Download Poster

Transportin’ With Transportin
A Nuclear Import Mechanism
Westosha Central SMART Team: Julia Alberth, Jonah Arbet, Nick Bielski, Monica Ceisel, Sam Colletti, Evan Kirsch, Mitchell Kirsch, Sean Quist, A.J. Reeves and Julia Williams
Teacher: Jonathan Kao
Mentor: Mark T. McNally, Ph.D., Medical College of Wisconsin
I. Abstract
III. Transportin’s Anatomy
Proteins manufactured in the cytoplasm play an important role in nuclear processes such as
RNA splicing. Immediately after transcription, precursor (pre-) mRNA contains introns that are
removed in making mature mRNA. Splicing proteins like hnRNP A1 (A1), manufactured in the
cytoplasm, are transported into the nucleus and influence RNA splicing decisions. Some
proteins in eukaryotic cells use the receptor Transportin (Trn1) for import. Cytoplasmic Trn1 is
found in a configuration that allows for the pick-up of cargo proteins. A1 has a nuclear
localization signal (NLS) to which Trn1 can bind. Once bound, the Trn1/A1 complex enters the
nucleus through a nuclear pore. The protein Ran, when associated with GTP, binds to the
complex and causes a loop of approximately 60 amino acids to move and expel the
cargo. With the cargo delivered, Trn1 returns to the cytoplasm as a Trn1/RanGTP
complex. GTP is then hydrolyzed into GDP signaling Ran to release Trn1. The amino acid loop
returns to its original position allowing for another round of transport. The model
constructed using 3D printing technology by the Westosha Central HS Smart team in
cooperation with MSOE features Trn1 in complex with RanGTP, the mechanical state of cargo
unloading. The NLS of A1 can be modified such that Trn1 cannot bind and deliver A1 to the
nucleus. Failure of A1 to reach the nucleus results in altered splicing of mRNA, which can lead
to diseases like cancer. Therefore, targeting the interaction between Trn1 and its cargo may
provide an option for treating diseases.
V. NLS Rules
Ran Protein
Amino Acid
Loop
Free floating protein in
the nucleus that expels
cargo from Trn1.
This loop of
amino acids
expel cargo
proteins based
on the state of
RAN.
“Cargo”
Binding Site
This is the
region where
the NLS binds:
it is obscured in
this model.
Based on 1QBK.pdb
Fig. 3 The Transportin Protein2
II. Introduction
The central dogma of genetics states DNA is transcribed into RNA which is
translated into a protein. Transcription, the formation of RNA coded by a strand
of DNA, occurs in the nucleus while translation, the formation of proteins coded
by RNA, occurs in the cytoplasm. During transcription, RNA polymerase reads a
a gene and transcribes DNA into mRNA. Before the mRNA departs the nucleus,
it almost always modified via splicing (See Fig. 1).
Fig. 5. Results of the pull down assay5. The left lane of each gel panel shows the
results of pulldowns with Trn1 and a predicted NLS. The right lanes show the
results including RanGTP, which is known to disrupt Trn1/cargo interactions.
IV. Nuclear Import Cycle
1.
2.
3.
4.
5.
6.
Pre-mRNA
Intron
Exon
Transportin recognizes the NLS and binds to the cargo protein .
Transportin carries the protein through a nuclear pore and into the nucleus.
RanGTP binds to Trn1 causing an amino acid loop to expel the cargo.
Transportin/Ran complex returns to the cytoplasm.
GTP is hydrolyzed to GDP causing Ran to release from transportin.
The amino acid loop returns to its original position and transportin can bind to
new cargo.
Exon
Exon
1.
Exon
Cargo
Exon
Exon
Mature mRNA
Transportin
+
In the process of transcription, the entire gene is read. The resultant pre-mRNA
may contain sections that are not represented in the final mRNA. Splicing is the
process whereby these unwanted sections of RNA, introns, are removed and
the remaining RNA section (exons) are fused together to create a mature mRNA.
Alternative splicing is the process where exons are spliced in different
combinations, allowing one gene to code for many different proteins. hnRNP A1
(A1) is a protein that influences alternative splicing decisions. If A1 fails to be
imported to the nucleus, alternative splicing is changed and, consequently, the
protein repertoire of the cell is changed.
A1, like all proteins, is manufactured in the cytoplasm. It must be transported
into the nucleus to carry out its function. One mechanism by which nuclear
import is accomplished uses the protein Transportin (Trn1). Trn1 recognizes a
Nuclear Localization Signal (NLS) contained within some proteins that are
destined for the nucleus. Trn1 binds to the cargo protein and carries it to the
nucleus through a nuclear pore.
Cytoplasm
6.
Nucleus
2.
2.
VI. Biological Significance
Ran protein
Fig. 6 Modification of a NLS can lead to impaired nuclear import.
Phosphorylation of the NLS reduces the affinity between cargo and the
transport protein. As a result, the cargo does not bind and reach the
nucleus. Figure adapted from Jans et al., 2008.
4. 4.
5.
hnRNP
A1
In the case of A1 cargo, it
influences alternative
splicing of many RNAs
Conclusion:
•Solving the structure of Trn1 allows for prediction of sequences that interact
with the cargo binding site.
Nuclear pore
3.3.
release
Results:
•GST was always pulled down (linked to beads)
•GST alone does not bind to Trn1 (negative control)
•All predicted NLSs bind to Trn1 (first lane of each panel)
•RanGTP prevents interaction of NLS and Trn1 (second lane of each panel)
Splicing is regulated by proteins that need to get to the nucleus. If they fail to
get to the nucleus, regulation of splicing goes awry1. The amino acid loop of
Trn1 allows deposition of cargo molecules, like splicing proteins, into the
nucleus when RanGTP is bound. The NLS of A1 can be modified (see Fig. 6) such
that it will not bind to Trn1.
Intron
removed
Fig. 1 The Central Dogma of Genetics & Splicing7
The sequence of the NLS recognized by Trn1 varies from protein to protein. The
scientists Lee et al. attempted to predict NLS sequences that interact with
transportin using the determined crystal structure of Trn1. Predicted NLSs were
attached to a micro-bead as fusion proteins with GST, a protein used to attach
the NLS to the micro-bead, then run through a pull down assay and gel
electrophoresis.
Fig. 4 The Nuclear Import Pathway
The SMART Team Program (Students Modeling A Research Topic) is funded by a grant from NIH-SEPA 1R25OD010505-01 from NIH-CTSA UL1RR031973.
Failure of A1 to reach the nucleus can cause changes in alternative RNA splicing
and the production of inappropriate proteins. These proteins have the potential
to cause deleterious conditions including cancer3,6.
References
1.
Caceres et al. (2000). The MKK3/6-p38–signaling Cascade Alters the Subcellular Distribution of hnRNP A1 and Modulates Alternative Splicing Regulation.
The Journal of Cell Biology 149:307-316
2.
Chook, M., Blobel, G. (1999). Structure of the nuclear transport complex karyopherin-beta2-Ran x GppNHp. Nature 399:230-237
3.
David, C.J., Manley, J.L. (2010). Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged. Genes Dev. 24:2343-2364
4.
Jans et al. (2008). Regulated nucleocytoplasmic trafficking of viral gene products: A therapeutic target? BBA – Proteins and Proteomics 1784:213-227
5.
Lee et al. (2006). Rules for the nuclear locatlization sequence recognition by karyopherin beta 2. Cell 126:543-558
6.
Lopez-Bigas, N., Audit, B., Ouzounis, C., Parra, G., Guigo, R. (2005). Are splicing mutations the most frequent cause of hereditary disease? FEBS Letters
579:1900-1903
7.
http://plantphys.info/plant_physiology/enzymebasics.shtml