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Emelia SoddersBerglund Lab17 August 2012MBNL1 interaction with modified CUG/CCUG repeat RNA1Myotonic Dystrophy Inherited type of muscular dystrophy

Disease Symptoms:Muscular WeaknessMyotoniaInsulin insensitivityCardiac arrhythmiaCataracts

Myotonic dystrophy is a genetic disorder that affects the muscles and other body systems. Patients with the disease have a hard time relaxing their muscles after they have been contracted. Myotonic dystrophy is one of the most common forms of inherited muscle disease; it is estimated that one person in every 20,000 is affected with DM. Some other symptoms include muscular weakness, insulin insensitivity, and cardiac arrhythmia 2Myotonic Dystrophy is a disease caused by expanded repeatsZNF9(CCTG)nDM1: expansion of CTG repeats in the 3 UTR of the DMPK geneUnaffected: N=5-50 CUG repeatsAffected: n= 50-4000 CUG repeats

DM2: expansion of CCTG repeats in the first intron of ZNF9 geneUnaffected: n=7-24 CCUG repeatsAffected: n=75-11,000 CCUG repeatsDMPK(CTG)nThere are two subtypes of Myotonic dystrophy. DM type 1 (DM1) is caused by the expansion of a CTG repeat in the 3 UTR of the DMPK (dystrophia myotonica protein kinase) gene. DM type 2 (DM2) is caused by an expansion of CCTG repeats in the first intron of ZNF9 (zinc finger 9) gene which is what I am focusing on. Myotonic Dystrophy (DM) is caused by expression of expanded repeat mutation in the non coding region of the DMPK gene (DM1) or the ZNF9 gene (DM2) The mutation results in the expression of long CUG (DM1) or CCUG (DM2) repeat RNAs

3MBNL1 sequestered in CUG/CCUG repeat RNAMBNL1: Muscleblind-like 1 proteins

MBNL1 proteins are expressed at high levels in skeletal muscles and is known to be directly involved in causing Myotonic Dystrophy ZNF9MBNL

UCU GCCUGCCUGCCUGGUCCGUCCGUCCWhen MBNL is sequesterd in CCUG repeat RNA, it results in misregulated, alternative splicing of several pre-mRNAs. So when MBNL is bound to ccug repeat RNA, it results in splicing defects which leads to disease symptoms beacause the CCUG repeats can no longer access their target transcripts which leads to misregulation of alternative splicing

We have data that shows that MBNL is sequestered to CCUG repeat RNA4MBNL1 interacts with the Watson-Crick side of YGCY motifsMBNL1 binds a YGCY consensus sequenceThis crystal structure shows it interacts with the Watson-Crick side of the YGCY motifsThis structure contains minimal RNA binding domain and a short RNA oligonucleotide

5353CG UUCCGGUTeplova et al 200853We can see that MBNL binds to GCU nucleotides but the watson crick sides of those nucleotides so when we se gcu gcc gc anything we know that mbnl binds to that5Model: MBNL1 binds relaxed CUG/CCUG repeats between RNA strandsCUG/ CCUG repeats breathe relaxing their helices

MBNL proteins bound to CUG/CCUG repeats can no longer access their target transcriptsSo my hypothesis is that MBNL1 binds relaxed CCUG repeats between RNA strands. So when CUG/CCUG repeats are breathing and relaxing their helix that is when MBNL binds resulting in misregulation of alternative splicing. And so again using a modified base will push the equilibrium to the secondary helix and prevent MBNL from binding. CUG/ CCUG repeats cause DM by forming stem loops that bind and sequester MBNL proteins into nuclear foci

The secondary helix: hydrogen bonding between the base pairs and MBNL is coming in between the structure when the helix is breathing

6Goal: To determine how the addition of structure stabilizing modifications affects MBNL1s ability to bind CUG/CCUG repeat RNA Determine if MBNL1 has a reduced affinity for RNA with a stabilized structure

Use a modified base to stabilize the secondary structure

CCUG is not as stable as CUG repeat RNA in the sense that CUG has one mismatch two base pairs etc, and CCUG has two mismatches, two base pairs, etc. But CCUG will still form a heliz because of the UUCG cap on tome for correct base pair making.

7Pseudouridine () stabilizes RNA secondary structure Pseudouridine is an isomerization product of uridine

The extra NH group adds another H-bond donor

Pseudouridine has been shown to stabilize secondary structure of RNA

So pseudouridine is a modified base that will be incorporated to help stabilize the secondary structure of CCUG repeat RNA. It is a modiied form of uridine. Pseuoridine stabilized single and double strranded RNA by forming hydrogen bonds with a water molecule that also interacts with the sugar phosphate backbone. Pseudouridine is found in many structured RNA: tRNA, spliceosomal RNA (smRNA), rRNA

8Hypothesis: Addition of Pseudouridine will inhibit MBNL1 bindingReduce MBNL1s ability to bind CCUG repeat RNAZNF9

U GCCUGCUGCCGGCCGUCGUCCUC

We dont specifically know where pseudouridine is incorporated9In vitro Transcription can be used to generate RNA with modificationsTranscription: process that involves the transcribing of genetic information from DNA to RNAT7 Transcription is the simple in vitro assay that I can use to incorporate Pseudouridine into CUG/CCUG repeats 35Noncoding strand (Template)T7 RNA PolymeraseA portion of the DNA is transcribed to produce a complementary strand of RNA.During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in an RNA complement that includes uracil (U) in all instances where thymine (T) would have occurred in a DNA complement.

T7 is a single molecule that does the polymerase job10T7 Transcription ReactionTo start, we tried out transcribing an RNA with 6 CUG repeats and an RNA with 6 CCUG repeats

CCUG6+CCUG6-CUG6+CUG6-

First, I had trouble just getting CCUG repeat RNA. I had problems with the RNA degrading, obtaining DNA instead of RNA. And after following a completely different protocol and adding Rnasin to prevent the RNA from degrading I had results only for CUG6. So I knew my transcription Reaction was working, I just wasnt getting any results for CCUG6 which is what I am focusing on. I tested to see if CUG was DNA or RNA by adding Dnase to the transcription reactions, and it was indeed I was getting RNA for CUG. I tried multiple times with the same t7 transcription reaction and I wasnt getting any results so I listed out the things I could change in my transcription reaction.11Changing the ConditionsDifferent CCUG6 template and coding strandNucleotide ConcentrationVarying template concentration/ different amounts of template in reactionTime length Varying Magnesium buffer12Different CCUG6 Template and Coding StrandExpected size:CCUG: 46 nucleotidesCUG: 58 nucleotides

Changing the template strand to optimize the reaction made it successful

CUG6+CUG6-CCUG6+CCUG6-100 bp

I repeated the same protocol I was before but I used a different template and coding strand. 13

Radioactive CTPWe did the identical reaction with radioactive nucleotides so the RNA products could be used in Gel shift experiments

We did not run a radioactive ladder so we did not know the size of the bands

CUG6-CUG6+CCUG6-CCUG6+How to present radioactive CTP??? label my nucleotides with a p3214Gel Shift Assay

CCUG6CUG6CUG4I wanted to test MBNL1s binding ability to the RNA I transcribed using a gel shift assay Tested this to see if it bound to MBNLWe did a binding assay because I thought I was getting RNA with the last reaction, however if you can see there is very little binding with MBNL and CCUG6CUG4- purchased RNA synthesizedKnow full length RNA- know it has MBNL binding abilitiesFrom left to right: increasing protein conentration Shifted: because when protein and rna bind together they form a complex which is bigger than rna itself so it migrates more slowly 15Changing the Nucleotide conditions

I used CUG4 instead of a ladder to make sure CCUG6 was migrating more slowly than CUG4

Changed the nucleotide concentrations from 0.5 mM to 0.1 mMCUG6-CUG6+CCUG6-CCUG6+CUG4So then I changed the conditions and converted the nucletides to 5 mM wheras before they were 10 mM. I added 1 ul of each nucletde instead of 2.5 ul and an extra 2.5 ul of hot CTP. I also added CUG4 into my denaturing gel as a control and I got goood results

CUG4 instead of a ladder to make sure CCUG6 were migrated more slowly than cug 4

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T7 CCUG6/CUG6 Transcription Reaction with Radioactive CTP and Pseudouridine I did the same T7 transcription reaction using Pseudouridine modified nucleotidesCUG6+CUG6-CCUG6-CCUG6+0%50%100%Because the reaction was working, we did the transcription using pseudo modified nucleoties put pseudo symbol after percent

17Summary/ConclusionsSpent a lot of time and effort optimizing and transcribing CCUG repeats with Pseudouridine

Goal: To determine how the addition of structure stabilizing modifications affects MBNL1s ability to bind CCUG/CUG repeat RNA

18Future DirectionsGel Shift Assay with modified CCUG6 and CUG6 Repeat RNA to test MBNL1s binding abilityDetermine the stability of repeats with and without pseudouridine using thermal melt assays

19Acknowledgments Andy Berglund (Principle Investigator)Elaine DeLorimier (Mentor)

Leslie CoonrodStacey WagnerDylan FarnsworthJulia OddoRuth SiboniRodger WoelkerDave Youngentob

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