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Thesis Defense: CACTIN is an Essential Nuclear Protein in Arabidopsis and may be Associated with the Eukaryotic Spliceosome

committee meeting
by

Kate Baldwin

on 7 February 2014

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Transcript of Thesis Defense: CACTIN is an Essential Nuclear Protein in Arabidopsis and may be Associated with the Eukaryotic Spliceosome

Arabidopsis Mutant
CACTIN Localization
Rescues Mutant
Normal Phenotype
CACTIN is nuclear in
Toxoplasma

Humans
Spliceosome
Arabidopsis seedling cDNA library
Indiana University Y2H Facility
RINTIN
Thank You
Masson Lab
Thesis Committee
Donna Fernandez
Simon Gilroy
Judith Kimble
Marisa Otegui
With Materials/Help From:
Kate Baldwin
Cellular and Molecular Biology Graduate Program
Advisor:
Patrick Masson
CACTIN Students
Lizzy Dinh
Brian Hart
Putative CACTIN Interactors from Literature
Younger cells have more diffusely nuclear CACTIN-RFP
CACTIN pulled down with the spliceosome in human cells
These interactions are likely not pairwise
SR45, RSp31, PININ, SmB, nuclear ankyrin coiled coil protein, Luc7LA, Luc7LB, Luc7LC
The Gene
Conserved
Arabidopsis CACTIN
~50% similarity to human
~79 kDa
Conserved throughout eukaryotes
No domains of known function
RINTIN has come up in 2 spliceosome isolation assays
GFP-RINTIN colocalizes wtih CACTIN-RFP
Evaluating Colocalization
Negative Control: PhyB
Richard Barker
Nicole Gibbs
Tanner Kreitzmann
Rachel Kean
Caitlin Shirk
Coiled coil domain
Nuclear localization signal
R/S rich region
Single copy in most organisms
Arabidopsis CACTIN
Fly
CACTIN binds
Fly
RINTIN by Y2H
Arabi
CACTIN binds
Arabi
RINTIN by Y2H
Goal: What does CACTIN do?
No molecular function has been proposed.
Multiple papers have suggested that CACTIN can affect global gene expression.
Goal: What does CACTIN do?
Spliceosome
CACTIN has an R/S rich domain, a feature of many spliceosome proteins.
CACTIN shows a dynamic more-diffusely-nuclear vs speckle pattern like spliceosome components.
CACTIN colocalizes with known spliceosome components.
CACTIN has conserved binding relationships with a protein that binds the spliceosome in animals.
CACTIN may be actively involved in mRNA splicing or may associate with the spliceosome but actually affect transcription or RNA regulation.
has a distant paralog,
Phylogeny suggests different functions
Lost in many Fungi
Arabidopsis
Patrick Masson
Allison Strohm
Lizzy Dinh
Sathya Jali
Shih-Heng Su
Amy Jancewicz
Heterozygous plants give rise to no homozygous mutants.
Dynamic Spliceosome Component Localization
Less Active
CACTIN Yeast-2-Hybrid Screen
RINTIN binds the spliceosome in human and fly cells
Early Embryo Lethal
Nuclear Speckles
CACTIN colocalizes with spliceosome proteins
Native
Promoter

Inhibiting Respiration causes CACTIN to be more speckled
Donna
Fernandez
UW Madison
Erin
Cram
NEastern U
Ruth
Steward
Rutgers U
National
Science
Foundation
HATCH Grant
CALS
UW Madison
CACTIN-RFP
Negative Control
Phytochrome B-GFP
CACTIN

is an Essential Nuclear Protein in Arabidopsis and may be Associated with the Eukaryotic
Spliceosome
Thesis Defense Presentation
Friends
& Family

Important
Mutant:
Larva Lethal
(Steward Lab)
Morpholino:
Abnormalities
& Death
(Moynagh Lab)
Scientific Visual Communication
K8Baldwin.com
Fast, Effective, Affordable
Tell your friends and colleagues
Vierstra Lab
Robb, Nick, Aaron
TS Mutant:
Cell Cycle
Arrest
(Gubbles Lab)
Mutant:
Larva Lethal
(Cram Lab)
"Domain of Unknown Function"
Scott
Michaels
Indiana U
Human CACTIN
Active
For complete references,
see: Baldwin et al 2013
ArgSerArgSer etc.
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Full transcript