Probing the initiation and effectors’ phases of the somatic piRNA pathway in Drosophila
To understand the combining RNAi in cultured cells and analysis, it is important to know the roles of known Piwi- interacting RNA(piRNA) pathway elements particularly in the initiation and the effecter phases of transposon silencing. Squash is one component that is physically with Piwi (Brennecke et al.,2007). The main components involved in the process are Squash, Zucchini or Armitage. These elements pose different effects on the transposon, and piRNAs. When squash expression is reduced, its effect is directly seen on the transposon. This is because when its expression is reduced, it leads to modest transposon depression but no results are seen on the piRNAs, consistent with an effecter role (Brower, 2007)
Zucchini, on the other hand, can reduce both piRNAs and Piwi protein when altered or is tampered with. Alteration in Zucchini leads to the formation of a stable Piwi RISC known as RNA- induced silencing complex (Brennecke, 2008)Continuance mutation or loss of Zucchini within its catalyst domain led to amassing of vulnerable herald transcripts from flamenco, steady with a role for this alleged nuclease in piRNA biogenesis.
There are a number of mechanisms that help regulate gene expression. The regulation is done Eukaryotic which are small RNAs, these RNAs perform the regulatory role at both the transcriptional and post-transcriptional levels. These small RNAs are divided into classes in accordance to their particular Argonaute protein partner and according to their mechanism of biogenesis. Piwi-interacting RNAs play a role of binding Piwi –clade Argonaute proteins and they do this in the gonadal tissues. Here, the piRNAs silence mobile genetic elements thereby guarding the integrity of the genome.
The piRNA pathway is divided into a number of phases namely: initiation phase, effectors phase and adaptation phase. There are different things happening in the three phases and this is why the activities were divided into the phases.
There are a number of things that happen in the initiation phase:
Production of small RNAs called piRNAs. The piRNAs are produced from their generative loci known as piRNA clusters.
The piRNA give rise to long, presumably single-stranded precursor transcripts which are produced as a result of unknown biogenesis mechanism into small RNAs that are larger than canonical microRNAs.
The piRNAs are stabilized when they are associated with Piwi proteins from Piwi RISCs.
The Piwi RISCs have additional proteins that help in the facilitation of target recognition and silencing(Lau et al., 2009).
There are many things that happen in the effectors phase:
Identification of target which is made by the Piwi RISCs via the complementary base-pairing (Lau et al, 2006)
Conservation of the Argonaute catalytic triad which is also made by the Piwi. (Klenov et al, 2007).
This phase is restricted to germ cells and it constitutes the ping-pong cycle. In this period transposon microrna cleavage is directed by primary piRNAs to trigger the production of secondary piRNAs (Klattenhoff et al., 2009) Later on, the produced secondary RNA triggers the production of piRNA from the target, closing the loop that that makes it possible for the overall small RNA population to adjust to challenging by a particular transposon. It is also in this phase where the piRNA in germ cells can be transmitted to the next generation to prime piRNA responses in progeny.
However, when it comes to Drosophila follicle cells, initiation and effectors phase are the only relevant phases. In Drosophila follicle cells, the piRNA pathway relies on the coupling between a single Piwi protein and a principal piRNA cluster to silence gypsy family retrotrasnposons.
From researches made, it was found that there are various ways by which Piwi proteins silence targets by interfering with their transcription. It was found out that piRNA are mostly absent from somatic tissues, results underlying these changes are presumed to have occurred during the development and to have been epigenetically maintained in the adult. Drosophila Piwi protein was found in the nucleus and it interacts with HP1. HP1 is a core part of heterochromatin. The results of the research showed that the effecter mechanism is what takes place in the Drosophila whereby Piwi-associated small RNAs direct heterochromatin formation and silencing of targets.
It was also found that the loss of Piwi poses some results on the transposon expression in the somatic cells (Ghildiyal, 2009). It was found out that the absence of Piwi proteins in the somatic cells during development is the major cause of genetic mutation. In simple terms, it was found that the piRNA pathway is always required for transposon silencing.
It was also found out that Armitage is a part of the somatic piRNA pathway. Researchers found that there are as many as twelve proteins that have been linked to the fully elaborated piRNA pathway that operates in germ cells (Girard 2006). Many of them were found to show germ-cell specific expression patterns consistent with their selective biological effects (Brennecke 2007). In Armitage, mutilation occurs as a result of loss of both the functional nuclear accumulation of Piwi protein and decrease in Piwi-associated piRNAs. It was concluded that Armitage play a role in the somatic and germline compartments(Cook HA, 2004).
Another result showed that both Armitage and Zucchini work at the initial stage, or phase. Different experiments which were used to determine which phases do the two functions showed that both function at the initiation phase (Cox DN, 2000)
A proteomic analysis of Piwi RNPs was performed and it was found out that Armitage is a part of Piwi RISC. It was found that Piwi immunoprecipitates contained several peptides from Armitage. This was a clear indication that there is the presence of this protein in Piwi. A detection of an association of Squash with Piwi and Armitage was also made.
It was also discovered that Squash impacts the piRNA effectors phase. It was found out that a mutation in the squash showed little impact on the piRNA populations in mutant ovaries.
The discovery made was of high value in the field(Gunawardane 2007) This is because specialists in this field now know the different causes of genetic mutation and when the cause is known it is easy to apply appropriate solutions if a problem occurs (Grivna 2006).The results provide a road map to the specialists to know how to tackle genetic mutation.
From this article, it is clear that the three; Armitage, Piwi protein, and Squash in some point they associate with each other. This discovery is of great value in the field. People can now understand the whole thing of genetic mutilation.
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