This document discusses PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs that interact with PIWI proteins. It describes how piRNAs were discovered in Drosophila and their role in silencing transposons in the germline. The document outlines piRNA biogenesis, including their location in clusters in genomes and the "ping-pong" mechanism of biogenesis. It also discusses compartmentalization of the piRNA pathway and functions of piRNAs in maintaining genome integrity, transposon silencing, and fertility.

1. PI-RNA
Presented by :
Sasmita Behura
MBT – I
Roll No - 1361024

2. The discovery of PI-RNAs
 In male flies, small RNAs derived from the supressor of stellate locus on the
y-chromosome silence the x-linked stellate locus during spermatogenesis.
 In su (ste) mutants , small RNAs targeting stellate are lost causing stellate
protein crystals to form in the primary spermatocytes and reduced fertility .
 Subsequent small RNA profiling studies in Drosophila reveal that su(ste)
small RNAs only represent a small subset of a class of small RNAs with a size
range 24-26nt and named as repeat associated small interfering RNAs
(rasiRNAs).
 And in mammals it has been seen that piwi protein associate with a class of
endogenous RNAs termed as Pi-RNAs.
 Thus Droshophila rasiRNAs were renamed as Pi-RNAs .

3. Piwi- interacting RNAs(knowns of
the unknown)
• Piwi - interacting RNA is the largest class of small non-coding RNA
molecules expressed in both vertebrates and invertebrates.
• Piwi- proteins are predominantly found in germline cells and associate
with a novel class of small RNAs known as pi-RNAs.
• Different types of piwi- proteins are found i.e MILI, MIWI1 and MIWI2
in mice, ZIWI1 and ZIWI2 in zebra fish and Ago3 and Aub in mammals.
• Piwi is a class of gene was originally identified as encoding regulatory
proteins responsible for maintaining the stability of cell division in
germline cells.

4. Location of pi-rna
 Pi-RNAs are mainly found in clusters through out the genomes and
they may contains 10 up-to many thousands of pi- RNAs and piwi
protein mainly found in germ cell and gonodial cell.
 It is found in both mono-directional and bidirectional clusters
which length is up to 20-1000 kilobases with 24-33 nuleotides.

5. Biogenesis
 Exact biogenetic process of pi-RNA is not yet fully understood. But
there is a” ping-pong”-mechanism which gives an overall view about its
biogenesis.
 Pi-RNAs involved in the ping-pong cycle directs its attack on
transposon transcripts and this cycle only acts at the level of
transcription.
 Pi-RNA production is a dicer- independent process.
 In c . elegans pi-RNAs have a 5’- mono-phosphate and a 3’ modification
that acts to block either 2’ or 3’ oxygen but the reason behind this
modification isn’t clear. It may be there to increase pi-RNAs stability.

6. Ping-Pong mechanism
 Pi-RNAs consists of 2-strands i.e sense strand and antisense strand.
 Ago3(argonaute protein) bound to sense strand pi-RNAs and catalyzes
antisense strand cleavage at an A:U base-pair that generate the 5’ end
of antisense strand.
 Then 5’end of the resulting cleavage associate with Aub or piwi which
generate mature 23-30nt antisense pi-RNAs.
 Then the mature antisense pi-RNA argonaute complexes are then
cleaved sense strand pi-RNA which associate with Ago3.

7.  Then production of mature sense strand complete the cycle.
 3’ methylation occures at both antisense and sense strand which is
carried out by Hen1 RNA methyl transferse.
 Mutation in Drosophila Hen1 reduces the length of pi-RNAs.
 In Drosophila some genes like armitage and spindle E encodes putative
helicase that required for pi-RNAs production.
 As antisense pi-RNAs guide Aub or piwi to cleave the transposon m-
RNAs and trigger the production of new antisense pi-RNAs.
 So Ping-Pong model proposes a target dependent amplification of
antisense pi-RNAs which depends on Ago3.

9. Compartmentalization of pi-RNAs
 Actually most pi-RNA pathway proteins, localizes almost exclusively to
nurse cell nuclei so that pi-RNA production and function might be
compartmentalized.
 piwi protein family involves piwi,Aub and Ago3 which are found in
nuage/cytoplasm , which is an amorphous electron dense cloud that
surrounds the nurse cell nuclei.
 Pi-RNA argonaute complexes appear to be catalytically active effectors
and these localization studies thus suggest that piwi mediates nuclear
functions for the pi-RNA pathway, where as Ago3 and Aub drive
cytoplasmic funcion.

10. Localization of piwi class
Argonautes in the
Drosophila ovary
Model for compartmentalized
production and function of
piRNAs

12. Types of pi-RNAs in mammals

13. A,B- pi-RNA complex.

14. Argonaute protein
 A- Eukaryotic ago protein with 4-domains.
 A crystal structure of ago protein in Pyrococcus
furiosus.

15. Functions
 Pi-RNA complexes linked to both epigenetic ,post –translational gene
silencing and maintain germline DNA integrity.
 Pi-RNAs also regulate the expression of protein coding genes.
 Piwi target RNA cleavage by silencing.
 Piwi required for the self renewing division of germline cells during
oogenesis.
 Mutation in any members of pi-RNA pathway causes derepression of
transposons and also leads to the loss of fertility.
 Piwi – complexes are imported to nucleus and mediate heterochromatin
assembly.

16. Unknown factors about pi-RNAs
 It is still unknown that how a new pi-RNA response is initiated and
why some regions of our genome are so uniquely poised to proudce piRNAs.
 The transcriptional region of pi-RNA cluster is still unknown.
 There are also various proteins rather than piwi- family which are
involves in ping-pong mechanism but there mode of action is also not
understood.