Developing RNA Select and Querying MBNL1 Binding Specificity
David Youngentob
Rotation Spring 2012 Berglund Lab
CUG repeat Toxic RNA competes with splice sites for MBNL binding
And other pre-mRNAs ...
MBNL1 prefers to bind YGCY RNAs
(Cass et al, 2011)
Basic model of MBNL binding
NGCN repeats
MBNL
preRNAs
+
+
+
YGCY
>1000 putative RNA-BPs
Roughly 50 human SR-family and hnRNP family proteins
~200 RRM, ~50 KH family proteins
~700 ZnF domain proteins
90% of 25,000 human genes are believed to be alternatively spliced
Axiomatic importance of RNA-BPs in transcriptional processing and translational regulation
Specificity vs Affinity
Affinity is a measure of how strongly a protein is bound to a given ligand
RNA-BP
RNA oligos
RNA oligos
Specificity vs Affinity
RNA-BP
RNA oligos
Specificity is the the ratio of ∆G of binding between two potential ligands
-Describes the protein's ability to "distinguish" between potential ligands
Describing the binding specificity of an RNA-BP
RNA-BP
RNA oligos
The highest binding substrate is not necessarily the only one that is physiologically relevant
Understanding the ability of an RNA-BP to recognize and bind a diverse spectrum of RNAs can only be achieved under certain [P] parameters
Target RNAs contain short sequence specific binding sites (K-mers)
RNA-BP
RNA oligos
Most RNA-BP - RNA binding interactions are between the protein and a short sequence specific binding site within the RNA
K-mers display varying amounts of degeneracy
Positional Weight Matrices
Method of modeling binding specificity
Need sequences and binding affinities
RNA Select
What is it?
Experimental technique to rapidly describe the binding specificity of RNA-BPs via high throughput sequencing
Basic procedure outline
1. Co purification RNA-oligos bound with RNA-BP
2. High Throughput sequencing
3. Analysis of reads to identify sequence specific binding sites, derive affinities and propose Positional Weight Matrices (PWMs)
How do we choose binding reaction paramaters ?
K-mers bind across a wide range of relevant affinities
affinity
What [P] is prudent for an experimental binding reaction ?
K-mer - def - the sequence specific binding site within a target RNA
RNA Select binding reaction parameters
We can achieve low error and relevant binding in regime Ia
Added benefit of physiologically relevant reaction conditions
Why not 1b?
Deriving specificity
Characterizing specificity from competitive binding
S is proportional to q in Regime Ia
By this logic, S can be defined from sequencing data.... 1-1 relationship between read and nucleic acid molecule in bound complex
L>>P
How it works
Adaptor-Adaptor!
Maintaining efficient sequencing
Preventing 3'-adaptor-5'-adaptor dimer
ssDNA
A
A
RT Primer "locks" free 3'-adaptor as dsDNA
5' RNA 3'
3'-adaptor
T4 RNA Ligase 1
Maintaining efficient sequencing
Preventing 3'-adaptor-5'-adaptor dimer
A
ssRNA
A
5' adaptor
X
Question: How do MBNL1 ZnF pairs influence binding specificity?
Specific Aim: Determine the binding specificity of MBNL1 and the role of its two ZnF pairs
Approach:
Use RNA Select to describe binding specificity for MBNL1; MBNL1-ZnF-1,2 ; MBNL1-ZnF-3,4
Question: How do MBNL1 ZnF pairs influence binding specificity?
Specific Aim: Determine the binding specificity of MBNL1 and the role of its two ZnF pairs
Approach:
Use RNA Select to describe binding specificity for MBNL1; MBNL1-ZnF-1,2 ; MBNL1-ZnF-3,4
Question: How do MBNL1 ZnF pairs influence binding specificity?
Specific Aim: Determine the binding specificity of MBNL1 and the role of its two ZnF pairs
Approach:
Use RNA Select to describe binding specificity for MBNL1; MBNL1-ZnF-1,2 ; MBNL1-ZnF-3,4
Truncated MBNL is used for study
Truncated version of 382aa full sequence
Amino Acids 2-260
Contains all four ZnFs
Sufficient for splicing regulation (Grammatikakis et al, 2010)
MBNL1
Bench Work outline
Purify MBNL1, ZnF-1,2, ZnF-3,4 (yBBP, FOX-1 controls)
Perform RNA-Select
Purifying MBNL1, ZnF-1,2, -3,4
AKTA with GSTrap and His-Trap
GST
His
Avi
Prescission Protease site
MCS
pGEX-6P-avi.1
T7
PCR of Inserts
MBNL1
MBNL1
ZnF1
ZnF2
ZnF3
ZnF4
PCR
MBNL 1
PCR of Inserts
ZnF 1,2
MBNL1
ZnF1
ZnF2
ZnF3
ZnF4
PCR
MBNL 1
PCR of Inserts
ZnF 3,4
MBNL1
ZnF1
ZnF2
ZnF3
ZnF4
PCR
1 2
3 4
MBNL 1
PCR Results
1 2
3 4
MBNL 1
~800bp
~350bp
Speed cloning
Fast reactions; no cleanup allowed DNA mass calculations for ligation/transformation
Digestion (EcorI & XhoI)->
DePhosphatase of Vector->
Heat inactivation->
Ligation ->
Transformation
1 hour
30mins
20mins
20 mins
Overnight
________
~3 hours of one afternoon
Ready the next day
Successful cloning
MBNL1
ZnF-1,2
ZnF 3,4
We have these in freezer stock if anyone has a use for them !
Succesful purification !
MBNL1
80
30
25
GST
MBNL
Uncleaved
Plan for the next 7 weeks
FOX1-pGEX cloning
Protein purification --(size fractionation)
Test ligations for sRNA libraries
Perform RNA SELECT !
Thanks!
Andy
Rodger
Stacy
Amy
Ruth
Leslie
Elaine
Questions?
Advice?