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P a r t i c i p a n t s :<br />
Laura Fanti, professor; Lucia Piacentini, researcher;<br />
Barbara Perrini, Marcella Marchetti, post-doc fellows;<br />
Enzo Marchetti, Eleonora Vitagliano, technicians.<br />
C o l l a b o r a t i o n s :<br />
Martin-Luther University, Halle, Germany (Prof. Gunter Reuter);<br />
Università di Lecce (Prof. Maria Pia Bozzetti).<br />
Report of activity<br />
The aim of this research is to systematically search,<br />
by a combined genetic, cytological and molecular<br />
approach, genes involved in RNAi, heterochromatin<br />
formation and gene silencing in Drosophila.<br />
Recently, a new and fascinating relationship between<br />
RNAi and heterochromatin has been discovered. In<br />
Drosophila, it has been found that mutations in genes<br />
that are components of the RNA interference<br />
machinery, are also involved in heterochromatin formation.<br />
These genes are expressed in the male and<br />
female germlines, are implicated in germline stem<br />
cell self-renewal and have crucial functions in the<br />
early stages of development.<br />
Intriguingly, it has also been found that these mutations<br />
induce testicular transcription of the repetitive<br />
Stellate DNA sequences causing a complex meiotic<br />
phenotype. This meiotic phenotype is characterized by<br />
the presence of star or needle-shaped crystals in spermatocytes,<br />
that are mainly composed by the STEL-<br />
LATE protein, abnormal chromosome condensation at<br />
first metaphase, and irregularities in the transmission<br />
of the chromosomes during meiosis such as nondisjunction<br />
and meiotic drive. Stellate sequences are<br />
repetitive sequences organized in one cluster at the<br />
crystal locus of the entirely heterochromatic Y chromosome<br />
and two other clusters, respectively located in<br />
the heterochromatin and euchromatin of the X chromosome,<br />
that are repressed by the crystal + in male<br />
testes via an RNAi mechanism that uses the production<br />
of rasiRNA (repeat-associated small interfering RNA),<br />
also called piRNA (piwi-associated RNA) (see Figure).<br />
Principal investigator: Sergio Pimpinelli<br />
Professor of Genetics<br />
Dipartimento di Genetica e Biologia Molecolare<br />
Tel: (+39) 06 49912876; Fax: (+39) 06 4456866<br />
sergio.pimpinelli@uniroma1.it<br />
65<br />
Molecular genetics of eukaryotes - AREA 3<br />
Heterochromatin, gene silencing and “RNA interference” in<br />
Drosophila<br />
Search for gene involved in RNA interference<br />
To genetically dissect the RNAi machinery and its<br />
relationship with heterochromatin, we performed a<br />
screen of genes acting during spermatogenesis by<br />
using Stellate phenotype as <strong>report</strong>er. This screen led<br />
to the identification of 13 genes and 3 new X-linked<br />
mutants that induce the expression of the Stellate<br />
sequences in presence of a normal Y chromosome<br />
carrying the crystal + locus.<br />
We tested, so far, six mutants that show a strong Ste<br />
phenotype (fs(1)h, CycB, snf, shu, Xst30 and Xst49) and<br />
we found that all these mutations affect the biogenesis<br />
of Piwi-associated interfering RNAs (piRNAs)<br />
corresponding to the Stellate sequences and to the<br />
sequences of several retrotransposons. Our results<br />
have permitted to isolate and identify new genes that<br />
are involved in RNAi mechanisms.<br />
We are now performing further experiments to assess<br />
the potential role of these genes in the various steps<br />
of RNAi-response pathway. The results of this work<br />
will be described in a manuscript in preparation.<br />
The Hsp90 is involved in piRNAs biogenesis<br />
in Drosophila<br />
The Hsp90 chaperone proteins are conserved proteins<br />
that are involved in several cellular processes and<br />
development pathways. It has been shown in Drosophila<br />
that mutations in the Hsp90 encoding gene are capable<br />
to induce the expression of the Stellate repeated<br />
sequences in testes of mutant males. These sequences<br />
are normally repressed by the Y-linked crystal locus by<br />
an RNA interference (RNAi) machinery that produces<br />
silencing small rasiRNAs (or piRNAs).<br />
We have found that mutations in Hsp83 locus, which<br />
encodes the Hsp90 protein, induce the activation of<br />
Stellate by affecting the production of rasiRNAs<br />
(piRNAs). In addition, we show that this mutation<br />
activates the transcription and the mobilization of<br />
diverse class of mobile elements by affecting the biogenesis<br />
of the complementary piRNAs.<br />
Both set of data demonstrate for the first time, that<br />
Hsp90 chaperone is involved in negative regulation