Digital archive of theses discussed at the University of Pisa


Thesis etd-10062010-232016

Thesis type
Tesi di laurea specialistica
Thesis title
Biochemical studies of the putative nuclease Zucchini and of its role in the piRNA pathway.
Course of study
relatore Prof.ssa Rossi, Anna Maria
  • nuclease assay
  • piRNAs
  • piwi
  • zucchini
Graduation session start date
Release date
Small RNAs (c.a. 20-30 nt) in the last 20 years have emerged as factors that play a role in regulating gene expression. The non protein coding RNAs are involved in diverse events occurring in the cell such as development, differentiation, cancer, viral defense, DNA elimination, mobile elements silencing, etc.
The number and type of small RNAs is continuously increasing, but the common feature of this novel class of regulators remains invariant. The ncRNAs are loaded onto proteins, which they guide to the targets. These proteins are baptised as Argonaute proteins and represent the effectors that modulate gene expression at both transcriptional and post-transcriptional level by promoting heterochromatinization and transcripts degradation or translation repression, respectively.
The small RNAs guidance is mediated by base-pairing to the target and if the complementarity is perfect the target transcript is destined to be cleaved by the RnaseH PIWI domain of the Argonautes on which the siRNA is loaded; whereas if the bound is characterized by an imperfect complementarity (miRNAs) the transcript translation will be repressed. siRNAs (small interfering RNAs) and miRNAs (micro RNAs) are the best studied small RNAs, their biogenesis and mechanism of action is vastly understood.
piRNAs (piwi interacting RNAs) represent an emerging class of ncRNAs that, in contrast to the ubiquitous expression of the siRNAs and miRNAs, their presence is confined to the germline.
Moreover, the Argonaute proteins are subdivided into two groups: Argonaute (Ago) and Piwi proteins. Unlike Agos, Piwis are restricted to the germline and are loaded with piRNAs. Based on the evidence provided from the-state-of-art literature it is known that piRNAs play a role in maintaining the integrity of germ cells by repressing the mobility of transposons.
The biogenesis of miRNAs passes through a 2-step processing, the nuclear Rnase Drosha catalyses the cleavage of the pri-miRNA and gives birth to the pre-miRNA (c.a. 70 nt) which is subsequently transported to the cytoplasm by Exportin 5 and it it is further processed by the cytoplasmic Rnase Dicer. siRNAs are also processed by Dicer, but unlike miRNAs Drosha does not participate in their biogenesis, furthermore, they can have an exogenous origin, while miRNAs are endogenously originated.
piRNAs (24-30 nt) derive from both intergenic repeats and distinct clusters in the genome, they undergo a Dicer-independent unknown primary processing and it is thought that, subsequently, piRNAs are amplified by the so called ping-pong mechanism (or amplification loop). piRNAs loaded on the piwi proteins guide piwi to a transposon transcript target, which is cleaved at the 5' end by the piwi domain of the Piwi proteins, an unknown enzyme generates the 3' end of the de novo piRNA, which will be loaded on another piwi protein. The novel piRNA guides the 5' end cleavage of a piRNA cluster transcript, the 3' end cleavage will finally generate a new piRNA and the cycle starts again.
Zucchini is a putative nuclease believed to be involved in the piRNA pathway. It is already shown that in fly zucchini mutants, piRNAs are lost. Zucchini has a nuage localization and interacts with Aubergine (one of the piwi proteins). In flies, the 'nuage' is defined as a ring around the cytoplasmic face of the nuclei of the transcriptionally active nurse cells, in mouse it is regarded as the inter-mitochondrial cement that surrounds the nuclei of the nurse cells. Zucchini is a small protein characterized by a single phospolipase D domain, typically present in bacteria nucleases. Interestingly, zuc has an N-terminal Mitochondrial Localization Sequence (MLS domain) which anchors the protein at the outer membrane of the mitochondria. The aim of the thesis project is to purify zucchini and perform nuclease assays to understand if it really has a nuclease activity that promotes RNA cleavage.