Phosphoregulation of spliceosome formation in Arabidopsis
FORS 8980
April 28, 2011
by Eric Talevich
Institute of Bioinformatics
University of Georgia
Thanks
Conclusions
Pathway
Methods
Evolution
Agathman (2009): Spliceosome assembly: the A complex.
https://secure.wikimedia.org/wikipedia/en/wiki/File:A_complex.jpg
LAMMER
GxGxxG
Activation Loop
Domain architecture (Pfam)
KinBase: CLK family report
KinBase: Gene tree
Gene tree
of AFC2
orthologs
Globular kinase
Autophosphorylated
KinBase:
Sequence conservation in eukaryotic CLKs
Important kinase motifs:

GxGxxG (ATP-binding)
HRD -> HTD (catalytic)
DFG...APE (actloop)
(EH)LAMMER
The
spliceosome regulation
AFC2
2 splice variants
More SNPs outside the kinase core
2 phosphorylation sites
"A LAMMER-type protein kinase that co-precipitates with serine/arginine-rich (SR) proteins in vitro, interaction modulated by phosphorylation of the proteins."  -- TAIR
AFC2 and SR proteins
Alternative splicing is arranged by complex protein-protein interactions.




These interactions are regulated by protein phosphorylation.
LAMMER kinase phosphorylates the RS domains of multiple SR proteins
U1-70K, the RBD of an SR protein, and other proteins form the U1 snRNP
The U1 and U2 snRNPs form the the early complex of the spliceosome, bridging the 5' and 3' ends of the splice site
The Spliceosome
Remove introns from mRNA transcripts of protein-coding genes
Produces alternative splice forms under varying conditions
Composed of 4 subunits, several other proteins, and mRNA
Where are SR proteins phosphorylated?
SR proteins and U1-70K
Control
Interactions
Identify interactions between...
Each SR protein
Non-phosphorylated (-) and phosphorylated (+) SR proteins, incubated with
non-phosphorylated (-) and autophosphorylated (+) AFC2,
probed with
T7 antibody (T7 Ab) and S.protein (SP).
SRPK also phosphorylates SR proteins, but with different specificity
Hypophosphorylation by SRPK,  hyperphosphorylation by CLK, dephosphorylation by phosphatase (?) affect localization
Recent findings:
small nuclear
ribonucleoproteins (snRNPs):
U1, U2, U4/U6, U5
Early ("A") Complex
U1 subunit:
1 U1 snRNA molecule
3 U1 snRNP-specific proteins (U1-70K, U1-A, U1-C)
8+ other proteins
U1 recognizes the 5' splice site
U2AF recognizes 3' splice site
SR proteins facilitate
SR proteins are phosphorylated by a CLK/LAMMER kinase, AFC2
SR protein facts
Domain architecture
RNA-binding domain (RBD)
Arg/Ser-rich domain (RS)
Regulation
Involved in many parts of the spliceosome
Phosphorylation required for spliceosome assembly and site selection
Localization: shuttles between cytoplasm and nucleus (also phosphorylation-dependent)
Several copies per genome
* Arginine has a positive charge, phosphate is negative
Genomic features:
Golovkin M, Reddy ASN:
An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein.
The Journal of Biological Chemistry 1999, 274:36428-38
Golovkin M, Reddy ASN:
An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein.
The Journal of Biological Chemistry 1999, 274:36428-38
http://kinase.com/kinbase/
Sequences from OrthoMCL

Neighbor-joining tree constructed in SeaView

Colorized in Archaeopteryx (phylosoft.org)
PDB: 3LLT
Pflammer
Fluhr R:
Regulation of Splicing by Protein Phosphorylation.
In Nuclear pre-mRNA Processing in Plants.
Edited by Reddy ASN, Golovkin M Berlin, Heidelberg: Springer Berlin Heidelberg; Vol. 326 2008:119–138.


Yeast
two-hybrid
system