1. Data Available Genomic sequences Corresponding mRNA transcripts
SNP database

3. 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

4. RNA Editing Dataflow System (REDS)

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

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

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

8. REDS Results

9. Does REDS work?

17. 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

18. Shot of REDS with other foldback predictions

19. 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) 90 2
chr1
AK127250
NM_000851: glutathione S-transferase M5) 50 3
CR627441
NM_ : mesoderm induction early response 1 isoform 32 4
chr20
BC027448 30 5
chr3
BC029869
NM_001248: ectonucleoside triphosphate diphosphohydrolase 6
AL832131
NM_017895: (Asp-Glu-Ala-Asp) box polypeptide 27 28 7
chr10
AK123885
NM_207372: domain containing 4B 26 8
chr2
BC045801
Homo sapiens hypothetical protein LOC554226, mRNA 9
chr9
AF098483
NM_021144: Homo sapiens transcriptional coactivator p52 mRNA
NM_033222: SFRS1 interacting protein 1 isoform 2 25 10
AF098482 11
chr12
AK001499
NM_018164: hypothetical protein LOC55726 24 12
BC040032 23 13
AK074357
NM_021627: specific protease 2 14
chr21
AJ001866
NM_ : tetratricopeptide repeat domain 3
NM_003316: tetratricopeptide repeat domain 3 15
AK222568
NM_018101: cell division cycle associated 8 22 16
chr22
U06935
NM_003216: thyrotrophic embryonic factor 17
chr19
BX648389
NM_173481: hypothetical protein LOC126353
709603 18
709625 19
BX648547
NM_145858: crystallin, zeta-like 1 20
AK225162
NM_022895: hypothetical protein LOC64897 21
chr11
CR627226
NM_015065: exophilin 5
AK223179
NM_006675: tetraspanin 9
AK057985
NM_002972: binding factor 1 isoform a
BC094885
NM_014871: ubiquitin specific protease 52
AK122695
NM_001033: ribonucleoside-diphosphate reductase M1 chain
L41669
NM_152437: zinc finger protein 664 27
chr5
AK074318
NM_016340: domain-containing guanine nucleotide
chr7
X16323
NM_000601: hepatocyte growth factor isoform 1
NM_ : hepatocyte growth factor isoform 2 precursor
NM_ : hepatocyte growth factor isoform 3 precursor
NM_ : hepatocyte growth factor isoform 4 precursor
NM_ : hepatocyte growth factor isoform 5 precursor

20. Where REDS & Mfold Agree

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

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

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

24. Where REDS & MFold Conflict

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

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

27. 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?

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

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

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

31. 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.

32. 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

33. 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

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

35. 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

36. PCR Amplicons
V0 PCR here

37. Preparative Gels
15 – 20 V0

38. Ready for Sequencing V0

39. Analyzing Sequences
V0 cDNA

40. Validating/Confirming RNA Editing
V0 gDNA

41. 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?

42. 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

43. SNX subclones

44. 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

45. Summary of Experimental Data

46. 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