1. Experimental Identification of the
Complete RNA–Binding Domain of Human
LARP6 Proteins
Shahad Amdeen
Faculty Adviser: Dr. Karen A. Lewis

2. Transcription Translation
DNA mRNA Protein
The Central Dogma
• RNA functions as a conduit between the DNA genome sequence and protein synthesis for
gene expression
• Genetic information is translated into proteins through messenger RNA (mRNA)
• In cells, RNAs are associated with RNA binding proteins (RBPs) which have an effect on
RNA’s structure and interactions as well as on the biogenesis and stability of RNA

3. RNA Folding and Structure
3-D
Structure
3-D
Structure
RNA
Chaperone
RNA
Folding
• Structure = Function
• RNA Chaperone aid folding
• The specific mechanisms behind RNA
chaperones’ ability to facilitate proper RNA
folding are yet unknown

4. La-Protein Superfamily
• 250 Eukaryotic Species
• La Motif (LaM) + RNA Recognition Motif (RRM) = La Domain
• Highly conserved La domain or module
• La Related Proteins: (LARPs)
• LARP6

5. La-Related Protein Superfamily
LARP6 LARP7LARP4LARP1Genuine
La
• Highly conserved across eukaryotic species
• The function and the mode of RNA binding of the La module are still not
understood well

6. mRNA Metabolism
Binding of PolyA tail
La-Related Proteins
LARP6 LARP7LARP1 LARP4
Post Transcriptional
Regulation
Tumor Suppressant
3’ UUU tRNA binding
?
• Type I collagen mRNA
• Binds to 5’ stem loop at the untranslated region (UTR) of collagen mRNAs

7. RRM
Martino, L., Pennell, S., Kelly, G., Busi, B., Brown, P., Atkinson, R., Salisbury, N., Ooi, Z.,
See, K., Smerdon, S., Alfano, C., Bui, T., and Conte, M. (2015) Nucleic Acids Res. 1-16.
LaM
--COOHN-- LaM RRM LSA
70 300
RRM
LaM
Constructs used for
structure
determination
295
70 183
180

8. --COOHN-- LaM RRM LSA
70 300
RRM
LaM
Constructs used for
structure determination
295
70 183
180
74 313
300
70 295
313
Constructs assayed for RNA binding
and/or chaperone activity70
70
Construct Kd (DissociationConstant)
74-313 26 nM
70-300 48 nM
70-295 77 nM
70-313 ?

9. Goals
• I will be expressing HsLARP6 residues (70-300) and (70-313)
• This is important because it will lead to better understanding of the La Module
RNA binding activity
• My main goal is to identify the specificity of LARP6 binding to RNA ligands
• This will provide information regarding the regulation of gene expression through
the highly important proper RNA folding

10. How to accomplish this?
• Protein Expression
Overnight small culture  large expression culture + IPTG  grow for a while 
harvest cells  freeze
• Protein Purification
Lyse cells by sonication  spin down cell debris  Ni-NTA affinity chromatography
(elute with 300 mM imidazole)  Concentrate to small volume  size exclusion
chromatography  store in freezer

11. Electrophoretic Mobility Shift Assay
(EMSA)
• Rapid and sensitive method used for protein-nucleic acid interactions
• Based on the differential migration of RNA/protein complexes and free RNA during
native gel electrophoresis
• Complexes can be visualized by using a radiolabeled RNA probe
• Mobility of protein-nucleic acid
complex is less than that of the free
nucleic acid

12. Separating Free and Bound Ligand:
Native Gel Electrophoresis
KD
on
off
LP
LP
k
k



][
]][[
=
[P · L]
[L]total
• Fraction Bound
Separation of
[PL] and [L]
Detection of
labeled [L]total
P = Protein
L = Ligand
Koff = Off rate
Kon = On rate
•

13. Kd is dissociation constant
•
𝑃∗𝐿
𝐿 𝑇
=
𝑃 𝐹
Kd−𝑃 𝐹
• 𝑦 =
𝑥
𝐾𝑑−𝑥
• P*L = Protein bound to Ligand
• 𝐿 𝑇 = Total Ligand
• 𝑃𝐹 = Free Protein
• Kd = Dissociation Constant
𝑃𝐹
𝑃 ∗ 𝐿
𝐿 𝑇
Kd

14. Quantitative EMSA to measure RNA binding
Bound
Free
[HsLARP6]
Bind to Protein
Separate on GelBiotinylate
Detect with
Streptavidin
HRP
Transfer to
Membrane
and Crosslink
https://www.thermofisher.com/order/catalog/product/20160

15. SUMO-fusion setup
His10--- ----GGS----
70 300
SUMO
His10--- ----GGS----
70 313
SUMO
• SUMO  Small modifier that covalently bound to the protein to
provide protein stability.

16. Expression of His10-SUMO-LARP6 (70-300)

17. Purification of His10-SUMO-LARP6 (70-300)
• Nickel Affinity Purification Figure 1. Gel electrophoresis of affinity-purified
His-tagged protein
• 10% SDS-PAGE gel
• L=Ladder
• S=Supernatant
• P=Pellet
• FT=Flow Through
• W=Wash
• E=Elution
• Ended up with His10-SUMO protein only
• Molecular weight ~ 35 kDa
His10-SUMO

18. • Gel Filtration Purification F11 F12 F21F22F19 F24 F26L F13 F20 F23 F25
Figure 2. Gel electrophoresis of elution
fractions from size-exclusion column.
• 10% SDS-PAGE gel
• L=Ladder
• F=Fraction #
• Fractions # 21, 22, 23, and 24 were
concentrated
• Final protein concentration: 9 µM
• Sephadex S75 resin was used to separate the small molecular weight protein using the buffer 50 mM Tris,
pH 7.5, 100 mM NaCl, 1 mM DTT, 5% glycerol.
-2
2
6
10
14
18
22
26
0 20 40 60 80 100
AbsorbanceUnits(280nm)
Elution Volume (mL)
Void
His10-SUMO

19. Electrophoretic Mobility Shift Assay (EMSA)
• Rapid and sensitive method used for protein-nucleic acid interactions.
• Based on the differential migration of RNA/protein complexes and free RNA during native gel
electrophoresis.
• Complexes can be visualized by using a radiolabeled RNA probe.
• Mobility of protein-nucleic acid complex
is less than that of the free nucleic acid.
Thermo Scientific Pierce Protein:Nucleic Acid Interaction Technical Handbook (2010)

20. Nonspecific Binding of SUMO to RNA Ligands
His10-SUMO
HsCOL1a1
XmCOL1a1b
XmCOL2a1a
Figure 3. EMSAs measuring RNA binding by purified His10-SUMO
protein. Others in the laboratory express recombinant fish proteins with
a His10-SUMO tag at the N-terminus. To see if there was interaction
between the RNA ligands and the SUMO protein purified from the
attempted HsLARP6(70-300) purification, EMSAs were performed.
SUMO exhibits millimolar affinity for these RNAs, which is weak
enough to be considered nonspecific.

21. EMSA Results with full length LARP6
Free
[HsLARP6]
Bound
• Unfortunately, degradation of HsLARP6 from His10-SUMO occurred.

22. Conclusions
• SUMO-HsLARP6 (70-300) degraded as seen in affinity chromatography SDS-PAGE.
• Purification of His10-SUMO was successful, as judged by the correct molecular weight
of the His10-SUMO in both the gel and size exclusion column.
• His10-SUMO exhibits non-specific binding to some RNAs at higher concentrations.

23. Future Directions
• Evaluate His10-SUMO include evaluating buffer effects.
• Expose His10-SUMO to other RNA sequences.
• Expression & purification protocol of HsLARP6(70-300) and HsLARP6(70-313) need
optimization.
• Buffer optimization to prevent degradation of constructs.