Research progress of piRNA evolution

Xuedong Pan2012-3-28

What is piRNA

• Small RNAs: piRNA, siRNA, miRNA.• piRNA: derive from repetitive genomic

element, interact with PIWIAGO

family protein

Guide RNARISC(RNA induced silencing complex)

Germline-specific AGO

PIWI family( PIWI,AUB, AGO3)

piRNApiRISC: recognize

and silence complementary

RNA

Biogenesis of small RNAs

Dicer independent

Unkown;important

Proved; 5’U and 10 A

piRNA biogenesisLong precursor can generate a lot of piRNAs, thus is piRNA cluster

Not only piwi. Piwi interact with Tudor-domain proteins directly.

Tudor-domain proteins methylate arginine in piwi, which differs piwi with other AGOs. Piwi also

needs Armi.

Putative roles for piRNA during silencing of protein-coding genes

In drosophila testis, if Su on ChrY do not work, Ste on ChrX is deprepressed, which leads to infertility.

Piwi expression driven by TJ. piR from 3’UTR of tj together with piwi suppress Fas3.

In drosophila testis, AT-chrX code piR, piR interact with AUB, and little AGO3, to suppress vasa

Failure of nos mRNA deadenylation and translational repression by piR

piRNA at different developmental stages in mouse

Piwi family in mouse:

MIWI2, MIWI, MILI

TDR: tudor domain containing proteins. Bind piwi directly.

piR enriched in TE. Binds MILI

and MIWI2

piR not enriched in TE. Binds MIWI

and MILI

Conclusion: different developmental stage,

different protein and piR

Pachytene: cross over happens

Current Evolutionary studies demonstrate

• piRNA strongly repress retrotransposons, co-evole?

• piRNA evolves very fast among species

• piRNA loci locates in low recombination region

• piRNA show a signature of selective constraint in African populations

Molecular evolution: piRNA evolves super fast and do not lost

• Rapid repetitive element-mediated expansion of piRNA clusters in mammalian evolution (09 PNAS, Assis et. al, U Michigan)

1) 43% of all rodent piR clusters arose after rodent-primate divergence. While the highest known expansion rate for olfactory receptors is 33%.

2) Olfactory receptors and miRNAs are lost at the same rate with which they are acquired. However, not a single cluster loss was observed for piRNA.

Positive selection most likely

• RE(repetitive element) usually increases deletions more than insertions.

• 60 cluster acquisitions without a single loss• Long insertions are unlikely to be neutual• piRNAs are involved in transposon silencing.

• So, an arms race between expanding families of mammalian TE and piRNA cluster?

piRNA evolve by ectopic recombination

(A) Architecture of a typical cluster-harboring genomic region. Intergenic region between two protein-coding genes piRNA cluster. The inserted segment is depicted in brackets.

(B) The inserted segment is scanned against the genome to locate the source paralog.(C) Similarity between cluster- and source paralog-harboring regions includes preceding REs(D) Double-stranded break, leads to extension and reannealing of the broken strand

Protein-coding gene

Protein-coding gene

piRNA cluster

Repetitive element

Population dynamics of piRNA and TE(transposable element) in Drosophila

• Jian Lu and Andrew G. Clark, PNAS 2010• Activities of a large number of retrotransposons

are severely silenced by piRNAs. 1) quantified expression levels of 32 TE families 2) in ovaries of one wild-type and three piRNA

mutants• Other reports prove piRNA silence transposons.

Life cycles of transposable elements(TE)

a: IR/DR: inverted/direct repeats bind by transposasesb:Reverse transcription in cytoplasm, integration in nucleus by integrasesLTR: long terminal repeatGAG protein: form virus-like particlesc:ORF1 and ORF2 have reverse transcriptase domainmRNAs are integrated into genome by target-primed reverse transcription

piRNA repress TEs in RNA level

Forward simulation of piRTs and targetRTs

• Focus on retrotransposon only• Retrotransposons including piRTs: located

inside piRNA loci; targetRTs: remaining.• Model: largely come from Dolgin and

Charlesworth, Genetics, 2008Start prameters

Selection: fitness is exponential quadratic, decreasing function of TE copy number.

recombination: uniform distribution of crossover positionstransposition and excision(Poisson process throughout genome)

For 15000 generations

parameters• Ne = 10e6, constant-sized• One chromosome, 40Mb(close to chr2,3)• One chr one crossover per generation, r = 2.5e-8• Fitness of chr: ; n: the number

of retrotransposons; a = 10e-5; b = 5e-6• Excision rate is v. v = 0• For targetRTs, retrotransposition rate is u1 if

piRNA is not expressed in the cell, u2 if piRNA is expressed.

2 / 2an bnw e

Model piRNA’s suppression effect

• For targetRTs, retrotransposition rate is u1 if piRNA is not expressed in the cell, u2 if piRNA is expressed.

• Four scenarios: 1) piRNAs have no repression effect: u1 = u2

2) 3) 4) piRNAs can reduce retrotransposition rates to 10%, 1% and 0.1%(u2 = 0.1u1, 0.01u1, 0.001u1)

Model’s other assumptions

• Retrotransposons inserted into piRNA-generating regions will be suppressed.

• No sequence divergence between paralogous copies of retrotransposons, so that one piRNA can potentially repress all retrotransposons.

• Retrotransposons located inside piRNA loci lose the ability to retrotranspose

• Ectopic recombination is not allowed

Forward simulation for 15000 generations: active piR repress retrotransposons and

increase fitness

A) Number of retrotransposonsB) Fitness costs to the host* scenario1,2,3,4: piRNA’s repressing capabilities increases

Forward simulation for 15000 generations: if piR is active, piRTs increases with time

Proportion(%) of all retrotransposons that are piRTsScaled parameters: Ne = 500, a = 0.001, b= 0.0005, r = 2.5e-8, u1 = 0.01, v = 0

Scenario IV

Scenario I

Frequency spectra of piRT insertions:When piRNA takes effect, piRTs have a higher probability to be fixed

Frequency spectra of targetRT insertionsAlso has a higher probability to be fixed, because their deleterious effects are alleviated by piRNAs

Frequency spectra of piRT and targetRT from published genomic data: recombination matters

A vs. B: TE longer than 500bp

C vs. D: subset of A vs. B, recombination occured

No recombination occured

Combination of 2 datasets

TE and piR like low recombination regions

• piR loci enriched in ericentromeric or telomeric heterochromatin

• TEs significantly enriched in low recombination regions, even in euchromatin

• When recombination occurs, piRTs are deleterious because they can mediate ectopic recombination

** piRNA also enriches in centromeric and telomeric region. Telomeric lacZ insertion produces abundant piRNAs. Telomere is a picluster?

Reducing recombination rate of piRNA loci greatly reduces retrotransposons

Recombination rate = 2.5e-8 Recombination rate = 0

Human piRNAs are under selection in Africans and Repress TEs

• Sergio Lukic and Kevin Chen, MBE, 2011• Material and methods: human piR sequences(Girard et al. 2006) mouse piR sequences(Lau et al. 2006) Hapmap phase3 data methods: derived allele frequency spectrum;

BWA tool, mapping reads

piR evolved rapidly between human and chimp

• piR region vs. piR flanking region piR flanking region (1000bp each side of piR); nucleotide substitution between human and

chimp is not significantly different between piR region and piR flanking region

Selective constraint in African populations

ASW, YRI vs. CHB, CEU

Selective constraint in Africans is consistent with a much higher rate of transposon insertions in African compared with non-African populations(Ewing A, Kazazian H, Genome Res, 2010)

Interspecies: no constraintintraspecies: constraint in Africans;

mild constraint in non-African

• Explanation1: strength of selective constraint my simply differ between these two time scales.

• Explanation2: interspecies substitution rate, but not the derived allele frequency distribution, is affected by mutation rate biases.

younger TE, more piR targets

Younger TEs are more active, so more piRNAs which target them remain in current human genome; the pattern is the same for mouse

The pattern is the same in our data

ORF2 of LINE1 is depleted of piRNA matches

Red line: number of G/C-nucleotides per base on L1 mRNABlue line: density of sense piRNA matches to L1 Green line: density of antisense piRNA matches to L1

L1-ORF2 functions as reverse transcriptase

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