ppr technology...how to use ppr technology ppr repeat sequence-specific binding module effector...

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PPR technology

1

4ii

PPR motif (35 aa)v t Y n t l I s g l c k a G r l e e A l e l f e e M k e k - G i a P d v1 4 ii

- A - G - U - C - A - C - U - G - A - G - A -

PPR protein

RNA

Base

Human mitochondrial RNA pol.Ringel et al., Nature (2011)

FTN VNS VTD FPD(1,4,ii)

Rec. base

[Base recognition code]

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Nu

ER

mRNA

Protein

???

RNA virus

mitochondria

DNAncRNA

Pentatricopeptide repeat


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3. Genome editing (since 2010)• ゲノムを自在に書き換える。

Science Breakthrough of the year 2013, 2014, 2015

Break-through technologies for the cell engineering

NGS

G.E.AI

1K

1M

1G

1T

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1985 1990 1995 2000 2005 2010

←Human Genome (3 G bp)

Manual seq.

ABI377

ABI3700

ABI3730

GS20(454)

GAI(Solexa)

SOLiD

SOLiD3

Heliscope

GAII(Solexa)

FLX Titanium

PacBio

1. 大規模配列解析技術(since 2004)ヒトゲノム : 1日で解読

• 多様な生物のゲノム情報を理解• 病気などの異常によるゲノム情報変化を理解

AlphaGo by Google DeepMind

2. 人工知能 (since 2015)• ゲノム情報改変の理論を理解• 遺伝子回路の構築原理を理解


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AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGTAATACGACTCACTATAGGGCAAGCTTAAAAAGCCTTCCATTTTCTATTTTGATTTGTAGAAAACTAGTGTGCTTGGGAGTCCCTGATGATTAAATAAACCAAGATTTTACCATGACTGCAATTTTAGAGAGACGCGAAAGCGAAAGCCTATGGGGTCGCTTCTGTAACTGGATAACTAGCACTGAAAACCGTCTTTACATTGGATGGTTTGGTGTTTTGATGATCCCTACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCGACTCTAGAATTCCAACTGAGCGCCGGTCGCTACCATTACCAACTTGTCTGGTGTCAAAAATAATAGGCCTACTAGTCGGCCGTACGGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGGCCTTAAGGGCCTCGTTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTT

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DSB

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Donor DNA(ssDNA, 100b)

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Val83, Ser85, Asp87 and Thr89), with each protomer contributingthree residues. Because many of the interactions are redundant,recognition of A(–7) will be described in detail and only thedifferences found in the A+1 binding pocket will be highlighted.For clarity, the prime symbol denotes residues located on the oppositeprotomer of the PP7DFG dimer.

The upper surface of the A(–7) binding pocket is formed by thehydrophobic side chain of Val83¢ and the aliphatic portion of theLys58¢ side chain, which make van der Waals contacts with the base(Fig. 2b,c). In the A+1 pocket, the side chain amine of Lys58 hydrogenbonds to the 2¢OH of G+3 and likely stabilizes its C2¢-endo sugarpucker (Fig. 2c). Asp87, Thr89 and Ser85¢ form the middle level of thepocket and make sequence-specific contacts to the adenine base. Theside chain OH of Thr89 is within hydrogen-bonding distance of boththe adenine N7 and N6 exocyclic amines. The backbone carbonyl ofAsp87 is also within hydrogen-bonding distance of the N6 exocyclicamine. The third RNA-protein interaction at this level is a hydrogenbond between the OH of Ser85¢ and adenine N1. Arg54, whoseguanidinium group makes a cation-p stacking interaction with theadenine base, forms the base of the binding pocket. The position of theArg54 side chain is buttressed by hydrogen bonds to the side chain ofAsp87. In the A+1 pocket, the Arg54¢ guanidinium group makes twohydrogen bonds to O6 and N7 of G+3 to specifically recognize theguanine base. (Fig. 2c). The importance of Lys58 and Arg54 in RNArecognition is supported by experiments that demonstrate that muta-tion of these residues leads to severe repression defects4.

The PP7DFG–RNA complex is further stabilized by several hydro-gen bonds and electrostatic interactions outside of the adeninerecognition pockets. The backbone amide of Gly48¢ and the sidechain of Asn47¢ make hydrogen bonds to phosphate oxygens of A(–2)and U(–1), respectively. The U(–1) nucleotide is extended away fromthe loop into a pocket formed by Thr51¢, Ala52¢, Val91¢ and Thr81.The backbone amide and side chain OH of Thr81 make hydrogenbonds to the O2 and 2¢-OH of U(–1). Another potential hydrogenbond exists between the side chain carboxylic acid of Asp60 and theA+1 O2¢, which may stabilize its C2¢-endo sugar pucker. A similarinteraction is observed in the MS2 coat protein–RNA complex, whereGlu63 is hydrogen bonded to the U(–5) 2¢ OH13. Crystal-packing

differences result in the side chain of Arg24 stacking with the guaninebase of G+4 and contacting either its O4 or phosphate oxygens. Weakelectron density and alternate conformations of the Arg24 side chainsuggest that this interaction does not contribute substantially to theoverall affinity of complex. There are also several positively chargedresidues (Arg24¢, Arg39, Arg45¢) that may participate in favorableelectrostatic interactions with the phosphate backbone of the RNA.

Although the PP7 and MS2 coat proteins share similar proteinscaffolds, their RNA-binding surfaces have evolved to specificallyrecognize distinct RNA hairpins. The most notable difference betweenthe two structures is the location of the adenine-recognition pockets,which are important components of binding for both coat pro-teins12,14,15. In the PP7 coat protein, the pockets are aligned along

b c

5′5′ 3′3′

3′

5′

G - C

A - U

G - C

A - UA - U

C - G

A

AU G

A U

C - G

G - CG - C

G

G - C

G - C

G - C

U - A

C - GA - U

A - U

A

AU U

A

N

N

NN

C

C

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–12

–11

–10 –9

–8

–7

–6

–5

–4–3

–2–1

+1

+2+3

+1+2

+3

+4+5

+6

+7+8

+9

+10+11

+12+13

–2

–3–4

–5

–6

–7–8

–9–10

–11

–12

–1

MS2GA

1 10 20 30 40 50 60 70 80 90 100 110 120

QbPP7

a

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+1

3′5′

+1Figure 1 Coat-protein sequencealignment and overview of the MS2coat protein and PP7DFG complexeswith RNA. (a) Alignment of four ssRNAbacteriophage coat proteins. (b,c) MS2coat protein–RNA (2BU1) (b) andPP7DFG–RNA (c) complexes areshown as cartoons. In both structuresthe RNA hairpin (orange, adenine; red,guanine; violet, uridine; yellow,cytidine) binds across the extendedb-sheet surface formed by the coatprotein dimer (blue, green).

b

cVal83

V83

A(–7)

A(–2)

G(–3)

A(–4)

A(–5)

G(–6)

A(–9)

G(–11)

G(–12)

C(–8)

K58

G48

N47

S85

T89

T81

U(–1)

U+2

D87 R54

R54

D60

S85 D87

A+1

G+4

C+5

G+3

U+6

U+7

C+8

G+9

U+10

G+11

C+12

C+13

V83

T89

K58

Ser85

Lys58

Arg54

Asp87Thr89

Val83′ Ser85′

Asp87′

Arg54′

Thr89′

Lys58′

A(–7)

A+1

G+3

3′5′

a

C(–10)

Figure 2 RNA-protein interface. (a) Schematic representation of PP7DFGinteractions with the RNA hairpin. Black arrows, hydrogen bonds; blacklines, van der Waals and stacking interactions. (b,c) The A(–7) (b) andA+1 (c) recognition pockets are shown, with the adenosine nucleotides(orange) and PP7DFG residues (blue, green) as sticks. Potential hydrogenbonds are shown as dashed lines.

BR I E F COMMUNICAT IONS

104 VOLUME 15 NUMBER 1 JANUARY 2008 NATURE STRUCTURAL & MOLECULAR BIOLOGY

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PPR motif (35 aa)v t Y n t l I s g l c k a G r l e e A l e l f e e M k e k - G i a P d v1 4 ii

- A - G - U - C - A - C - U - G - A - G - A -

PPR protein

RNA

Base

Human mitochondrial RNA pol.Ringel et al., Nature (2011)

FTN VNS VTD FPD(1,4,ii)

Rec. base

[Base recognition code]

�Design and use of artificial RNA binding protein[PCT/JP2012/077274� patented in Japan, US.]

�Design and use of artificial DNA binding protein[PCT/JP2014/061329� patented in Japan, US]

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PPR technologyPentatricopeptide repeat


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What is the PPR proteinHuman (7)Yeast (2)

Prokaryote (0)Algae (0)

ArchaeaBacteria �0�

Plant �500�

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How to use PPR technology

PPR repeat

Sequence-specific binding module

Effector module Application

DNase

RNase

Splicing factor

Genome editing

Knock-down

Splicing controlwo/ module RNA remodeling

Block XX.(ex. miRNA, RBP)

AAV Nanoparticle

Translation factor Translation up

dPPR tech.(DNA-binding)

rPPR tech.(DNA-binding)

Plasmid


Nu

ER

DNA

mRNA

Protein

ncRNA

???

Pre-mRNA

mRNA

lncRNA

miRNA

RNA virus

RNA virus

mitochondria

Mitochondrial RNA

PPR

Possible rPPR-based applications


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Beyond genome editing

mRNAtRNA

rRNA

ncRNAcircRNA

pre-mRNA

dsRNA

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Thank you for your attention

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ppr technology...how to use ppr technology ppr repeat sequence-specific binding module effector...

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