download report - Istituto Pasteur

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