UIN:___________
CERTIFICATE OF THESIS APPROVAL
University of Illinois at Urbana-Champaign
May 2017
We hereby recommend that the thesis by:
Alexander B Willis
Entitled:
THE ROLE OF THE EIF3 COMPLEX IN SPECIALIZED TRANSCRIPT SPECIFIC
TRANSLATION
Be accepted in partial fulfillment of the requirements for the degree of:
Bachelor of Science
Biochemistry
Signature:
______________________________
Director of Research
1
The Role of the eIF3 complex in Specialized Transcript
Specific Translation
By
Alexander Willis
Thesis
for the
Degree of Bachelor of Science
in
Biochemistry
College of Liberal Arts and Sciences
University of Illinois,
Urbana-Champaign, Illinois
2017
2
Table of Contents
Page Number
Abstract
3
Introduction
3
Methods and Materials
6
Results & Discussion
8
Figures & Tables
9
Bibliography
10
3
Abstract
Translational initiation is a complex process in eukaryotes that necessitates a
multitude of proteins known as initiation factors (eIFs). eIF3, a protein whose core
structure is conserved throughout eukaryotes, plays a number of key roles in the
translational process including binding free 40S ribosome and serving as a scaffold for
other initiation factor binding, aiding in scanning for the start codon, assisting in ribosome
recycling, and importantly binding certain mRNA structures in 5’ UTRs to promote cap
independent translation. In these studies, the six-subunit eIF3 complex was purified from
chromosomally eIF3b-TAP-tagged saccharomyces cerevisiae(Tandem Affinity
Chromatography). This complex will be used in addition with other initiation factors to
perform translation initiation assays on mRNAs identified via high throughput screening as
being candidates for non-canonical transcript specific translation.
Introduction
To sustain life, cells are required to maintain precise temporal control over the
translational process, in which mRNA is translated by the ribosome into protein. To
accomplish this eukaryotes require over a dozen factors, termed eukaryotic initiation
factors (eIF’s), that recruit the ribosome to the 5’ end of the mRNA and allow it to scan for
the proper start codon.1 Initiation of translation was thought to be dependent on
recognition of the 5’ 7-methyl-guanosine cap, but later it was discovered that structured
mRNA elements can recruit the ribosome directly to the start codon. The eIF3 complex has
been found a key mechanistic and regulatory component of both cap-dependent and
certain types of cap-independent translation, as well as ribosome recycling after
translation. These studies, yet to be completed, will focus on the role of eIF3 in specialized
4
translational initiation of transcripts identified as cap independent by high throughput
screening.
The eIF3 complex is composed of 6 subunits in saccharomyces cerevisiae(a,b,c,g,i,j)
while its mammalian homologue contains 13(a-l).2,3 The 8 subunit “core” of the
mammalian form shares structural homology with yeast eIF3. Both exhibit a five lobed
structure that interacts with the 40S ribosomal subunit mostly on the solvent side, on the
E-site-side of the 40S, and wraps around its 60S interface, serving as a scaffold for initiation
factor and mRNA binding, and positioning initiation factors on both sides of the mRNA
channel.4 Clear structural data is lacking due to the dynamic nature of the eIF3-40S
relationship, but EM data combined with modeling and evolutionary connection with the
26S proteasome lid have helped to model the interactions.5
eIF3 has been shown to stabilize the binding of the ternary complex (TC), composed
of eIF2-Met-tRNA-GTP, to the ribosome to form the 43S preinitiation complex.6 While no
direct contact between eIF3 and eIF2 has been observed, both proteins can simultaneously
bind eIF5 as part of the multifactor complex, conserved in yeast, plants, and humans,
composed of eIF’s 1, 3, 5, and 2.7 8 This multifactor complex can be isolated from cell
extracts and is an important intermediate for translation initiation in vivo. Parallels have
been drawn between it and eIF3 specifically and the mediator complex that stimulates
transcriptional initiation and polymerase recruitment.9 With the ternary complex bound,
the 43S ribosome complex can scan for the proper start codon. Scanning is made possible
by eIF1 and eIF1A, which put the ribosome in a favorable conformation for scanning, and
by eIF3 which interacts with mRNA upstream of the E site and may extend the mRNA
binding channel. Upon proper base pairing with the initiator tRNA, eIF1 is displaced which
5
allows eIF5 to stimulate eIF2’s GTPase activity. This GTP hydrolysis is the commitment
step of translational initiation, and the conformational change allows eIF5b to mediate
joining of the 60S subunit and displacement of eIF1, eIF1A, eIF2-GDP, and eIF3.10
After translation, the ribosome remains bound to the mRNA, in a post-termination
complex (post-TCs) with proteins known as release factors. eIF3 together with eIF1 and
eIF1A separate the ribosome, yielding the 60S subunit, and the 40S, still bound to the
mRNA and tRNA. The transient subunit of eIF3, eIF3j, binds with negative cooperativity to
mRNA binding, and promotes release. These subunits likely remain bound to the 40S to
prevent subunit reassociation until the ternary complex can again be bound.11
Along with its roles in cap dependent translation and ribosome recycling, eIF3 also
plays a role in a number of cap independent translational initiation pathways. These sites
where the ribosome can bind and initiate translation independent of the cap and cap
binding protein (eIF4E) are known as IRES’s(internal ribosome entry sites). First
discovered in viruses as a means to bypass cellular defense by global translation reduction,
these mRNAs have dense secondary and even tertiary structure in their 5’ UTR’s that bind
initiation factors and allow for the ribosome to be inserted directly at the start codon.12
The RBM (RNA-binding motif) in the N-terminus of eIF3a can differentially bind the HCVIRES and 40S subunit.13 However, IRES’s are not limited to viral mRNAs and are often
found natively in cellular transcripts. A RNA-Protein crosslinking experiment in
mammalian cells and subsequent immunoprecipition of eIF3 showed it bound to nearly
500 different transcripts, with most transcripts encoding proteins involved in the cell cycle,
and proliferation as well as differentiation and apoptosis.14 This is logical as cells would
need these genes to be expressed during stress conditions where translation is globally
6
repressed, usually through reduction in eIF4E(cap binding) or eIF2(initiator tRNA loader)
which are both tied to stress and cellular metabolism pathways.15 Remarkably, it was
found capable of acting as a translational activator or repressor.16 These results
demonstrate the integral role of eIF3 in recruiting the ribosome to crucial genes during
starvation when cap dependent translation is globally repressed, and moreover the
importance of cap-independent translational methods in maintaining homeostasis.
The eIF3 complex was natively purified from eIF3b TAP-chromosomally-tagged
(Tandem Affinity Purification). It consists of a IgG binding domain separated downstream
by a TEV Protease Cleavage site from a Calmodulin Binding Peptide.17 From the binding to
eIF3b the whole eIF3 complex was purified. First cell lysate is bound to IgG beads, then the
protein is cleaved off the beads with TEV protease, where it can be subsequently bound by
Calmodulin Resin to improve purity. This complex will be used with other factors purified
in the Jin lab, and will be used in upcoming translational initiation assays with specific
transcripts identified as candidates for cap-independent translation by high throughput
screening.
Materials and Methods
Purification
1 𝜇L of the eIF3b TAP chromosomally tagged saccharomyces cerevisiae strain was
spread on a YEPD plate and incubated at 28°C overnight. One of these colonies was picked
and placed in 50mL of YEPD to serve as a starter culture, it was incubated for a day on the
shaker at 28°C. This culture was then evenly added to 6L of YEPD broth and grown for 2
days until OD600 ~ 1.8 (when cells turned white). The cells were pelleted by spinning for
10 minutes at 4K rpm. The supernatant could then be poured off.
7
The cells were resuspended in one pellet volume of Lysis Buffer (100 mM HEPES
KOH pH 8.0, 200mM KCl, 2mM Mg(OAc)2, 10% volume glycerol, 2mM DTT, .1mM PMSF,
.1mM BZA). Next, the cells were lysed by the addition of liquid nitrogen and the use of a
blender until the cells were ground to a fine powder. After thawing the lysate, the cells
were spun at 18k rpm for 30 minutes. The clear supernatant was then transferred to new
50mL tubes. 600 𝜇𝐿 of 1M Trizane base was added to each 50mL of lysate.
250uL of IgG sepharose was washed 3 times with TAP Purification Buffer(20mM
HEPES KOH pH 8.0, 150mM KCl, 1mM Mg(OAc)2, .1% NP 40, 1mM DTT). The lysate was
then added to the beads and placed in the 4°C room overnight to bind. Next, the beads
were spun at 3K rpm and the supernatants carefully removed down to about 1mL. The
resin was resuspended in the remaining supernatant, and transferred to a Biorad column.
The beads were then washed with ~10mL TAP Purification Buffer 3 times.
200 𝜇L of TEV Protease was then mixed with 2mL of TAP Purification Buffer. This
mixture was added to the IgG beads in the Biorad column and incubated rotating at 4°C
overnight. The next day, the flow through was collected along with 3 bead volume washes
of TAP purification buffer. These were pooled together and then dialyzed against 1L of
Calmodulin Binding Buffer(20mM HEPES KOH pH 8.0, 150mM KCl, 1mM Mg(OAc)2, 2mM
CaCl2, .1% NP40, 1mM DTT).
After dialysis, the FT and washes were added to 200 𝜇𝐿 Calmodulin Resin, and
incubated overnight. The flow through is collected, along with 3 washes of the resin with
~2mL of Calmodulin Binding Buffer each time(washes incubated for at least 10 min.). .5mL
of Calmodulin Elution Buffer(20mM HEPES KOH pH 8.0, 150mM KCl, 1mM Mg(OAc)2, 5mM
EGTA, .1% NP40, 1mM DTT) was then added to the resin and incubated for ~45 minutes,
8
then the elution supernatant fraction was collected. This step was repeated 3 times.
Samples were taken of each elution fraction and wash fractions were taken for analysis on
12% SDS Gels, and the gels were stained with coomasie blue.
Due to a high amount of proteins copurified with the eIF3 complex initially, the
protein was dialyzed back against Calmodulin Binding Buffer, rebound to Calmodulin
Resin. The resin was divided into 4 equal aliquots, and was this time washed with 4
different Calmodulin Binding Buffers with increasing ionic strength (.25M, .3M, .35M, and
.4M KCl respectively) to try to wash off proteins bound to the complex. The elutions were
then compared via SDS PAGE(figure 1) and .25M KCl Binding Buffer was selected for
subsequent runs as it showed to reduce unwanted protein copurifications without
decreasing the affinity of the eIF3 complex for the resin.
Results and Discussion
By increasing the ionic strength of the calmodulin wash buffer, unwanted interactions with
other proteins were greatly reduced in all four trials. However, increasing the salt
concentration beyond 300mM appeared to reduce binding to the calmodulin resin and
most of the complex was washed away(Figure 1). The 250mM and 300mM wash were
combined and eluted, and the resulting complex was free of previous impurities(Figure 2).
The assays were planned be run with other protein factors purified by other members on
the lab on transcripts identified as cap independent targets by high throughput screening
to try to further establish eIF3’s job in stimulating the initiation of cap-independent
translation. However, by the time subsequent purifications were performed, time
restrictions forced these experiments to remain unfinished.
9
Figures and Tables
Figure 1: SDS-Analysis of Salt Washes in Decreasing Undesired Protein Interactions
Calmodulin resin was washed with Calmodulin Binding Buffers of varying KCl
concentration, and resin and wash samples were compared on a 12% polyacrylamide gel.
Lane 1: Ladder, Lane 2: 250mM KCl resin, Lane 3: 250 mM Wash, Lane 4: 300mM KCl resin,
Lane 5: 350mM KCl resin, Lane 6: 300mM Wash, Lane 7: 350mM Wash, Lane 8: 400mM KCl
Resin, Lane 9: 400mM Wash
10
a - 110.4 kD
b + tag – 93.1
kD(88+5)
c – 93.2 kD
i- 38.7 kD
g- 30.5 kD
j – 29.5kD
Figure 2 : eIF3 Complex Final Elution: The final elution of the combined 250mM and
300mM fractions, showing subunits a, b, c, g, I, and j, run on 12% polyacrylamide gel.
Figure 3: eIF3 Complex, second purification: Final elution, shows bands for subunits a,
b, c, i, and j/g, run on a 12% polyacrylamide gel.
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