1. The document discusses natural antisense transcripts (NATs) in the protozoan parasite Entamoeba histolytica, which has a compact genome. 2. Strand-specific RNA-seq reveals pervasive NAT transcription, with over 60% of coding genes partially covered by NATs. NAT transcription initiation and polyadenylation sites are correlated with genomic motifs. 3. Simulations show that positional constraints on NAT transcription are encoded in the genome, with NAT transcription initiation preferentially occurring at mRNA stop codons and NAT polyadenylation preferentially near mRNA start codons. Codon usage bias and enrichment of polyadenylation signals on the antisense strand also influence NAT expression.

2. my desk is my bed

3. my desk is my bed

4. my desk is my bed

5. Human’s innovation......
in a crowded city,
where space is limited.

6. Nature’s innovations......
in a compact genome,
where space is also limited.

7. A Tale of Two Strands
sharing of transcriptional motifs between
the two strands of a compact genome
Paris, France

8. Entamoeba histolytica
a protozoan parasite

9. It has a compact genome
genome size
coding genes
A+T content
20Mb
9000
75%
coding region
60%
retrotransposon
25%

10. It has a compact genome
genome size
coding genes
A+T content
20Mb
9000
75%
intron frequency
0.2
per gene
intergenic distance
150bp
5’UTR and 3’UTR
15bp

11. codon restricted evolution on both strands
in a compact genome
real estate is limited

12. codon restricted evolution on both strands
Natural
Antisense
Transcripts
?
?
[ NAT ]
end
motifs
start
motifs

13. 1
2
4
3

14. 1
RNA-Seq
4
NAT
Locations
2
3

15. 2
1
RNA-Seq
4
NAT
Locations
Motif
Identification
3

16. 2
1
RNA-Seq
4
NAT
Locations
Motif
Identification
Genomic
Determinants
Simulations
3

17. 2
1
RNA-Seq
NAT
Locations
Motif
Identification
Implications
Genomic
Determinants
Simulations
4
3

18. 1
RNA-Seq
strand-specific
RNA-Seq
transcription start
site mapping
poly(A) site mapping

19. 1
RNA-Seq
strand-specific
RNA-Seq
transcription start
site mapping
poly(A) site mapping

20. Pervasive NAT transcription
Partially
covered
by NAT
~61%
coding genes are
covered by NAT
47%
Not
covered
by NAT
39%
14%
Fully covered
by NAT

21. NATs at 3’end of genes
Partially
covered
by NAT
47%
5’biased
unbiased
3’biased

22. NATs at 3’end of genes
+ve strand
RNA-Seq
log 2 coverage
EHI_016120
-ve strand
RNA-Seq
log 2 coverage
NAT
EHI_016130
NAT
NAT
EHI_016140

23. 1
RNA-Seq
Artifact-corrected
strand-specific
RNA-Seq
transcription start
site mapping
poly(A) site mapping

24. n=4186
NAT TSS
Hotspot around stop codon
mRNA TSS
Hotspot around -15nt of start codon
n=1991

25. Stop
codon
NAT TSS
Hotspot at 1st base of stop codon

26. mRNA TSS

27. mRNA TSS
Stop
codon
Reverse
Complement
stop codon

28. Stop codon resembles an TSS initiator motif"
on the antisense strand
mRNA TSS
Sequence around
mRNA TSS
n=4186
NAT TSS ???
Reverse complement
of sequence around
stop codon
n=7312
reverse
complement
stop codon

29. Stop codon resembles an TSS initiator motif"
on the antisense strand
Sequence around
mRNA TSS
n=4186
Reverse complement
of sequence around
stop codon
n=7312
initiator motif

30. 1
RNA-Seq
Artifact-corrected
strand-specific
RNA-Seq
transcription start
site mapping
poly(A) site mapping

31. n=7312
mRNA poly(A) site
at around +20nt of stop codon

32. NAT poly(A) site
most poly(A) before ~500bp
and decreases gradually along CDS
n=3128

33. NAT poly(A) site
Hotspot at around -20nt of start codon
n=7312

34. mRNA
TSS
?????
n=7312

35. 2
1
RNA-Seq
4
NAT
Locations
Motif
Identification
3

36. 2
Motif
Identification
poly(A) motifs
TSS motifs
Correlations

37. 2
Motif
Identification
poly(A) motifs
TSS motifs
correlations

38. 1
2
Poly(A) Signal Seq
(PAS), p<0.00001
3
Cleavage
Downstream Seq Element
Sequence
(T-rich DSE) , p<0.00001
Element
(CSE)
mRNA
Poly(A) site
n=4603
AAAAAA

39. 1
2
3
Cleavage
Downstream Seq Element
Sequence
(T-rich DSE) , p<0.00001
Element
(CSE)
Poly(A) Signal Seq
(PAS), p<0.00001
mRNA
Poly(A) site
n=4603
1
2
3
NAT
Poly(A) site
n=1991

40. 3
Downstream Seq Element
(T-rich DSE) , p<0.00001
mRNA
Poly(A) site
n=4603
3
NAT
Poly(A) site
n=1991

41. 2
Motif
Identification
Poly(A) Motifs
TSS Motifs
Correlations

42. 1
A-rich region
(A-Box), p<0.00001
2
Upstream promoter core
(core), p<0.00001
Initiator
Motif
(Inr)
3
mRNA
TSS
n=3865

43. 1
A-rich region
(A-Box), p<0.00001
2
Upstream promoter core
(core), p<0.00001
Initiator
Motif
(Inr)
3
mRNA
TSS
n=3865
1
2
3
NAT
TSS
n=1991

44. Positional
Constrains
of NAT are encoded in
the genome or not?

45. 2
Motif
Identification
Poly(A) motifs
TSS motifs
Correlations

46. NAT TSS
Reverse
Complement
stop codon

47. mRNA
Downstream seq element
(T-rich DSE) , p<0.00001
Poly(A) site
motif
NAT
Promoter
motif
A-rich Box
(A-Box), p<0.00001

48. NAT poly(A) site
Hotspot at around -20nt of start codon
n=7312

49. A-rich Box
(A-Box), p<0.00001
mRNA
Promoter
motif
NAT
Downstream seq element
(T-rich DSE) , p<0.00001
Poly(A) site
motif

50. mRNA
TSS
NAT
Poly(A)
mRNA
Poly(A)
NAT
TSS

51. NAT poly(A) site
Peak at ~500bp and decrease gradually
n=3128

52. n=3128
Observed poly(A) sites are
~5 times more frequent in the
Antisense Strand
n=3128

53. 2
1
RNA-Seq
4
NAT
Locations
Motif
Identification
Genomic
Determinants
Simulations
3

54. Rationale
Results
Simulations
3

55. Rationale
Results
Simulations
3

56. Defining the positional weighting matrix
1
A-rich Box
(A-Box), p<0.00001
2
Upstream promoter core
(core), p<0.00001
Initiator
Motif
(Inr)
3
mRNA
TSS
n=3865

57. Defining the positional weighting matrix
Weight factor for positions of
3 motifs

58. Calculate the TSScore
Individual motif score
= positional weighting * log (MAST p-value)

59. Calculate the TSScore
TSScore = A-Box PW * log ( A-Box MAST p-value )
+ core PW * log ( core MAST p-value )
+ Inr PW * log ( Inr MAST p-value )

60. Rationale
Results
Simulations
3

61. RNA-Seq
Observed mRNA TSS distribution
TSScore
Simulated mRNA TSS distribution

62. RNA-Seq
Observed NAT TSS distribution
TSScore
Simulated NAT TSS distribution

63. Constrains for
NAT transcription initiation is
encoded in genome

64. How about
Poly(A) site?

65. Simulation
PAScore

66. RNA-Seq
Observed poly(A) site
PAScore
Simulated poly(A) site

67. PAScore
Simulated poly(A) site
Codon constrains
on sense strand
More poly(A) motifs
on antisense strand ?

68. Codon usage bias

69. Codon bias towards 3rd position A codons

70. Codon bias towards 3rd position A codons

71. Codon bias towards 3rd position A codons

72. Enrichment of T-rich motif on antisense strand
Codon bias towards NNA
Enrichment of T-rich motifs

73. Enrichment of T-rich motif on antisense strand
Antisense vs Sense strand
Enriched in
Antisense strand
Enriched in
Sense strand

74. Enrichment of T-rich motif on antisense strand
Observed poly(A) sites are
~5 times more frequent in the
antisense strand

75. T-rich motifs are depleted on long NAT
T-rich motifs
depleted?
Long NAT
Short NAT

76. T-rich motifs are depleted on long NAT
Short vs Long NAT
Enriched in
Short NAT
Enriched in
Long NAT

77. T-rich motifs are depleted on long NAT
T-rich motifs "
depleted
T-rich motifs
enriched
Long NAT
Short NAT

78. 2
1
RNA-Seq
NAT
Locations
Motif
Identification
Implications
Genomic
Determinants
Simulations
4
3

79. mRNA with short 3’UTR
average 15nt
AAAAAA
stop codon
T-rich DSE
Initiator motif
A-box
NAT
pervasive
transcription at 3’end

80. Codon bias towards NNA
Enrichment of T-rich
DSE for poly(A)
AAAAAA
NAT transcription is limited to 3’end by
enriched poly(A) site motifs

81. Natural selection for long NAT?
Reduced codon bias towards NNA
Depletion of T-rich DSE for poly(A)
NAT transcription is lengthen
and possibly functional

82. neighbor
mRNA
mRNA
Promoter motifs
A-box
Partition ??
T-rich DSE
AAAAAA
NAT poly(A) site hotspot.
Partition between genes?

83. A Tale of Two Strands
sharing of transcriptional motifs between
the two strands of a compact genome
Paris, France