1. Introduction
La-related proteins (LARPs) are a group of RNA-binding
proteins that have critical functions in the cell, and use a “La
Module” domain to bind RNA substrates. Limited knowledge is
known on the subfamily of LARP6 proteins. This project
optimized native polyacrylamide gel electrophoresis with
biotinylated RNA to measure RNA-LARP6 binding.
Future studies of binding will rely on the accurate
identification of functional boundaries of the La Module in
LARP6. Studies have examined the function of amino acids 74-
3131 and 70-3002. However, no studies have examined amino
acids 70-313. These inclusive boundaries will provide
information critical for the creation of chimeric La Modules that
combine subdomains from different species.
Before these two questions could be explored, the lab was
experiencing possible RNase contamination. Previous RNA-
protein binding assays showed that when protein was added,
the RNA bands in those lanes disappeared. Therefore, the first
step was to test the hypothesis of RNase contamination.
Andrew Kocian, Eli Lee Peña, and Dr. Karen Lewis
Department of Chemistry & Biochemistry
Texas State University, San Marcos, TX 78666
Evaluating RNA Binding Activity of the Protein LARP6
Results
Approach
Electrophoretic mobility
shift assay (EMSA)
Cloning of La Module
to be expressed
Optimize EMSA Express Protein
Determine functional
boundaries of protein binding
Conclusions
Acknowledgments
References
1. Is there RNase contamination?
2. Establish methods to quantitatively measure binding
activity of LARP6.
3. What functional boundaries of the HsLARP6 La Module should be used for
future phylogenetic comparison studies?
Figure 1: The RNA ligand that human LARP6 binds was conjugated to a biotin tag at the
3’ end. The RNA is found in the 5’ untranslated region of collagen type I mRNAs.
detection system is presented here as well. Luminol oxidation by peroxide diagram by
LifeTechnologies. (A) The structure of biotin. (B) The RNA hairpin without the biotin tag.
(C) Model of the RNA hairpin with the biotin label attached via a cytidine at the 3’ end.
C C A C A A A G A C A U G U C U A G
U G U A C A G A U CG G U G U U U C
A
G U C
U
A UC
G
G
U
G
5’
3’
C
C C A C A A A G A C A U G U C U A G
U G U A C A G A U CG G U G U U U C
A
G U C
U
A UC
G
G
U
G
5’
T4 RNA Ligase
Figure 2: Detecting the biotinylated RNA. (A) The biotin tag is bound by a fusion protein
composed of streptavidin (SA) and horseradish peroxidase (HRP). (B) HRP catalyzes the
oxidation of luminol by H2O2 to create an unstable compound (3’aminophthalate), which
decays by emitting light. (C) Previous gel shift data, which shows only free RNA.
Free RNA
LARP6-RNA
+-LARP6 protein
++Biotinylated RNA
+-
++
A
B
C
RNase contamination was not identified in assays. We were able to show that the
biotinylation method works for visualizing RNA and LARP6 binding. Continued
optimization of gel conditions will further improve the resolution of the EMSAs.
Now that both La Module constructs have been expressed, the next step is to
purify the proteins, and then execute an EMSA with our optimized conditions. The
construct that best mimics full-length LARP6 will be used to design the La Module
boundaries for future studies with the homologs in fish.
Additionally, once the boundaries are established, interspecies chimeric La
Modules can be created. Studies involving different species will provide vital
information regarding the function and importance of the La Module sequence in
these different species.
1. Hussain et al (2013) Nucleic Acids Res. 41:8715-8725.
2. Martino (2015) Nucleic Acids Res 43:645-60.
3. Altschuler et al, (2013) J Nucleic Acids Invest 4:19-28.
This work was supported by the National Science Foundation (1156579) and the
Texas State University Department of Chemistry and Biochemistry.
Figure 3: RNase assays. Lab protein stocks were checked for RNases by native
PAGE. (A) Determining the detection limit of ethidium bromide staining of RNA on
native PAGE. (B and C) Ethidium bromide stain of RNA incubated with either (B)
RNA polymerase or (C) human LARP6 and then analyzed by native PAGE. No RNase
activity was detected in either of the protein stocks.
A
[LARP6]
LARP6RNA
Free RNA
HEPES Binding Buffer
6.7% acrylamide, 25 min, 1X TBE running buffer
Semidry transfer in 1X TBE
A
[LARP6]
LARP6RNA
Free RNA
Tris-HCl Binding Buffer
6% acrylamide, 15 min, 1X TBE + 5% glycerol running buffer
Semidry transfer in 1X TBE
B
Tris-HCl Binding Buffer
6% acrylamide, 25 min, 1X TBE+ 5% glycerol running buffer
Semidry transfer in 1X TBE
LARP6RNA
Free RNA
[LARP6]
C
Figure 4: Analysis of various EMSA conditions. (A) EMSA of LARP6 binding to
biotinylated RNA confirms that this method can be used for visualization. (B and C)
EMSAs using varying parameters, as described below the gels. Optimization of gel
conditions is necessary for quantitation and reliable data acquisition. Biotinylation of
RNA was repeated after (C) to improve the quality of future EMSA experiments.
Site-directed
mutagenesis
BamHI Digest
BamHI/XhoI Digest
Ligation
Transform
Plate on kanamycin
Extract DNA from colonies
basepairs
1650
650
850
650
850 600
850
850
5000
12,000
[RNA Polymerase]B
base pairs
12,000
3000
~700
[LARP6]C
base pairs
12,000
3000
~700
μL of 8μM RNA
Transfer in 1X TBE
enabled detection of
LARP6-RNA band
New binding buffer
yielded sharper bands
and well shifts.
Longer run time suggests
multiple shifted bands, but
RNA signal is weak.
B
Remove endogenous restriction site
Clone #
1 2 3 4 5 6 7 8 9 base pairs
C
PCR amplify inserts that code for desired La module boundaries
70-313 70-300
1650
basepairs
D
Ligate and recover new plasmid constructs
PCR to detect insert
E
Screen constructs for protein expression
A
Diagram of LARP6 protein domains
Figure 5: Development of reagents to evaluate the role of residues 300-313 in RNA binding. (A) The La Module,
showing the two domains that are necessary and sufficient for minimal RNA binding activity. (B) The removal of
BamHI site. This step allows for insertion of inserts with desired La Module boundaries. (C) Amplification of the
inserts. The inserts and plasmid are cut with enzymes BamHI and XhoI to enable ligation. (D) After ligation and
antibiotic-based selection, the DNA was extracted and subjected to PCR to check for the presence of insert. (E)
Protein expression of four clones that code for amino acids 70-313 (left) or 70-300 (right) of LARP6. Protein of
interest is expected to be present around 55 kDa, and increase in intensity over time.
La Module
La motif RRM
Not digested insert
basepairs
70-313 70-300
Digested
1 2 3 4 1 2 3 4
70-31370-300
100
12,000
1650
850
180
72
55
43
34
26
17
kDa
180
72
55
43
34
26
17
0
kDa hr
2 145
Clone 1 Clone 2 Clone 3 Clone 4
LifeTechnologies
70-313
Clone 1 Clone 2 Clone 3 Clone 4
70 300 313
hr
180
72
55
43
34
26
17
kDa
180
72
55
43
26
17
kDa
70-300
hr
34