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The heterogeneous nuclear ribonucleoprotein (hnRNP),
Nab2p, is essential for export of the poly(A) RNA through the nuclear
pore complex. Like several other hnRNPs, Nab2p is methylated by
the arginine methyltransferase, Hmt1p. We have also identified an
interaction between Nab2p and the RING finger E3 ligase Siz1p. Previously,
Air1p (arginine methyltransferase interacting RING finger protein)
was found to negatively regulate Hmt1p-mediated methylation of another
yeast hnRNP, Npl3p, in vitro.1 The goal of this project was to elucidate
the relationship between Nab2p, Hmt1p, and Siz1p, and test if methylation
of Nab2p is regulated by Siz1p analogous to the above situation.
Protein-protein interactions between Nab2p, Hmt1p, and Siz1p were
tested using the yeast two-hybrid assay.
Eukaryotic cells contain a membrane bound nucleus,
separating chromosomal DNA from the translation machinery in the
cytoplasm. Thus, after DNA is transcribed into messenger RNA (mRNA),
the mRNA must be transported out of the nucleus into the cytoplasm
for translation. This exchange is mediated by nuclear pore complexes.
Poly(A) RNA is exported as a complex of RNA and many proteins. A
large family of evolutionarily conserved heterogeneous nuclear ribonucleoproteins
(hnRNPs) helps escort poly(A) RNAs during nuclear export. In particular,
Nab2p is necessary for efficient bulk mRNA export.3 Nab2p also plays
an essential role in mRNA metabolism and contains four functional
domains.3 Among them, the RGG repeats box is the site of methylation
by the arginine methyltransferase, Hmt1p. The unique N-terminal
domain interacts with a RING finger E3 ligase for SUMO (small ubiquitin-related
modifier), Siz1p. Siz1p was identified in a yeast two-hybrid screen
as a Nab2p interactor.4 Siz1p and its homologue Siz2p are two proteins
required for most SUMO conjugation in yeast. The goal of this project
is to examine the relationships between Nab2p, Hmt1p, and Siz1p.
We hypothesize that sumoylation may regulate the methylation of
Nab2p, through the interaction of Hmt1p and Siz1p.
Yeast Two Hybrid Assay (Y2H):
A system designed to identify and analyze protein-protein interactions
wherein two proteins of interest are expressed as fusion proteins
with a transcriptional activating domain (AD, prey), and a DNA-binding
domain (DBD, bait). If the two proteins interact a reporter gene
is activated.
Yeast Two-Hybrid Knockout Strains
To determine whether Siz1p is required for the interactions examined,
we created a yeast two-hybrid strain lacking the SIZ1 gene. The
SIZ1/SIZ2 gene was replaced by a kanamycin resistance gene (KANR)
in yeast two-hybrid cells by PCR amplification and homolgous recombination.
Cells deleted for SIZ1/SIZ2 were able to grow on Kan (G418) plates,
while wildtype cells did not. PCR amplification showing that the
KANR gene was specifically recombined in place of SIZ1/SIZ2.
Site-Directed Mutagenesis of Hmt1p
To created an enzymatically inactice form of Hmt1p, we created a
set of primers that contained an Hmt1p G68R active site mutation
and used site-directed muagenesis to incorporate this mutation into
Hmt1p in both Y2H vectors. This mutation eliminated a KpnI restriction
site so we were able to utilize a KpnI digest to screen for mutants
of Hmt1p*( * ) .
Yeast Two-Hybrid Analysis
Pairwise yeast two-hybrid analysis was performed to map binding
of Nab2p fragments to Siz1p and Hmt1p in wildtype cells and cells
deleted for HMT1 or SIZ1. Protein-protein interaction model based
on our experimental results.
Siz1-GFP and Siz2-GFP Cloning
Siz1-GFP and Siz2-GFP were cloned by PCR amplification of SIZ1/SIZ2
and ligation into a GFP vector. Ten clones were analyzed by restriction
digestion for the presence of the correct insert. One clone for
each mutant was sequenced and further analyzed. Expression of Siz1-GFP
and Siz2-GFP was analyzed by Western blot and fluorescence microscopy.
Siz1p does not interact with Hmt1p in our Y2H assay. In the absence
of Hmt1p, there is no difference in binding of Nab2p to Siz1p. In
the absence of Siz1p, there is no difference in binding of Nab2p
to Hmt1p. There is no discernable interaction between Siz1p and
Hmt1p-DBD. Hmt1p binds to itself and may act as a dimer in vivo.
There is no evidence that Siz1p regulates methylation of Nab2p.
Future Studies
-Test other protein interactions with Nab2p in deleted SIZ1 cells.
-Sequence alternate Siz1-GFP and Siz2-GFP clones and examine them
by fluorescence microscopy and Western blot analysis.
-Elucidate the potential role of Siz2p in the regulation of Nab2p
function.
This material is based upon work supported by the Howard Hughes
Medical Institute under Grant No. 52003727 and by Anita Corbett
under Grant No. 520046. I'd like to thank Michelle Harreman and
Alex Lehner for supplying yeast strains and Alexander V. Strunnikov
for supplying the Siz1 plasmid.
References
(1) Inoue, K., Mizuno, T., Wada, K., Hagiwara, M. 'Novel RING Finger
Proteins, Air1p and Air2p, Interact with Hmt1p and Inhibit the Arginine
Methylation of Npl3p.' The Journal of Biological Chemistry 275 (2000):
32793-99.
(2) Corbett, A. H. 'Nuclear Pores and Nuclear Import/Export.' Encyclopedia
of Biological Chemistry 00 (2004): 1-6.
(3) Green, D. M., Marfatia, K. A., Crafton, E. B., Zhang, S., Cheng,
X., Corbett, A. H. 'Nab2p Is Required for Poly(A) RNA Export in
Saccharomyces cerevisiae and Is Regulated by Arginine Methylation
via Hmt1p.' The Journal of Biological Chemistry 277 (2002): 7752-7760.
(4) Ordanic-Kodani, Maja. NRSA Application. Unpublished Data.
A critical process in any cell is gene expression. One way to
understand this complex process is to research the process of RNA
transcription and translation. My lab traces the process of mRNA
export out of the nucleus into the cytoplasm via the nuclear pore
complex. I focused my research on a specific protein, Nab2p, which
helps escort mRNA into the cytoplasm. Previous research found that
one domain of Nab2p interacts with the enzyme Siz1p. It was also
known that the enzyme Hmt1p added a methyl group to another domain
of Nab2p. My project examined the relationship between these three
proteins and we hypothesized that the methylation of Nab2p by Hmt1p
was dependent upon the presence of Siz1p. We used a simple assay,
the yeast two-hybrid system, to detect interactions between Hmt1p
and Nab2p and Siz1p and Hmt1p. This process allows us to screen
for possible interactors by either the appearence of blue or white
colonies on specific yeast plates. Blue colonies support interactions,
whereas white colonies show no interactions. After creating the
tools to perform the yeast-two hybrid assay, I tested a number of
different interactions. We were able to confirm interaction between
Siz1p and Nab2p, although we saw no interaction between Nab2p and
Hmt1p, or Siz1p and Hmt1p. We also found that Hmt1p interacts with
itself since it forms a dimer in the cell. This helped solve a technical
problem in the lab since there was no previous positive control
for Hmt1p interaction. In conclusion there is no evidence from my
analysis to suggest that Siz1p regulates methylation of Nab2p, however
further research must be performed.
Yeast Two-Hybrid Assay, Replicaplating PCR, Western Blot, Site-Directed
Mutagenesis, Cloning through PCR, and ligation.
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