Transcript The Importance of the Linker Between Zinc Finger Sets in MBNL

The Importance of the Linker
Between Zinc Finger Sets in MBNL
KIMBERLY JONES
AUGUST 20, 2010
SPUR PROGRAM 2010
UNIVERSITY OF OREGON
BERGLUND LAB
INSTITUTE OF MOLECULAR BIOLOGY
P.I.-ANDY BERGLUND, PHD
MENTORS-AMY MAHADY, MS
& DANIELLE CASS, PHD
Berglund Lab Studies Myotonic Dystrophy
 Most common adult onset form of muscular
dystrophy
 Effects 1 in ~8000 people

Symptoms include:
Myopathy
 Myotonia
 Cataracts
 Cardiac Arrhythmia
 Etc.

Myotonic Dystrophy Foundation, 2010
Genetic Basis of Myotonic Dystrophy
DMPK gene
3’ UTR
(CTG)n
Transcription
DMPK pre-mRNA
Warf, 2009
Autosomal dominant genetic disorder caused
by a CTG repeat in the 3’-UTR of the DMPK
gene.
This leads to a toxic, hairpin, CUG-repeat
pre-mRNA which sequesters
muscleblind-like (MBNL), a known regulator
of alternative splicing.
This is what likely gives rise to the
characteristic symptoms.
Ex.: Insulin Resistance due to insulin
receptor misplicing
Myotonia due to chloride ion channel
misplicing
Muscleblind-like
MBNL and Mbl Structure
MBNL
MBNL (Muscleblind-like) consists of 4 zinc fingers in 2 domains
(ZnF1/2 and ZnF3/4) with a long, central linker between zinc finger
domains.
Mbl
Mbl (Muscleblind) is an MBNL orthologue in Droshophila (fruitfly).
It consists of 2 zinc fingers in 1 domain.
Zinc Fingers Comprise the Binding Regions
of Proteins in the Muscleblind Family
o Binding is facilitated by base
stacking and hydrogen
bonding interactions.
o These proteins show YGCY
RNA motif preference, where Y
is a pyrimidine (either U or C).
o Zinc Finger consists of 3 cysteines and
1 histidine coordinated around a zinc ion.
o This holds the protein in a folded position
to allow for proper RNA binding.
o Two zinc fingers make up a domain, as
ZnF1/2 and ZnF3/4.
Teplova M, Patel DJ. Figures 1 and 3. Diagrams.
Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1.
Nature Structural & Molecular Biology. Dec. 2008; 15(12) p.1344
Zinc Fingers are Highly Conserved
ZnF1
ZnF3
ZnF2
ZnF4
Teplova M, Patel DJ. Figure 1. Diagrams.
Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1.
Nature Structural & Molecular Biology. Dec. 2008; 15(12) p.1344
Not Much is Known about MBNL’s Central
Linker
MBNL
114 amino acids
• Contains segments of conserved residues
• Not known to be structured
• Not known whether it contributes to RNA binding
Voelker, 2010
Conformation of MBNL/Mbl when
Binding RNA
The current model assumes that MBNL folds
using its central linker anti-parallel to itself to
bind RNAs that contain short U spacer segments
between GC binding regions. This flexibility would
also allow it to bind to RNAs with longer U spacer
segments.
Since Mbl only has one binding domain and no
flexible linker region, it binds only to RNAs with
longer U spacer regions between GC binding regions.
MBNL/Mbl Binding to Specific RNAs was Tested
 Previously the following RNAs were tested with
MBNL and Mbl:
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RNA 1: UUUUUUUUUGCUGCUUUUUUUUU
RNA 11: UUUUGCUUUUUUUUUUUGCUUUU
RNA 2GG: UUUGGUUUUUUUUUUUUUGGUUU
Binding of MBNL and Mbl
MBNL
Mbl
MBNL and Mbl both bind RNA 11.
 MBNL binds RNA 1 very tightly,
while Mbl binds it very loosely to
not at all. However more data is
needed for Mbl since the error is
larger than the Kd.
 Both proteins show no binding to
RNA 2GG, as expected for a
negative control.

Cass, 2010
Experimental Question
 If Mbl is doubled, will the resulting construct show
similar binding behavior to MBNL, or will it require
a longer central linker?

More specifically, will the doubled Mbl construct be able to
bind RNAs that contain short U spacers between GC binding
regions with or without the longer central linker?
Experimental Approach
 Make and purify 2 protein constructs using Mbl:
 2xMbl: Double Mbl
 2xMbl-HL: Double Mbl plus a portion of human MBNL central
linker
 Run binding gel shift assays using 3 different RNAs
 RNA 1: UUUUUUUUUGCUGCUUUUUUUUU
 RNA 11: UUUUGCUUUUUUUUUUUGCUUUU
 RNA 2GG: UUUGGUUUUUUUUUUUUUGGUUU
 Calculate the Kds of each protein for each RNA
Protein and RNA Constructs
Linker taken from center of
human MBNL central linker.
Proteins:
2xMbl
52 amino acids
2xMbl-HL
104 amino acids
RNAs:
RNA 1:
U9GCUGCU9
Single U spacer between GC binding sites
RNA 11: U4GCU11GCU4
Eleven U’s spacer between GC binding sites
RNA 2GG: U3GGU13GGU3
GC changed to GG for negative control
Purified Proteins
kDa
MBNL ~28.4kDa
35
Mbl ~11.9kDa
25
2xMbl ~24kDa
Contamination?
15
10
2xMbl-HL~29.2kDa
Predictions of Binding Behavior
 Since 2xMbl has double the number of zinc fingers
(binding sites) as Mbl, it should show stronger binding
than Mbl to both RNA 11 and 1. However, it should show
less binding than MBNL.
 2xMbl-HL contains double the zinc fingers like 2xMbl
but also contains a longer central linker, thus it should
bind stronger than 2xMbl and more similarly to MBNL
for both RNA 11 and 1.
 Note: This assumes that ZnF1/2 acts similarly to ZnF3/4.
Results: Binding Gel Shifts
2xMbl
1: 0.2nM
µM
11: 0.2nM
µM
2GG: 0.2nM
µM
Protein:RNA
Complex
Free RNA
2xMbl-HL
1: 0.3nM
µM
11: 0.3nM
µM
2GG: 0.3nM
µM
Protein:RNA
Complex
Free RNA
RNA 1:
U9GCUGCU9
RNA 11: U4GCU10GCU4
RNA 2GG: U3GGU13GGU3
Protein Concentration: 0-1.2µM
Deriving the Dissociation Constant (Kd)
Kd equation
A dissociation constant (Kd) is a
measure of equilibrium of how
likely a complex is to dissociate
into separate parts.
This tells us how tightly the
protein binds to the RNA.
The lower the Kd, the tighter the
protein binds to the RNA and
vice versa.
Results: Dissociation Constants (Kds)
MBNL
Mbl
2xMbl
2xMbl-HL
2xMbl-HL shows tighter binding
to RNA 11 than MBNL, while
2xMbl binds weaker. But more
data is needed to confirm its
results due to the error being
larger than the actual Kd.
 2xMbl-HL shows similar binding
to RNA 1 as MBNL.
 2xMbl binds more strongly to
RNA1 than Mbl; however more
data is needed to confirm results
due to the error being larger than
the actual Kd for Mbl.
 All proteins showed no binding to
RNA 2GG, as expected for a
negative control.

Conclusions
 Both 2xMbl and 2xMbl-HL bind RNA 11 and RNA 1.
o More data is needed for 2xMbl on RNA 11 due to larger error than
Kd. This is also the case for Mbl on RNA 1.
 The data show that the 52 amino acid linker in 2xMbl
is sufficient for binding to short U spacer RNA 1.
 The central linker seems to play an important role,
with increased length giving increased binding
affinity.
 The tighter binding of 2xMbl-HL to both RNAs
seems to support the binding model for MBNL
described already.
 ZnF1/2 appears to act similarly to ZnF3/4.
Future Directions/Current Work
2xMbl
Work with other RNAs to further determine
sequence specificity of binding sites in 2xMbl and
2xMbl-HL.
2xMbl-HL
 Mutate MBNL portion of central linker in 2xMbl-HL,
specifically the most conserved residues to determine
if the sequence of the linker is important for
increasing the binding affinity.
Acknowledgements
 Berglund Lab
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Andy Berglund, PhD-P.I.
Amy Mahady, MS-Mentor
Danielle Cass, PhD-Mentor
Paul Barber-Former Mentor
Jamie
Rodger
Julia
Leslie
Devika
Julien
Elaine
Alex
Brandi
Phil
 SPUR Program & the
University of Oregon

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Peter O’Day
Blakely Strand
Questions?
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MBNL Linker Portion in 2xMbl-HL
Portion of MBNL central linker in 2xMbl-HL
The Central Linker is only Partly Conserved
Data for Kd Results
2xMBL
RNA
NV11
NV1
1GC
2GG
2GC
NVC
NVA4
2GGCG
Kd 1
Kd 2
34.147
17.554
19.596
NB
Kd 3
41.503
21.517
138.94
NB
115.27
NQ
NQ
NB
NB
57.209
NQ
NQ
NB
NQ
NQ
NB
MBL
RNA
NV11
NV1
1GC
2GG
2GC
NVC
NVA4
2GGCG
2xAGCA
2xCGCC
Kd 1
Kd 2
2.1551
18.617
1253.1
NB
Kd 3
43.902
160.88
397.04
NB
1.0307
106.75
36.757
621.25
9.0358
0.46269
NB
66.119
2.0344
2xMBL-L
Relative to
Avg
Std Dev
2GC
RNA
122.96 66.20333333 49.29013179 1.537934537 NV11
49.183
29.418 17.23130178
0.53764543 NV1
756.41
304.982 395.4758867
12.339509 1GC
NB
NB
NB
2GG
62.719 78.39933333 32.04956428
1 2GC
NQ
NQ
NQ
NVC
NQ
NQ
NQ
NVA4
NB
NB
NB
2GGCG
2xAGCA
2xCGCC
MBNL
Relative to
Avg
Std Dev
2Gc
RNA
23.02855 29.51951608 22.34263122 NV11
89.7485 100.595132 87.07528864 NV1
825.07 605.3258311 800.4948094 1GC
NB
NB
NB
2GG
1.0307
#DIV/0!
1 2GC
106.75
#DIV/0!
103.5703891 NVC
36.757
#DIV/0!
35.66217134 NVA4
621.25
#DIV/0!
602.7457068 2GGCG
37.5774 40.36391781 36.45813525 2xAGCA
1.248545 1.111366799
1.21135636 2xCGCC
Kd 1
Kd 2
1.5163
0.73774
0.076092
NB
Kd 3
1.0675
0.070082
0.31891
NB
4.9456
150.75
272.33
628.17
38.794
17.898
Kd 1
NB
Kd 2
NB
28.145
62.975
148.3
458.92
Relative to
Avg
Std Dev 2Gc
1.708966667
0.8 0.086260149
19.085
5.8674555
8.9 0.296160011
73.105 23.3512505
34.4 1.178655143
NB
NB
NB
39.047
19.811775
16.6
1
96.17533333
47.6 4.854453139
197.67
65.8 9.977399804
966.8633333
738.1 48.80245881
0
0
Kd 3
Kd 4
Avg
NB
NB
NB
0.80705
0.35456
NB
1.538
10.246
35.291
23.124
2.8387
2.5431
3.577
19.905
7.1095
74.801
172.38
1813.5
319.23
32.286
0.1339
0.176
0.52409
Kd 4
1.6129
20.479
1.8266
3.7542
0.32355
Relative to
2GC
#DIV/0!
#DIV/0!
#DIV/0!
NB
#DIV/0!
#DIV/0!
#DIV/0!
#DIV/0!
#DIV/0!
#DIV/0!