Data Available Genomic sequences Corresponding mRNA transcripts SNP database

What we are searching for A/G discrepancy between gDNA & mRNA Discrepancy encodes nonsynonymous amino acid change Site is not SNP Possible self-complementarity within pre-mRNA

RNA Editing Dataflow System (REDS)

Stage 1: Finding Differences Scans the genome and mRNA for discrepancies

Stage 2: Filter SNPs Removes all validated SNPs from the search

Stage 3: Perform Foldback Searches results for self-complementary sequences

REDS Results

Does REDS work?

Validating REDS Returns all known sites possible within set parameters Exceptions: mRNA not in database used by REDS Largest foldback is less than 5 base pairs Finds self-complementarity present in known RNA secondary structures these results are just a few among 100s of possible self-complementarity sites

Shot of REDS with other foldback predictions

Using REDS Results: Ranked by Basepairing length Hit Chr. mRNA acc. # Reference sequence # Site position Continuous basepairing length 1 chr14 BC034554 NM_001085: serpin peptidase inhibitor, clade A, member 3) 94155441 90 2 chr1 AK127250 NM_000851: glutathione S-transferase M5) 110057883 50 3 CR627441 NM_001077700: mesoderm induction early response 1 isoform 67223383 32 4 chr20 BC027448 4071869 30 5 chr3 BC029869 NM_001248: ectonucleoside triphosphate diphosphohydrolase 40408547 6 AL832131 NM_017895: (Asp-Glu-Ala-Asp) box polypeptide 27 47289277 28 7 chr10 AK123885 NM_207372: domain containing 4B 82338403 26 8 chr2 BC045801 Homo sapiens hypothetical protein LOC554226, mRNA 132622172 9 chr9 AF098483 NM_021144: Homo sapiens transcriptional coactivator p52 mRNA NM_033222: SFRS1 interacting protein 1 isoform 2 15464194 25 10 AF098482 11 chr12 AK001499 NM_018164: hypothetical protein LOC55726 26980918 24 12 BC040032 15476021 23 13 AK074357 NM_021627: specific protease 2 186809754 14 chr21 AJ001866 NM_001001894: tetratricopeptide repeat domain 3 NM_003316: tetratricopeptide repeat domain 3 37389527 15 AK222568 NM_018101: cell division cycle associated 8 37940054 22 16 chr22 U06935 NM_003216: thyrotrophic embryonic factor 40113303 17 chr19 BX648389 NM_173481: hypothetical protein LOC126353 709603 18 709625 19 BX648547 NM_145858: crystallin, zeta-like 1 33916232 20 AK225162 NM_022895: hypothetical protein LOC64897 119926554 21 chr11 CR627226 NM_015065: exophilin 5 107917676 AK223179 NM_006675: tetraspanin 9 3261243 AK057985 NM_002972: binding factor 1 isoform a 49247963 BC094885 NM_014871: ubiquitin specific protease 52 54999370 AK122695 NM_001033: ribonucleoside-diphosphate reductase M1 chain 4116107 L41669 NM_152437: zinc finger protein 664 123062981 27 chr5 AK074318 NM_016340: domain-containing guanine nucleotide 130816647 chr7 X16323 NM_000601: hepatocyte growth factor isoform 1 NM_001010931: hepatocyte growth factor isoform 2 precursor NM_001010932: hepatocyte growth factor isoform 3 precursor NM_001010933: hepatocyte growth factor isoform 4 precursor NM_001010934: hepatocyte growth factor isoform 5 precursor 81197020

Where REDS & Mfold Agree

Hit 6: 28 base pairs AL832131 ((Asp-Glu-Ala-Asp) box polypeptide 27) mfold with 2500nt

Hit 20: 22 base pairs AK225162 (hypothetical protein LOC64897) mfold with 2500nt

Hit 21: 22 base pairs CR627226 (exophilin 5) mfold with 2500nt

Where REDS & MFold Conflict

AK074357 (specific protease 2) Hit 13: 23 base pairs AK074357 (specific protease 2) mfold with 2500nt

Hit 17 & 18: 22 bp BX648389 (hypothetical protein LOC126353) mfold with 2500nt

Reconciling the Discrepancies REDS predicts self-complementarity Mfold uses energy minimization calculations What would be an accurate way of determining the utility of the folding heuristic?

Hit 7: 26 base pairs - mfold with 2500nt AK123885 (domain containing 4B)

Hit 15: 22 base pairs AK222568 (cell division cycle associated 8) mfold with 2500nt

Hit 16: 22 base pairs U06935 (thyrotrophic embryonic factor) mfold with 2500nt

Experimental Validation Reverse transcription on mammalian brain RNA. PCR on cDNA and gDNA pairs. Amplicon purification and isolation for sequencing. Comparison of cDNA and gDNA electropherograms. Possible subcloning of amplicon.

Reverse Transcription Synthesizing a cDNA library to be used as a PCR template Editing is most upregulated in the brain single-stranded RNA is reverse transcribed into complementary DNA (cDNA) by using total cellular RNA or poly(A) RNA, a reverse transcriptase enzyme, a primer, dNTPs and an RNase inhibitor 32

Amplifying a DNA fragment via enzymatic replication PCR Amplifying a DNA fragment via enzymatic replication isolating and exponentially amplifying a fragment of DNA, via enzymatic replication, without using a living organism 33

Designing Primers for PCR Basic Criteria for Primers: Equal amount of all 4 nucleotides About 20-25 base pairs product should extend over 2+ exons BLAST sequence to check uniqueness Product should be 200-800 nucleotides long

Purifying and Isolating an Amplicon Gel Electrophoresis expected size? Change PCR parameters NO YES Phenol Chloroform Extraction to remove proteins Preparative Gel expected size? Product lost during purification NO YES Isolate desired bands for Agarose Gel Purification Gel Electrophoresis expected size? NO Isolated incorrect band Low product yield YES Determine concentration & Ready to be Sequenced 35

PCR Amplicons V0 PCR here

Preparative Gels 15 – 20 V0

Ready for Sequencing V0 15 - 20

Analyzing Sequences V0 cDNA

Validating/Confirming RNA Editing V0 gDNA

Further Characterization … By Sublconing What percentage of gene transcripts are edited? What effect does editing have on alternative splicing or translation? … By knockout studies What functional impact does the modification have in the organism?

Subcloning Moving our amplicon of interest into a destination vector isolating and exponentially amplifying a fragment of DNA, via enzymatic replication, without using a living organism 42

SNX subclones

Our Searches Lab Reference # Gene Name REDS Hit # Analysis Status 3 SNX n/a Subcloned – no further evaluation 4 FZD Subcloning 5 SMAP Amplified incorrect sequence 6 V0 Confirmed 5 sites; to be subcloned 7 UNK Ready for sequencing 8 SHDC To be amplified 9 PSIP 12 Sequenced – subclone? 10 SPP 13 Sequenced – no further evaluation 11 TSP 22 HYP 20 AGA 14 HY2P 17, 18 15 EXPH 21 16 PANK2 29 17 BP4 34 18 GTF2I 40 19 ABP4 50 ACA 51

Summary of Experimental Data

Conclusions 2 found by REDS without Stage 3 do not appear to be edited 16 predicted with Stage 3  1 with 5 sites confirmed to date, maybe more Demonstrates importance of strong foldback structure Experimental REDS data set sorted by longest continuous base pairing ranks known sites low other contributing factors that should be weighted more heavily in ranking Negative sequencing results are disappointing but will be useful in making REDS tool a more accurate predictor of targets