Amanda Reese Erin Vrana

Mike Walker

Acknowledgements

Munoz Amanda Reese Erin Vrana Mike Walker Acknowledgements Funding Sources NIAID: 1K22AI101039-01 (SEL) Penn State

Funding Sources NIAID: 1K22AI101039-01 (SEL) Penn State Start Up Finds (SEL)

Reese Erin Vrana Mike Walker Acknowledgements Funding Sources NIAID: 1K22AI101039-01 (SEL) Penn State Start Up Finds
Reese Erin Vrana Mike Walker Acknowledgements Funding Sources NIAID: 1K22AI101039-01 (SEL) Penn State Start Up Finds