Expression of the X-linked repeated Stellate (Ste) genes, which code for a protein with 38% similarity to the beta-subunit of casein kinase II, is suppressed by the Su(Ste) locus on the Y chromosome. The structure and evolution of the Y-linked repeats in the region of the Su(Ste) locus were studied. The 2800 bp repeats consist of three main elements: the region of homology to the Ste genes, an adjacent AT-rich, Y-specific segment, and mobile element 1360 inserted in the Ste sequence. Amplification of repeats was followed by point mutations, deletions, and insertions of mobile elements. DNA sequencing shows that these repeats may be considered as Ste pseudogenes or as damaged variants of a putative gene(s) encoding a protein quite different from the Ste protein as a result of an alternative splicing pattern. A comparison of 5 variants of the Y-Su(Ste) repeats shows a number of recombination events between amplified and diverged sequences that could be due to either multiple unequal mitotic sister-chromatid exchanges or to gene conversion. It is a first demonstration on a molecular level of these processes occurring in heterochromatic non-rDNA tandemly organized sequences in an eukaryotic genome (Balakireva, 1992).
The organization and transcription is reported of diverged tandemly repeated Y-linked Su(Ste) genes that are considered as suppressors of testis-expressed X-linked-repeated Stellate genes that encode a protein sharing extensive homology with beta-subunit of casein kinase 2. Clustering of restriction variants is confirmed. Size variants of Su(Ste) repeats appear to be nonhomogeneously distributed among the P1 phage clones. Different ways of Su(Ste) RNA processing because of the appearance of new splice sites and polyadenylation signals were detected. The high extent of homology between Stellate and Su(Ste) repeats suggests the possibility of Stellate suppression by antisense transcription of Su(Ste) elements. The detection of only 'sense' Su(Ste) cDNAs in testis cDNA library allows this proposal to be rejected. The genomic and cDNA clones are shown to be equally diverged. This indicates widespread rather than restricted transcription capacity of these repeats (Kalmykova, 1998).
The homeless gene of Drosophila encodes a member of the DE-H family of ATPase and RNA helicase proteins. Loss-of-function homeless mutations were previously found to cause female sterility with numerous defects in oogenesis, including improper formation of both the anterior-posterior and dorsal-ventral axes and failure to transport and localize key RNAs required for axis formation. One homeless mutation was also found to affect male meiosis, causing elevated X-Y nondisjunction. The role of homeless in male meiosis has been further analyzed. homeless mutations cause a variety of defects in male meiosis including nondisjunction of the X-Y and 2-2 pair, Y chromosome marker loss, meiotic drive, chromosome fragmentation, chromatin bridges at anaphase, and tripolar meiosis. In addition, homeless mutations interact with an X chromosomal factor to cause complete male sterility. These phenotypes are similar to those caused by deletion of the Suppressor of Stellate locus. Like Su(Ste) deficiencies, homeless mutants also exhibit crystals in primary spermatocytes and derepression of the X-linked Stellate locus. To determine whether the regulatory role of hls is specific for Stellate or includes other repeated sequences as well, testis RNA levels were compared for nine transposable elements; all but one, copia, are expressed at the same levels in hls mutants and wild type. Copia, however, is strongly derepressed in hls mutant males. It is concluded that hls functions along with Su(Ste) and other recently described genes to repress the Stellate locus in spermatocytes, and that it may also play a role in repressing certain other repeated sequences (Stapleton, 2001).
The RNA helicase Armitage is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. armi has been shown to be required for RNAi. armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced silencing complex (RISC), which mediates target RNA destruction in RNAi. These results suggest that armi is required for RISC maturation (Tomari, 2004).
Silencing of the X-linked Ste gene by the highly homologous Y-linked Su(Ste) locus is an example of endogenous RNAi. In Drosophila testes, symmetrical transcription of Su(Ste) produces dsRNA, which is processed into siRNAs (Gvozdev, 2003). Su(Ste) siRNAs direct the degradation of Ste mRNA. Inappropriate expression of Ste protein in testes is diagnostic of disruption of the RNAi pathway. Both the Argonaute protein, aub, and the putative DEAD-box helicase, spn-E, have been shown to be required for RNAi in Drosophila oocytes. Both mutants fail to silence Ste, as evidenced by the accumulation of Ste protein crystals in the testes of aub and spn-E mutants. No Ste protein is detected in wild-type testes. Strikingly, Ste protein accumulates in testes of two different armi alleles, armi1 and armi72.1. Neither allele is expected to be a true null because armi1 is caused by a P element insertion 5' to the open reading frame, whereas armi72.1, which was created by an imprecise excision of the armi1 P element, corresponds to a deletion of sequences in the 5' untranslated region. Ste silencing is re-established in males homozygous for the revertant chromosome, armirev 39.2 (henceforth, armirev; Cook, 2004), which was generated by excision of the armi1 P element. These data suggest a role for Armi in Drosophila RNAi (Tomari, 2004).
Immunofluorescent detection of Ste protein in testes implicates both armi alleles in endogenous RNAi, but provides only a qualitative measure of allele strength. Since Ste protein in males reduces their fertility , the percent of embryos that hatch when mutant males are mated to wild-type (Oregon R) females provides a more quantitative measure of Ste dysregulation. Hatch rates were measured for the offspring of wild-type, armi1, armi72.1, and spn-E1 homozygous males mated to Oregon R females. For spn-E1 males, 82% of the progeny hatched. Seventy-five percent of the progeny of armi1 males hatched, but only 45% for armi72.1. In contrast, 97% of the offspring of wild-type males hatched. Thus, armi72.1 is a stronger allele than armi1, at least with respect to the requirement for armi in testes (Tomari, 2004).
In contrast to wild-type, lysates prepared from armi72.1 ovaries do not support siRNA-directed target cleavage in vitro: no cleavage product was observed in the armi72.1 lysate after 2 hr. This result was observed for more than ten independently prepared lysates. To determine if the RNAi defect was allele specific, ovaries from armi1 were tested. Phenotypically, this allele is weaker than armi72.1 in its effects on both male fertility and oogenesis. For armi72.1 females, 92% of the eggs lacked dorsal appendages, compared to 67% for armi1 eggs, and some armi1 eggs had wild-type or partially fused dorsal appendages. Consistent with its weaker phenotype, the armi1 allele showed a small amount of RNAi activity in vitro. The two alleles were analyzed together at least four times using independently prepared lysates. In all assays, total protein concentration was adjusted to be equal. Lysate from the revertant allele, armirev, which has wild-type dorsal appendages, showed robust RNAi, demonstrating that the RNAi defect in the mutants is caused by mutation of armi, not an unlinked gene (Tomari, 2004).
The rate of target cleavage was much slower for armi1 than for wild-type . Since the rate of target cleavage in this assay usually reflects the concentration of RISC, it was hypothesized that armi mutants are defective in RISC assembly. To test this hypothesis, a method to measure RISC was developed that requires less lysate than previously described techniques. Double-stranded siRNA was incubated with ovary lysate in a standard RNAi reaction. To detect RISC, a 5' 32P-radiolabled, 2'-O-methyl oligonucleotide complementary to the antisense strand of the siRNA was added. Like target RNAs, 2'-O-methyl oligonucleotides can bind to RISC containing a complementary siRNA, but unlike RNA targets, they cannot be cleaved and binding is essentially irreversible (Hutvagner, 2004). RISC/2'-O-methyl oligonucleotide complexes were then resolved by electrophoresis through an agarose gel (Tomari, 2004).
To validate the method, RISC formation was examined in embryo lysate. Four distinct complexes (C1, C2, C3, C4) were formed when siRNA was added to the reaction. Formation of these complexes required ATP and was disrupted by pre-treatment of the lysate with the alkylating agent N-ethylmaleimide (NEM), but it was refractory to NEM treatment after RISC assembly; these are all properties of RNAi itself. No complex was observed with an siRNA unrelated to the 2'-O-methyl oligonucleotide. The amount of complex formed by different siRNA sequences correlated well with their capacity to mediate cleavage. The four complexes were also detected in wild-type ovary lysate, suggesting that the same RNAi machinery is used during oogenesis and early embryogenesis. The lower amount of RISC formed in ovary compared to embryo lysates can be explained by the lower overall protein concentration of ovary lysates (Tomari, 2004).
The 2'-O-methyl oligonucleotide/native gel assay was used to analyze RISC assembly in armi mutant ovary lysates. armi mutants are deficient in RISC assembly. The extent of the deficiency correlated with allele strength: less C3/C4 complex formed in lysate from the strong armi72.1 allele than from armi1. Compared to the phenotypically wild-type armirev, >10-fold less RISC was produced in armi72.1 (Tomari, 2004).
The defect in RISC assembly in armi mutants is similar to that observed in lysates from aubHN2 ovaries.
aub mutants do not support RNAi following egg activation and
fail to silence the Ste locus in testes, and lysates from
aubHN2 ovaries do not support RNAi in vitro. Aub is one of five Drosophila Argonaute
proteins, core constituents of RISC. It is therefore not surprising
that Aub is required for RISC assembly. Since RISC assembly in vitro
was not detectable in aubHN2 lysates, the data
suggest that Aub is the primary Argonaute protein recruited to
exogenous siRNA in Drosophila ovaries. In contrast, ovaries
from nanosBN, a maternal effect mutant not
implicated in RNAi, are fully competent for both RISC assembly and
siRNA-directed target RNA cleavage (Tomari, 2004).
To date, few natural cases of RNA-silencing-mediated regulation have been
described. Repression has been analyzed of testis-expressed Stellate
genes by the homologous Suppressors of Stellate [Su(Ste)]
repeats that produce sense and antisense short RNAs. The Stellate
promoter is dispensable for suppression, but local disturbance of
complementarity between the Stellate transcript and the
Su(Ste) repeats impairs silencing. Using in situ RNA
hybridization, temporal control was found of the expression and spatial
distribution of sense and antisense Stellate and Su(Ste)
transcripts in germinal cells. Antisense Su(Ste) transcripts
accumulate in the nuclei of early spermatocytes before the appearance of sense
transcripts. The sense and antisense transcripts are colocalized in the nuclei
of mature spermatocytes, placing the initial step of silencing in the nucleus
and suggesting formation of double-stranded RNA. Mutations in the
aubergine and spindle-E genes, members of the Argonaute and RNA
helicase gene families, respectively, impair silencing by eliminating the short
Su(Ste) RNA, but have no effect on microRNA production. Thus,
different small RNA-containing complexes operate in the male germ line (Aravin,
2004).
Stellate gene transcription yields only sense transcripts, while
Su(Ste) repeats yield both sense and antisense transcripts.
Expression of Stellate and sense Su(Ste) transcripts is
repressed in wild-type males but antisense Su(Ste) transcripts
escape silencing despite their complementarity to short RNAs. Antisense RNAs
accumulate in the nucleoplasm and are not transported into the cytoplasm. This
result supports the proposal that nonpolyadenylated antisense RNAs escape the
cytoplasmic degradation machinery because they are sequestered in the nucleus
(Aravin, 2004).
Sense transcripts are localized in nuclei of mature wild-type primary
spermatocytes. In cry1Y males, in which the Stellate
genes are derepressed, these transcripts are found only in the cytoplasm. These
results correspond to the accumulation of the Stellate-coded protein as
crystalline aggregates in the cytoplasm of mature primary spermatocytes of
cry1Y males. The total amount of Stellate and
Su(Ste) sense transcripts is greatly increased in
cry1Y males. The absence of a nuclear signal in
cry1Y males, contrasts with the presence of sense transcripts
in wild-type nuclei, therefore suggests that these transcripts are never
released from the wild-type nucleus. Nuclear retention of sense transcripts in
the wild type might be explained by the interaction between sense and antisense
transcripts. Nuclear localization of sense and antisense transcripts has also
been observed for bidirectionally transcribed white transgenes, which
induce RNAi of the endogenous white gene (Aravin, 2004).
The distinct sharp dots observed in the nuclei for both sense and antisense
RNAs in mature primary spermatocytes may correspond to the accumulation of the
native transcript at the sites of transcription. The signals are often located
at the border between the chromatin (DAPI stained) and the nucleoplasmic areas
of the nucleus, where actively transcribed loci are thought to be located.
Restricted nuclear signals corresponding to the sites of transcription have been
observed for a number of genes, whereas transcripts in the process of export
from the nucleus are usually below the detection sensitivity of the standard in
situ hybridization technique. The colocalization of the sense and antisense
transcripts suggests the formation of dsRNA in the nucleus, thus placing the
initiation of Stellate silencing in the nucleus. It is proposed that
these nuclear dsRNA species may involve hybrids between sense and antisense
Su(Ste) transcripts, as well as between sense Stellate and
antisense Su(Ste) transcripts, and that these hybrids are
essential for Stellate silencing by Su(Ste) (Aravin, 2004).
A strong correlation is observed between Stellate silencing and the
presence in testes of sense and antisense 25- to 27-nt RNAs homologous to
Stellate and Su(Ste) sequences. The short RNAs are absent
when Stellate genes are derepressed as a consequence of either a
Su(Ste) locus deletion or mutations in the aub and
spn-E genes. The cloning of short RNA from D. melanogaster testes
also demonstrates the presence of short RNAs that are derived from
Su(Ste) and are highly homologous to Stellate. A rigid size
restriction of 21 to 23 nt has, however, been observed for siRNA in various in
vitro studies of D. melanogaster RNAi. Examination of Dicer activity with
different dsRNAs suggests a strong specificity of processing to 21- to 23-nt
fragments in both Drosophila embryo extracts and cell culture.
Furthermore, investigation of the functional anatomy of chemically synthesized
siRNAs in embryo extracts defined the optimal length of siRNAs as 21 to 23 nt,
while RNAs longer than 24 nt have practically no cognate-mRNA cleavage activity.
It has been proposed that only RNAs that meet this size requirement can be
loaded into the RISC. However, examples of the existence of two size classes of
short RNAs (21 or 22 nt and 24 to 26 nt) involved in silencing have also been
reported. Two different size variants of short RNAs were observed during
artificial silencing in plants, with the short variant responsible for
posttranscriptional gene silencing and the long one most likely participating in
DNA methylation and spreading of the silencing signal. Furthermore, only RNAs
from the long class have been detected that correspond to endogenous plant
transposable elements. Two size classes of short RNAs are produced from dsRNA in
plant extracts, and the activity of different Dicer proteins was shown to be
responsible for producing each class. Cloning of endogenous short RNAs from
D. melanogaster has also identified two size classes of short RNAs, with
the short class (21 to 23 nt) including microRNAs and the long class (24 to 26
nt) comprising sequences derived from transcripts of transposable elements and
other repetitive heterochromatic sequences (Aravin, 2004).
The larger size of the short Su(Ste) RNA may be explained by
specific sequences affecting dsRNA processing by Dicer or by the presence in
testes of specific factors that change the cleavage interval of dsRNA. However,
exogenous Su(Ste) dsRNA is cleaved into 21- to 23-nt siRNA in
testis extracts, most likely reflecting the activity of the same Dicer protein
that acts in somatic tissues. The hypothesis is favored that the 25- to 27-nt
Su(Ste) RNAs detected in vivo are produced by a mechanism at least
partially different from conventional siRNA production. A clue to the origin of
the short Su(Ste) RNAs comes from the finding that
Su(Ste) dsRNA formation occurs in the nucleus, unlike that of
artificial RNAi, in which dsRNA is believed to be processed in the cytoplasm.
Both conventional-size siRNA and a longer short RNA have been observed during
viroid replication in the plant nucleus. Two size classes of short RNAs may be
produced in D. melanogaster by different Dicer proteins, as has been
demonstrated in plants. Alternatively, specific nuclear factors may affect how a
single Dicer protein processes dsRNA in the nucleus (Aravin, 2004).
Mutations in the aub and spn-E genes lead to elimination of
short Su(Ste) RNA in testes. However, neither mutation affects
processing of exogenously provided dsRNA to 21- to 23-nt siRNA in testis
extracts. It has been observed that both aub and spn-E mutations
block RNAi in oocytes produced by injected dsRNA. It has been proposed that both
proteins affect RNAi because of their involvement in translational control, but
the results suggest that Aub and Spn-E may be involved in the production and/or
stabilization of siRNA. Similarly, the rde-1 and mut-7 genes of
Caenorhabditis elegans are required for the production of siRNA in vivo
but are dispensable for dsRNA processing in vitro. The corresponding proteins
are required for long-term stabilization of siRNA rather than for dsRNA
processing (Aravin, 2004).
The aub and spn-E mutations eliminate the short
Su(Ste) RNA without affecting the abundance of two different
microRNAs in testes. It is proposed that distinct protein complexes mediate
production and/or stabilization of short Su(Ste) RNA and microRNAs
in testes. Similarly, different members of the Argonaute family participate in
artificial RNAi and in microRNA processing in C. elegans and plants,
despite the central role of Dicer in both processes (Aravin, 2004).
Homologous silencing mediated by short RNA may occur by posttranscriptional
degradation of mRNA and by DNA and chromatin modification leading to
transcriptional repression. The nuclear antisense RNA accumulation and dsRNA
formation that was found in this study raises the question of whether
posttranscriptional or transcriptional mechanisms of silencing operate in
Stellate repression. For animals, it is generally believed that
artificial RNAi caused by dsRNA leads to posttranscriptional degradation of
mRNA. However, it has been shown that dsRNA or short RNA can affect
transcription and chromatin structure of homologous sequences in plants and
Saccharomyces cerevisiae. In plants, for example, transcriptional
silencing of reporter constructs can be caused if the dsRNA produced by hairpin
constructs or virus infection is homologous to the untranscribed promoter region
of the target gene, while posttranscriptional degradation of the corresponding
mRNA occurs if there is homology between the dsRNA and the transcribed sequence
(Aravin, 2004).
Constructs containing the Stellate coding sequence driven by a
heterologous promoter are regulated by Su(Ste) repeats in the same
manner as native Stellate genes or reporter constructs with
Stellate sequence fused to lacZ. In contrast, expression of the
endogenous ßNac-like genes, having a putative promoter region with
high levels of sequence similarity (95%) to Stellate but an unrelated
transcribed sequence, shows no response to the deletion of
Su(Ste). In the present study, it was also found that nucleotide
substitutions in the transcribed region of a Stellate fragment homologous
to the Su(Ste) sequence lead to a release of silencing. Thus,
homology to Su(Ste) in the untranscribed region is dispensable for
repression, while local disturbance of complementarity in the transcribed
sequence impairs silencing. The possibility that regulatory sequences important
for transcriptional silencing may be present in the transcribed region cannot be
ruled out, but the results are more simply explained by a posttranscriptional
Stellate silencing mechanism (Aravin, 2004).
The two blocks of tandemly repeated Stellate genes are located in
intercalary and constitutive heterochromatin of the X chromosome, and
Su(Ste) repeats are located in the heterochromatic Y chromosome.
siRNA-mediated transcriptional repression of centromeric heterochromatin repeats
has been recently demonstrated in S. cerevisiae. The results do not
exclude participation of transcriptional repression of genomic Stellate
repeats acting in concert with a posttranscriptional mechanism. Similarly, both
transcriptional and posttranscriptional mechanisms have been shown to operate in
the repression of multicopy transgenes associated with the presence of
homologous short RNA in D. melanogaster. Thus, both transcriptional and
posttranscriptional mechanisms might act in Stellate silencing, and
further studies will be directed to understanding the contribution of each of
them (Aravin, 2004).
microRNAs (miRNAs) are a large family of 21- to 22-nucleotide non-coding RNAs that interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. miRNAs are excised in a stepwise process from primary miRNA
(pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves
pri-miRNAs to release hairpin-shaped precursor miRNAs (pre-miRNAs). These
pre-miRNAs are then exported to the cytoplasm and further processed by Dicer to
mature miRNAs. Drosophila Dicer-1 interacts with Loquacious, a
double-stranded RNA-binding domain protein. Depletion of Loquacious results in
pre-miRNA accumulation in Drosophila S2 cells, as is the case for depletion of
Dicer-1. Immuno-affinity purification experiments revealed that along with
Dicer-1, Loquacious resides in a functional pre-miRNA processing complex, and
stimulates and directs the specific pre-miRNA processing activity. Efficient miRNA-directed silencing of a reporter transgene, complete repression
of white by a dsRNA trigger, and silencing of the endogenous
Stellate locus by Suppressor of Stellate, all require Loqs. In
loqsf00791 mutant ovaries, germ-line stem cells are not
appropriately maintained. Loqs associates with Dcr-1, the Drosophila
RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known
Drosophila RNase-III endonuclease is paired with a dsRBD protein that
facilitates its function in small RNA biogenesis. These results
support a model in which Loquacious mediates miRNA biogenesis and, thereby, the
expression of genes regulated by miRNAs (Forstemann, 2005; Saito, 2005).
loqsf00791 males are incompletely fertile. When Oregon R
females were mated to loqsf00791 homozygous mutant males,
only 17% of embryos hatched; for
loqsf00791 heterozygous males, 47% of embryos hatched.
Ninety percent of embryos hatched for
wild-type Oregon R males. Genes required for RNA silencing often reduce male
fertility, because the X-linked gene Ste is epigenetically silenced in
testes by dsRNA derived from the bi-directionally transcribed Suppressor of
Stellate (Su(Ste)) locus. Ste silencing is
genetically similar, but not identical, to RNAi, in that like RNAi it requires
the function of the gene armitage (armi), but unlike RNAi does not require
r2d2. In the absence of Ste
silencing, Stellate protein accumulates as protein crystals in the testes.
loqsf00791 mutants contain Stellate crystals in their testes, much like
armi72.1 mutants, identifying a second role for
loqs in silencing by endogenous RNA triggers, distinct from its
function in miRNA biogenesis (Forstemann, 2005).
In the Drosophila germline, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Whereas small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appears not to require Dicer-1, which makes microRNAs (miRNAs), or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2',3' hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. These data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways (Vagin, 2006).
In plants and animals, RNA silencing pathways defend against viruses, regulate endogenous gene expression, and protect the genome against selfish genetic elements such as retrotransposons and repetitive sequences. Common to all RNA silencing pathways are RNAs 19 to 30 nucleotides (nt) long that specify the target RNAs to be repressed. In RNA interference (RNAi), siRNAs are produced from long exogenous double-stranded RNA (dsRNA). In contrast, ~22-nt miRNAs are endonucleolytically processed from endogenous RNA polymerase II transcripts. Dicer ribonuclease III (RNase III) enzymes produce both siRNAs and miRNAs. In flies, Dicer-2 (Dcr-2) generates siRNAs, whereas the Dicer-1 (Dcr-1)–Loquacious (Loqs) complex produces miRNAs. After their production, small silencing RNAs bind Argonaute proteins to form the functional RNA silencing effector complexes believed to mediate all RNA silencing processes (Vagin, 2006 and references therein).
In Drosophila, processive dicing of long dsRNA and the accumulation of sense and antisense siRNAs without reference to the orientation of the target mRNA are hallmarks of RNAi in vitro. Total small RNA was prepared from the heads of adult males expressing a dsRNA hairpin that silences the white gene via the RNAi pathway. white silencing requires Dcr-2, R2D2, and Ago2. siRNAs were detected with a microarray containing TM (melting temperature)–normalized probes, 22 nt long, for all sense and antisense siRNAs that theoretically can be produced by dicing the white exon 3 hairpin. Both sense and antisense white siRNAs were detected in wild-type flies but not in dcr-2L811fsX homozygous mutant flies. The Dcr-2–dependent siRNAs were produced with a periodicity of ~22 nt, consistent with the phased processing of the dsRNA hairpin from the end formed by the 6-nt loop predicted to remain after splicing of its intron-containing primary transcript (Vagin, 2006).
Drosophila repeat-associated small interfering RNAs (rasiRNAs) can be distinguished from siRNAs by their longer length, 24 to 29 nt. rasiRNAs have been proposed to be diced from long dsRNA triggers, such as the ~50 copies of the bidirectionally transcribed Suppressor of Stellate [Su(Ste)] locus on the Y chromosome that in testes silence the ~200 copies of the protein-coding gene Stellate (Ste) found on the X chromosome (Vagin, 2006).
Microarray analysis of total small RNA isolated from fly testes revealed that Su(Ste) rasiRNAs detectably accumulate only from the antisense strand, with little or no phasing. As expected, Su(Ste) rasiRNAs were not detected in testes from males lacking the Su(Ste) loci (cry1Y). Su(Ste) rasiRNAs were also absent from armitage (armi) mutant testes, which fail to silence Ste and do not support RNAi in vitro. armi encodes a non–DEAD-box helicase homologous to the Arabidopsis thaliana protein SDE3, which is required for RNA silencing triggered by transgenes and some viruses, and depletion by RNAi of the mammalian Armi homolog Mov10 blocks siRNA-directed RNAi in cultured human cells. Normal accumulation of Su(Ste) rasiRNA and robust Ste silencing also require the putative helicase Spindle-E (Spn-E), a member of the DExH family of adenosine triphosphatases (Vagin, 2006).
The accumulation in vivo of only antisense rasiRNAs from Su(Ste) implies that sense Su(Ste) rasiRNAs either are not produced or are selectively destroyed. Either process would make Ste silencing mechanistically different from RNAi. In support of this view, mutations in the central components of the Drosophila RNAi pathway—dcr-2, r2d2, and ago2—did not diminish Su(Ste) rasiRNA accumulation. Deletion of the Su(Ste) silencing trigger (cry1Y) caused a factor of ~65 increase in Ste mRNA, but null or strong hypomorphic mutations in the three key RNAi proteins did not (Vagin, 2006).
Fly Argonaute proteins can be subdivided into the Ago (Ago1 and Ago2) and Piwi [Aubergine (Aub), Piwi, and Ago3] subfamilies. Unlike ago1 and ago2, the aub, piwi, and ago3 mRNAs are enriched in the germline. Aub is required for Ste silencing and Su(Ste) rasiRNA accumulation. In aubHN2/aubQC42 trans-heterozygous mutants, Su(Ste) rasiRNAs were not detected by microarray or Northern analysis, and Su(Ste)-triggered silencing of Ste mRNA was lost completely. Even aubHN2/+ heterozygotes accumulated less of the most abundant Su(Ste) rasiRNA than did the wild type. That the Ago subfamily protein Ago2 is not required for Ste silencing, whereas the Piwi subfamily protein Aub is essential for it, supports the view that Ste is silenced by a pathway distinct from RNAi. Intriguingly, Su(Ste) rasiRNAs hyperaccumulated in piwi mutant testes, where Ste is silenced normally (Vagin, 2006).
Mutations in aub also cause an increase in sense, but not antisense, Su(Ste) RNA; these results suggest that antisense Su(Ste) rasiRNAs can silence both Ste mRNA and sense Su(Ste) RNA, but that no Su(Ste) rasiRNAs exist that can target the antisense Su(Ste) transcript. The finding that Su(Ste) rasiRNAs are predominantly or exclusively antisense is essentially in agreement with the results of small RNA cloning experiments, in which four of five Su(Ste) rasiRNAs sequenced were in the antisense orientation, but is at odds with earlier reports detecting both sense and antisense Su(Ste) rasiRNAs by non-quantitative Northern hybridization (Vagin, 2006).
Is germline RNA silencing of selfish genetic elements generally distinct from the RNAi and miRNA pathways? The expression of a panel of germline-expressed selfish genetic elementswas examined in mutants defective for eight RNA silencing proteins: three long terminal repeat (LTR)-containing retrotransposons (roo, mdg1, and gypsy); two non-LTR retrotransposons (I-element and HeT-A, a component of the Drosophila telomere), and a repetitive locus (mst40). All selfish genetic elements tested behaved like Ste: Loss of the RNAi proteins Dcr-2, R2D2, or Ago2 had little or no effect on retrotransposon or repetitive element silencing. Instead, silencing required the putative helicases Spn-E and Armi plus one or both of the Piwi subfamily Argonaute proteins, Aub and Piwi. Silencing did not require Loqs, the dsRNA-binding protein required to produce miRNAs (Vagin, 2006).
The null allele dcr-1Q1147X is homozygous lethal, making it impossible to procure dcr-1 mutant ovaries from dcr-1Q1147X/dcr-1Q1147X adult females. Therefore, clones of dcr-1Q1147X/dcr-1Q1147X cells were generated in the ovary by mitotic recombination in flies heterozygous for the dominant female-sterile mutation ovoD1. RNA levels, relative to rp49 mRNA, were measured for three retrotransposons (roo, HeT-A, and mdg1) and one repetitive sequence (mst40) in dcr-1/dcr-1 recombinant ovary clones and in ovoD1/TM3 and dcr-1/ovoD1 nonrecombinant ovaries. The ovoD1 mutation blocks oogenesis at stage 4, after the onset of HeT-A and roo rasiRNA production. Retrotransposon or repetitive sequence transcript abundance was unaltered or decreased in dcr-1/dcr-1 relative to ovoD1/TM3 and dcr-1/ovoD1 controls. It is concluded that Dcr-1 is dispensable for silencing these selfish genetic elements in the Drosophila female germline (Vagin, 2006).
roo is the most abundant LTR retrotransposon in flies. roo silencing was analyzed in the female germline with the use of microarrays containing 30-nt probes, tiled at 5-nt resolution, for all ~18,000 possible roo rasiRNAs; the data was corroborated at 1-nt resolution for those rasiRNAs derived from LTR sequences. As observed for Su(Ste) but not for white RNAi, roo rasiRNAs were nonhomogeneously distributed along the roo sequence and accumulated primarily from the antisense strand. In fact, the most abundant sense rasiRNA peak corresponded to a set of probes containing 16 contiguous uracil residues, which suggests that these probes nonspecifically detected fragments of the mRNA polyadenylate [poly(A)] tail. Most of the remaining sense peaks were unaltered in armi mutant ovaries, in which roo expression is increased; this result implies that they do not contribute to roo silencing. No phasing was detected in the distribution of roo rasiRNAs (Vagin, 2006).
As for Su(Ste), wild-type accumulation of antisense roo rasiRNA required the putative helicases Armi and Spn-E and the Piwi subfamily Argonaute proteins Piwi and Aub, but not the RNAi proteins Dcr-2, R2D2, and Ago2. Moreover, accumulation of roo rasiRNA was not measurably altered in loqs f00791, an allele that strongly disrupts miRNA production in the female germline (Vagin, 2006).
Loss of Dcr-2 or Dcr-1 did not increase retrotransposon or repetitive element expression, which suggests that neither enzyme acts in rasiRNA-directed silencing. Moreover, loss of Dcr-2 had no detectable effect on Su(Ste) rasiRNA in testes or roo rasiRNA in ovaries. The amount of roo rasiRNA and miR-311 was measured in dcr-1/dcr-1 ovary clones generated by mitotic recombination. Comparison of recombinant (dcr-1/dcr-1) and nonrecombinant (ovoD1/TM3 and dcr-1/ovoD1) ovaries by Northern analysis revealed that roo rasiRNA accumulation was unperturbed by the null dcr-1Q1147X mutation. Pre–miR-311 increased and miR-311 declined by a factor of ~3 in the dcr-1/dcr-1 clones, consistent with about two-thirds of the tissue corresponding to mitotic dcr-1/dcr-1 recombinant cells. Yet, although most of the tissue lacked dcr-1 function, improved, rather than diminished, silencing was observed for the four selfish genetic elements examined. Moreover, the dsRNA-binding protein Loqs, which acts with Dcr-1 to produce miRNAs, was also dispensable for roo rasiRNA production and selfish genetic element silencing. Although the possibility that dcr-1 and dcr-2 can fully substitute for each other in the production of rasiRNA in the ovary cannot be excluded, biochemical evidence suggests that none of the three RNase III enzymes in flies—Dcr-1, Dcr-2, and Drosha—can cleave long dsRNA into small RNAs 24 to 30 nt long (Vagin, 2006).
Animal siRNA and miRNA contain 5' phosphate and 2',3' hydroxy termini. Enzymatic and chemical probing was used to infer the terminal structure of roo and Su(Ste) rasiRNAs. RNA from ovaries or testes was treated with calf intestinal phosphatase (CIP) or CIP followed by polynucleotide kinase plus ATP. CIP treatment caused roo and Su(Ste) rasiRNA to migrate more slowly in polyacrylamide gel electrophoresis, consistent with the loss of one or more terminal phosphate groups. Subsequent incubation with polynucleotide kinase and ATP restored the original gel mobility of the rasiRNAs, indicating that they contained a single 5' or 3' phosphate before CIP treatment. The roo rasiRNA served as a substrate for ligation of a 23-nt 5' RNA adapter by T4 RNA ligase, a process that requires a 5' phosphate; pretreatment with CIP blocked ligation, thus establishing that the monophosphate lies at the 5' end. The rasiRNA must also contain at least one terminal hydroxyl group, because it could be joined by T4 RNA ligase to a preadenylated 17-nt 3' RNA adapter. Notably, the 3' ligation reaction was less efficient for the roo rasiRNA than for a miRNA in the same reaction (Vagin, 2006).
RNA from ovaries or testes was reacted with NaIO4, then subjected to ß-elimination, to determine whether the rasiRNA had either a single 2' or 3' terminal hydroxy group or had terminal hydroxy groups at both the 2' and 3' positions, as do animal siRNA and miRNA. Only RNAs containing both 2' and 3' hydroxy groups react with NaIO4; ß-elimination shortens NaIO4-reacted RNA by one nucleotide, leaving a 3' monophosphate terminus, which adds one negative charge. Consequently, NaIO4-reacted, ß-eliminated RNAs migrate faster in polyacrylamide gel electrophoresis than does the original unreacted RNA. Both roo and Su(Ste) rasiRNA lack either a 2' or a 3' hydroxyl group, because they failed to react with NaIO4; miRNAs in the same samples reacted with NaIO4. Together, these results show that rasiRNAs contain one modified and one unmodified hydroxyl. Because T4 RNA ligase can make both 3'-5' and 2'-5' bonds, the blocked position cannot currently be determined. Some plant small silencing RNAs contain a 2'-O-methyl modification at their 3' terminus (Vagin, 2006).
Drosophila and mammalian siRNA and miRNA function through members of the Ago subfamily of Argonaute proteins, but Su(Ste) and roo rasiRNAs require at least one member of the Piwi subfamily for their function and accumulation. To determine whether roo rasiRNAs physically associate with Piwi and Aub, ovary lysate were prepared from wildtype flies or transgenic flies expressing either myc-tagged Piwi or green fluorescent protein (GFP)–tagged Aub protein; they were immunoprecipitated with monoclonal antibodies (mAbs) to myc, GFP, or Ago1; and then the supernatant and antibody-bound small RNAs were analyzed by Northern blotting. Six different roo rasiRNAs were analyzed. All were associated with Piwi but not with Ago1, the Drosophila Argonaute protein typically associated with miRNAs; miR-8, miR-311, and bantam immunoprecipitated with Ago1 mAb. No rasiRNAs immunoprecipitated with the myc mAb when lysate was used from flies lacking the myc-Piwi transgene (Vagin, 2006).
Although aub mutant ovaries silenced roo mRNA normally, they showed reduced accumulation of roo rasiRNA relative to aub/+ heterozygotes, which suggests that roo rasiRNAs associate with both Piwi and Aub. The supernatant and antibody-bound small RNAs were analyzed after GFP mAb immunoprecipitation of ovary lysate from GFP-Aub transgenic flies and flies lacking the transgene. roo rasiRNA was recovered only when the immunoprecipitation was performed with the GFP mAb in ovary lysate from GFP-Aub transgenic flies. The simplest interpretation of these data is that roo rasiRNAs physically associate with both Piwi and Aub, although it remains possible that the roo rasiRNAs are loaded only into Piwi and that Aub associates with Piwi in a stable complex. The association of roo rasiRNA with both Piwi and Aub suggests that piwi and aub are partially redundant, as does the modest reduction in roo silencing in piwi but not in aub mutants. Alternatively, roo silencing might proceed through Piwi alone, but the two proteins could function in the same pathway to silence selfish genetic elements (Vagin, 2006).
These data suggest that in flies, rasiRNAs are produced by a mechanism that requires neither Dcr-1 nor Dcr-2, yet the patterns of rasiRNAs that direct roo and Ste silencing are as stereotyped as the distinctive siRNA population generated from the white hairpin by Dcr-2 or the unique miRNA species made from each pre-miRNA by Dcr-1. A key challenge for the future will be to determine what enzyme makes rasiRNAs and what sequence or structural features of the unknown rasiRNA precursor lead to the accumulation of a stereotyped pattern of predominantly antisense rasiRNAs (Vagin, 2006).
Small silencing RNAs repress gene expression by a set of related mechanisms collectively called RNA-silencing pathways. In the RNA interference (RNAi) pathway, small interfering mRNA (siRNAs) defend cells from invasion by foreign nucleic acids, such as those produced by viruses. In contrast, microRNAs (miRNAs) sculpt endogenous mRNA expression. A third class of small RNAs, Piwi-interacting RNAs (piRNAs), defends the genome from transposons. This study reports that Drosophila piRNAs contain a 2'-O-methyl group on their 3' termini; this is a modification previously reported for plant miRNAs and siRNAs and mouse and rat piRNAs. Plant small-RNA methylation is catalyzed by the protein HEN1. Drosophila melanogaster Hen1 (DmHen1), the Drosophila homolog of HEN1, termed Pimet (piRNA methyltransferase) by Saito (2007) in a parallel study, methylates the termini of siRNAs and piRNAs. Without DmHen1, the length and abundance of piRNAs are decreased, and piRNA function is perturbed. Unlike plant HEN1, DmHen1 acts on single strands, not duplexes, explaining how it can use as substrates both siRNAs, which derive from double-stranded precursors, and piRNAs which do not. 2'-O-methylation of siRNAs may be the final step in assembly of the RNAi-enzyme complex, RISC, occurring after the Argonaute-bound siRNA duplex is converted to single-stranded RNA (Horwich, 2007; Saito, 2007).
The 3' termini of two types of highly abundant piRNAs were examined in the germline of flies heterozygous or homozygous for hen1f00810. In testes, the Suppressor of Stellate [Su(Ste)] locus produces 24-27 nucleotide rasiRNAs, a subclass of piRNAs that directs silencing of the selfish genetic element Stellate. Su(Ste) rasiRNAs, like other Drosophila piRNAs, are modified at their 3' termini and therefore do not react with NaIO4. In contrast, Su(Ste) rasiRNAs from hen1f00810/hen1f00810 mutant testes reacted with NaIO4 and could therefore be β-eliminated to remove the last nucleotide of the RNA, thereby increasing their gel mobility and indicating that in the absence of DmHen1 protein, they are not modified. Similarly, rasiRNAs that guide silencing of roo, the most abundant retrotransposon in Drosophila melanogaster, were not modified in hen1f00810 homozygous ovaries. The Su(Ste) and roo rasiRNAs were also shorter in the hen1f00810 homozygotes. In contrast, the length and amount of miR-8, which is expressed in both the male and female germline, was unaltered in hen1f00810 homozygotes. For both Su(Ste) and roo, rasiRNAs were on average shorter and less modified even in hen1f00810 heterozygotes, compared to the wild-type, suggesting that the abundance of DmHen1 protein limits the stability or production of piRNAs in
In vitro experiments have shown that Stellate can interact with the catalytic ß subunit of casein kinase 2 enzyme, altering CK2 activity (Bozzetti, 1995).
Males of Drosophila melanogaster lacking the Y chromosome-linked Su(Ste) locus show multiple meiotic alterations, including chromosome disorganization and prominent crystal formation in primary spermatocytes. These alterations are due to the derepression of the X chromosome-linked Stellate sequences. To understand how the derepression of the Stellate elements gives rise to these abnormalities, the protein encoded by the Stellate sequences was expressed in bacteria and an antibody against the fusion protein was produced. Immunostaining of Su(Ste) minus testes has clearly shown that the Stellate protein is a major component of the crystals. The Ste protein is absent in XY males but is a major component of the crystalline aggregates in Su(Ste) mutant males. (Bozzetti, 1995).
Drosophila melanogaster males deficient for the crystal (cry) locus of the Y chromosome that carries between 15 and 60 copies of the X-linked Stellate (Ste) gene are semisterile, have elevated levels of nondisjunction, produce distorted sperm genotype ratios (meiotic drive), and evince hyperactive transcription of Ste in the testes. Ste seems to be the active element in this system, and it has been proposed that the ancestral Ste gene was 'selfish' and increased in frequency because it caused meiotic drive. This hypothetical evolutionary history is based on the idea that Ste overexpression, and not the lack of cry, causes the meiotic drive of cry- males. To test whether this is true, a Ste-deleted X chromosome was constructed and the phenotype of Ste-/cry- males was examined. If hyperactivity of Ste were necessary for the transmission defects seen in cry- males, cry- males completely deficient for Ste would be normal. Although it is impossible to construct a completely Ste- genotype, it was found that Ste-/cry- males have exactly the same phenotype as Ste+/cry- males. The deletion of all X chromosome Ste copies not only does not eliminate meiotic drive and nondisjunction, but it also does not even reduce them below the levels produced when the X carries 15 copies of Ste (Belloni, 2002).
Meiotic drive was proposed originally as an evolutionary mechanism, but it has been difficult to demonstrate that any example of drive serves this role. Hurst (1992 and 1996) has suggested that the cry-Ste system could be an evolutionarily important case. Paraphrasing his reasoning:
This is an attractive hypothesis, but its correctness depends on the validity of the first two points. It is not obvious, however, that hyperactivity of Ste necessarily implies that it is the element that causes the drive, nor does the lack of crystals in spermatocytes of D. simulans XO males necessarily mean that cry is not present somewhere in that species' genome (Belloni, 2002).
A Ste-deleted X chromosome was created to test the first of these premises. There is not the slightest hint that Ste is necessary for the meiotic drive that occurs in cry- males. It is the absence of cry, and not hyperactivation of Ste, that causes meiotic drive. To get around this conclusion would require that the Ste activity induced in the Stellate depleted BSY-born Stellate copies is sufficient to provoke drive and that there is no response to doubling its Ste copy number and testes-specific Ste transcription, even though there is a linear response to further increases in Ste copy number. For now, it seems preferable to think that deletion of cry triggers the downstream problems of sperm function, but that the downstream targets, if they include Ste at all, must include other elements as well. There is no lack for candidates: the cis-responsive element that has been identified in translocation-provoked meiotic drive, recessive autosomal mutants that accumulate Ste protein, or the transposable elements that are reported to respond to cry deletion (Belloni, 2002).
If the role of cry+ is not merely the suppression of a parasitic Ste element, what does it do? Perhaps cry+ and the other genes whose mutants mimic cry- form part of a general gene-silencing system or part of a system aimed particularly at silencing repeated elements that must act before spermiogenesis can proceed. If so, meiotic drive is not the result of a normal checkpoint control that eliminates aneuploid products, but is instead one symptom of the dysgenesis produced by the testicular expression of genes (Ste and others) that should have been shut off by the end of meiosis. Working out the cellular pathway that, disrupted, leads to meiotic drive remains a problem yet to be resolved (Belloni, 2002).
Construction of the Ste-deleted chromosomes also allowed the direct measurement of the activity of the transposed heterochromatic block of Ste copies present in the BSY chromosome. Testes-specific transcription of those copies is induced by deleting cry, but the heterochromatic copies appear to be substantially less active in the testes than are the euchromatic copies. While not directly relevant to the question that led to these experiments, the tightly regulated level of the 1400-nt ubiquitous transcript (independent of cry, independent of copy number, and independent of heterochromatic or euchromatic origin) needs to be considered as attempts are made to understand the findings that implicate an iRNA in the regulation of Ste. How, in the very same tissue, is production of one Ste transcript inhibited by cry+, while the other is not only not inhibited, but is produced in a copy-number-compensated fashion (Belloni, 2002)?
At the same time as these experiments were undertaken, Palumbo and Bozzetti (personal communication to Belloni, 2002) independently started a species survey of Ste and cry sequences. That project examines the second of the key assumptions in the evolutionary hypothesis, and their results are no more coherent with the selfish evolution of Ste than are those reported here. For example, although there is no crystal production in spermatocytes of D. simulans XO males, the D. simulans Y chromosome does carry Ste-like repeats (Belloni, 2002).
Numerous situations in D. melanogaster cause distorted sperm recovery, including rDNA deletions, segregation of some translocations, segregation of univalents, compound autosomes, deletion of cry, and mutation of other genes that interact in the cry-Ste system. Hence, meiotic drive, in the sense of distorted sperm recovery rather than in the sense of an evolutionary force, seems to be a general downstream response to meiotic problems. Whether any situations that activate this process are of evolutionary importance remains to be seen (Belloni, 2002).
Repeated elements are remarkably important for male meiosis and spermiogenesis in Drosophila melanogaster. Pairing of the X and Y chromosomes is mediated by the ribosomal RNA genes of the Y chromosome and X chromosome heterochromatin, and spermiogenesis depends on the fertility factors of the Y chromosome. Intriguingly, a peculiar genetic system of interaction between the Y-linked crystal locus and the X-linked Stellate elements seem to be also involved in male meiosis and spermiogenesis. Deletion of the crystal element of the Y, via an interaction with the Stellate elements of the X, causes meiotic abnormalities, gamete-genotype dependent failure of sperm development (meiotic drive), and deposition of protein crystals in spermatocytes. The current hypothesis is that the meiotic abnormalities observed in cry- males is due to an induced overexpression of the normally repressed Ste elements. An implication of this hypothesis is that the strength of the abnormalities would depend on the amount of the Ste copies. To test this point the relationship of Ste copy number and organization to meiotic behavior in cry- males has been genetically and cytologically examined. Heterochromatic as well as euchromatic Ste repeats are functional and the abnormality in chromosome condensation and the frequency of nondisjunction are related to Ste copy number. Moreover, meiosis is disrupted after synapsis and cry-induced meiotic drive is probably not mediated by Ste (Palumbo, 1994).
The sting mutation (see aubergine), caused by a P element inserted into polytene region 32D, was isolated by a screen for male sterile insertions in Drosophila melanogaster. This sterility is correlated with the presence of crystals in spermatocytes and spermatids that are structurally indistinguishable from those produced in males carrying a deficiency of the Y-linked crystal (cry) locus. In addition, their morphology is needle-like in Ste+ flies and star-shaped in Ste flies, once again as observed in cry- males. The sti mutation leads to meiotic drive of the sex chromosomes, and the strength of the phenomenon is correlated with the copy number of the repetitive Ste locus. The same correlation is also true for the penetrance of the male sterile mutation. A presumptive sti null allele results in male sterility and lethal maternal effect. The gene was cloned and shown to code for a putative protein that is 866 amino acids long. A C-terminal domain of 82 amino acids is identified that is well conserved in proteins from different organisms. The gene is expressed only in the germline of both sexes. The interaction of sting with the Ste locus can also be demonstrated at the molecular level. While an unprocessed 8-kb Ste primary transcript is expressed in wild-type males, in X/Y homozygous sti males, as in X/Y cry- males, a 0.7-kb mRNA is produced (Schmidt, 1999).
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date revised: 16 January 2008
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