The study of P-element repression in Drosophila led to the discovery of the telomeric Trans-Silencing Effect (TSE), a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequence or TAS) has the capacity to repress in trans in the female germline, a homologous transposon, or transgene located in euchromatin. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. This study reports that TSE displays an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted factor. This silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var)205 encoding Heterochromatin Protein 1 and Su(var)3-7) and the repeat-associated small interfering RNA (or rasiRNA) silencing pathway (aubergine, homeless, armitage, and piwi). In contrast, TSE is not sensitive to mutations affecting r2d2, which is involved in the small interfering RNA (or siRNA) silencing pathway, nor is it sensitive to a mutation in loquacious, which is involved in the micro RNA (or miRNA) silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a repeat-associated small interfering RNA pathway linked to heterochromatin formation, which was co-opted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway (Josse, 2007; full text of article).
Repression of transposable elements (TEs) involves complex mechanisms that can be linked to either small RNA silencing pathways or chromatin structure modifications depending on the species and/or the TE family. Drosophila species are particularly relevant to the study of these repression mechanisms since some families of TEs are recent invaders, allowing genetic analysis to be carried out on strains with or without these TEs. In some cases, crossing these two types of strains induces hybrid dysgenesis, a syndrome of genetic abnormalities resulting from TE mobility. In D. virilis, repression of hybrid dysgenesis has been correlated to RNA silencing since small RNAs of the retroelement Penelope, responsible for dysgenesis, were detected in nondysgenic embryos but not in dysgenic embryos. In D. melanogaster, repression of retrotransposons can be established by noncoding fragments of the corresponding element (I factor, ZAM, and Idefix) and can be in some cases (gypsy, mdg1, copia, Het-A, TART, and ZAM, Idefix) sensitive to mutations in genes from the Argonaute family involved in small RNA silencing pathways. In the same species, strong repression of the DNA P TE, by a cellular state that has been called 'P cytotype', can be established by one or two telomeric P elements inserted in heterochromatic 'Telomeric Associated Sequences' (TAS) at the 1A cytological site corresponding to the left end of the X chromosome. This includes repression of dysgenic sterility resulting from P transposition. This P cytotype is sensitive to mutations affecting both Heterochromatin Protein 1 (HP1) (Ronsseray, 1996) and the Argonaute family member AUBERGINE (Reiss, 2004). P repression corresponds to a new picture of TE repression shown, using an assay directly linked to transposition, to be affected by heterochromatin and small RNA silencing mutants (Josse, 2007).
In the course of the study of P cytotype, a new silencing phenomenon has been discovered. Indeed, a P-lacZ transgene or a single defective P element inserted in TAS can repress expression of euchromatic P-lacZ insertions in the female germline in trans, if a certain length of homology exists between telomeric and euchromatic insertions. This homology-dependent silencing phenomenon has been termed Trans-Silencing Effect (TSE) (Roche, 1998). Telomeric transgenes, but not centromeric transgenes, can be silencers and all euchromatic P-lacZ insertions tested can be targets. TSE is restricted to the female germline and has a maternal effect since repression occurs only when the telomeric transgene is maternally inherited (Ronsseray, 2001). Further, when TSE is not complete, variegating germline lacZ repression is observed from one egg chamber to another, suggesting a chromatin-based mechanism of repression. Recently, an extensive analysis of small RNAs complexed with PIWI family proteins (AUBERGINE, PIWI, and AGO3) was performed in the Drosophila female germline. The latter study showed that most of the RNA sequences associated to these proteins derive from TEs. TSE corresponds likely to such a situation (Josse, 2007).
This study analyzed the genetic properties of TSE and shows that it has an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted stimulating component. Further, in order to investigate the mechanism behind TSE, a candidate gene analysis was performed to identify genes whose mutations impair TSE. It was found that TSE is strongly affected both by mutations in genes involved in heterochromatin formation and in the recently discovered small RNA silencing pathway called 'repeat-associated small interfering RNAs' (rasiRNA) pathway. In contrast, this study shows that TSE is not sensitive to genes specific to the classical RNA interference pathway linked to small interfering RNAs (siRNA) or to the micro RNA (miRNA) pathway. This suggests thus that TSE involves a rasiRNA pathway linked to heterochromatin formation and that such a mechanism, working in the germline, may underlie epigenetic transmission of repression through meiosis (Josse, 2007).
Drosophila syncitial blastoderm embryo lysate has been used widely to study the RNAi pathway. However, armi flies lay few eggs, making it difficult to collect enough embryos to make lysate. To surmount this problem, lysates were prepared from ovaries manually dissected from wild-type or mutant females. Approximately 10 μl of lysate can be prepared from ∼50 ovaries (Tomari, 2004).
The well-characterized siRNA-directed mRNA cleavage assay (Elbashir, 2001a, 2001b) was used to evaluate the capacity of ovary lysate to support RNAi in vitro. Incubation in ovary lysate of a 5' 32P-cap-radiolabeled firefly luciferase mRNA target with a complementary siRNA duplex yielded the 5' cleavage product diagnostic of RNAi. siRNAs containing 5' hydroxyl groups are rapidly phosphorylated in vitro and in vivo, but modifications that block phosphorylation eliminate siRNA activity. Replacing the 5' hydroxyl of the antisense siRNA strand with a 5' methoxy group completely blocks RNAi in the ovary lysate. In Drosophila, siRNAs bearing a single 2'-deoxy nucleotide at the 5' end are poor substrates for the kinase that phosphorylates 5' hydroxy siRNAs (Nykanen, 2001). A comparison of initial cleavage rates shows that in ovary lysate, target cleavage was slower for siRNAs with a 2'-deoxy nucleotide at the 5' end of the antisense strand than for standard siRNAs. Furthermore, the rate of target cleavage was fastest when the siRNA was phosphorylated before its addition to the reaction. A similar enhancement from pre-phosphorylation was reported for siRNA injected into Drosophila embryos (Boutla, 2001). It is concluded that lysates from Drosophila ovaries faithfully recapitulate RNAi directed by siRNA duplexes (Tomari, 2004).
Data suggest that both armi and aub are required genetically for RISC assembly, but they provide no insight into the molecular basis of their RISC assembly defect(s). At what step(s) in RISC assembly are armi and aub blocked? In order to answer this question, protein-siRNA intermediates in the RISC assembly pathway were identified. The 2'-O-methyl oligonucleotide/native gel method detects only complexes competent to bind target RNA (mature RISC). Therefore, a native gel assay designed to detect intermediates in the assembly of RISC was used. The siRNA was radiolabeled, allowing detection of complexes containing either single-stranded or double-stranded siRNA and functionally asymmetric siRNAs (Schwarz, 2003) were used to distinguish between complexes containing single- and double-stranded siRNA (Tomari, 2004).
RISC contains only a single siRNA strand. Functionally asymmetric siRNAs load only one of the two strands of an siRNA duplex into RISC and degrade the other strand (Schwarz, 2003); the relative stability of the 5' ends of the two strands determines which is loaded into RISC. siRNA 1 loads its antisense strand into RISC, whereas siRNA 2 loads the sense strand (Schwarz, 2003). The two siRNA duplexes are identical, except that siRNA 2 contains a C-to-U substitution at position 1, which inverts the asymmetry (Schwarz, 2003). For both siRNAs, the antisense strand was 3' 32P-radiolabeled and will always be present in complexes that contain double-stranded siRNA. However, RISC will contain the 32P-radiolabeled antisense strand only for siRNA 1. siRNA 2 will also make RISC, but it will contain the nonradioactive sense strand (Tomari, 2004).
When either siRNA 1 or siRNA 2 was used to assemble RISC in embryo lysate, two complexes (B and A) were detected in the native gel assay; a third complex was detected only with siRNA 1. This third complex therefore contains single-stranded siRNA and corresponds to RISC. Complexes B and A are good candidates for RISC assembly intermediates. Formation of all three complexes was dramatically reduced when the antisense siRNA strand contained a 5' methoxy group, a modification that blocks RNAi (Nykanen, 2001). When the antisense strand of the siRNA contained a single 5'-deoxy nucleotide, making it a poor substrate for phosphorylation in the lysate (Nykanen, 2001), assembly of all three complexes was reduced. Phosphorylating the 5' deoxy-substituted siRNA before the reaction restored complex assembly. Formation of complex A and of RISC required ATP. In contrast, complex B assembled efficiently in the absence of ATP, but only if the siRNA was phosphorylated prior to the reaction (Tomari, 2004).
Complexes B, A, and RISC also formed in ovary lysates. As for embryo lysate, complexes B and A contained double-stranded siRNA, whereas RISC contained single-stranded. No complexes formed in ovary lysate when siRNA 5' phosphorylation was blocked and complex assembly was reduced when siRNA phosphorylation was slow (Tomari, 2004).
To determine the relationship of complexes B, A, and RISC, the kinetics of complex formation were monitored and the data was analyzed by kinetic modeling. Of all possible models relating free siRNA, B, A, and RISC, only the simple linear pathway siRNA → B → A → RISC fit well to the model. The modeled rate constants for the pathway are consistent with the observation that formation of complex B is ATP independent, but RISC is ATP dependent (Tomari, 2004).
A 'chase' experiment was also performed to confirm the prediction that complex B is a precursor to RISC (via A). Complex B was assembled by incubating 32P-radiolabeled siRNA in embryo lysate for 5 min, then a 20-fold excess of unlabeled siRNA was added to prevent further incorporation of 32P-siRNA into complex. Then the incubation was continued and the formation of complexes was monitored. Complex B disappeared with time; A increased with time then peaked at ∼60 min, and RISC accumulated throughout the experiment. The amount of radiolabeled free siRNA was essentially unchanged throughout the experiment, demonstrating that the unlabeled siRNA effectively blocked incorporation of 32P-free siRNA into complex. Thus, B was chased into RISC, likely via A. Together, this kinetic modeling and chase experiment provides support for a RISC assembly pathway in which the siRNA passes through two successive, double-stranded siRNA-containing complexes, B and A, in order to be transformed into the single-stranded siRNA-containing RISC (Tomari, 2004).
Liu and colleagues have proposed that a heterodimeric complex, comprising Dicer-2 (Dcr-2) and the dsRNA binding protein R2D2, loads siRNA into RISC (Liu, 2003). Complex A contains the Dcr-2/R2D2 heterodimer. R2D2 and Dcr-2 are readily crosslinked to 32P-radiolabeled siRNA with UV light (Liu, 2003). An siRNA was synthesized containing a single photocrosslinkable nucleoside base (5-iodouracil) at position 20. The 32P-5-iodouracil siRNA was incubated with embryo lysate to assemble complexes, then irradiated with 302 nm light, which initiates protein-RNA crosslinking only at the 5-iodo-substituted nucleoside. Proteins covalently linked to the 32P-radiolabeled siRNA were resolved by SDS-PAGE. Two proteins -- ∼200 kDa and ∼40 kDa -- efficiently crosslinked to the siRNA. Both crosslinked proteins were coimmunoprecipitated with either α-Dcr-2 or α-R2D2 serum, but not with normal rabbit serum. Neither crosslink was observed in ovary lysates prepared from r2d2 homozygous mutant females, a result expected because Dcr-2 is unstable in the absence of R2D2 (Liu, 2003; Tomari, 2004).
The crosslinking was repeated, and the reaction analyzed by native gel electrophoresis to resolve complexes B, A, and RISC. Each complex was eluted from the gel and analyzed by SDS-PAGE. The R2D2 and Dcr-2 crosslinks were present in complexes A and RISC, but not B. In a parallel experiment, complexes B, A, and RISC were isolated (without crosslinking) and analyzed by Western blotting with either α-Dcr-2 or α-R2D2 antibodies. Again, complexes A and RISC, but not B, contained both Dcr-2 and R2D2. Finally, complex assembly was tested in ovary lysates prepared from r2d2 homozygous mutant females. Only complex B formed in these lysates. It is concluded that complex A contains the previously identified Dcr-2/R2D2 heterodimer (Liu, 2003), and that both Dcr-2 and R2D2 remain associated with at least a subpopulation of RISC, consistent with earlier reports that Dcr-2 in flies and both DCR-1 and the nematode homolog of R2D2, RDE-4, coimmunoprecipitate with Argonaute proteins (Tomari, 2004).
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).
Long-lasting forms of memory require protein synthesis, but how the pattern of synthesis is related to the storage of a memory has not been determined. This study shows that neural activity directs the mRNA of the Drosophila Ca2+, Calcium/Calmodulin-dependent Kinase II (CaMKII), to postsynaptic sites, where it is rapidly translated. These features of CaMKII synthesis are recapitulated during the induction of a long-term memory and produce patterns of local protein synthesis specific to the memory. mRNA transport and synaptic protein synthesis are regulated by components of the RISC pathway, including the SDE3 helicase Armitage, which is specifically required for long-lasting memory. Armitage is localized to synapses and lost in a memory-specific pattern that is inversely related to the pattern of synaptic protein synthesis. Therefore, it is proposed that degradative control of the RISC pathway underlies the pattern of synaptic protein synthesis associated with a stable memory (Ashraf, 2006).
The CaMKII 3'UTR is necessary and sufficient for the robust localization of CaMKII to dendritic arbors: Since the mammalian aCaMKII is found at synapses, where its synthesis is regulated by neural activity, the attention of this study turned to the CaMKII gene of Drosophila. Drosophila CaMKII has a role in neuromuscular synaptic plasticity and memory in the courtship-conditioning paradigm. CaMKII is localized to both pre- and postsynaptic sites in the adult brain. Focus was placed on the olfactory system because of its well-described neural components, circuitry, and paradigms for the establishment of memory. This system consists of sensory neurons and interneurons that form an early receptive and processing circuit with synapses organized in bilaterally symmetric centers known as the antennal lobes. The first-order interneurons (Projection Neurons; PNs) collect sensory input in a stereotyped array of multisynaptic structures known as glomeruli, where the PN dendritic synapses collect cholinergic input via nicotinic acetylcholine receptors. The PNs direct-output to two brain centers via branching axons that project to the 'calyx' of the mushroom body and to the lateral horn. These terminals release acetylcholine from choline acyltransferase (ChAT)-positive boutons. The PN dendrites also form reciprocal synapses with local interneurons. On the PN dendrites, CaMKII was localized in postsynaptic puncta with the markers Discs Large (DLG) and ARD (a nAChR β-subunit. CaMKII was also concentrated at the PN presynaptic boutons in the calyx and lateral horn. Thus, within the same neuron, CaMKII is concentrated at both pre- and postsynaptic sites (Ashraf, 2006).
The mouse aCaMKII mRNA displays dendritic localization and activity-dependent synaptic translation, features conferred by sequences in its 3'UTR. To determine whether this is the case for Drosophila CaMKII, the 3'UTR was inserted downstream of the EYFP coding sequence in the reporter, UAS-EYFP3'UTR. An additional pair of constructs was made bearing a translational fusion of EYFP to CaMKII, with the 3'UTRCaMKII present (UAS-CaMKII::EYFP3'UTR) or absent (UAS-CaMKII::EYFPNUT) (Ashraf, 2006).
When expressed specifically in PNs using the GAL4, UAS binary system, EYFP3'UTR fluorescence was strikingly localized to synapses on the PN dendritic and axonal termini and colocalized with ChAT at the presynaptic boutons and with the nAChR subunit ARD on dendritic branches in glomeruli. This distribution roughly matched that of CaMKII protein, though EYFP was somewhat more diffuse along the dendritic branches; presumably EYFP does not bind to the postsynaptic apparatus as CaMKII does. In contrast, a cytoplasmic EYFP reporter lacking CaMKII sequences was distributed poorly to the axons and dendrites (Ashraf, 2006).
The CaMKII::EYFP fusion protein synthesized from mRNA harboring the 3'UTR (CaMKII::EYFP3'UTR) displayed synaptic localization in axons and dendrites like that of EYFP3'UTR but was notably more concentrated in synaptic puncta. The same fusion protein made from mRNA lacking the 3'UTR (CaMKII::EYFPNUT) was strongly localized to axonal presynaptic sites but found at a very low level on the antennal lobe dendrites, where it was localized to synaptic puncta. Thus the CaMKII 3'UTR is necessary and sufficient for the robust localization of CaMKII to dendritic arbors but not required for axonal localization (Ashraf, 2006).
Localization and rapid induction of CaMKII in dendrites is due to 3'UTR-dependent synaptic protein synthesis; To determine how neural activity might affect CaMKII expression, brains were explanted into bath culture with acetylcholine (ACh) or nicotine (an agonist of nAChRs). After 20 min, the tissue was examined by anti-CaMKII immunohistochemistry and quantitative confocal microscopy. On average, when cholinergic synapses were activated, CaMKII immunofluorescence in the antennal lobe increased by ~3- to 4-fold. In a time-course experiment, an increase in CaMKII level was detected within 5-10 min of nicotine exposure. The increase was widespread in the brain and reflected in a ~4-fold increase of CaMKII protein on Western blot analysis. The CaMKII increase was also specific, as the levels of synaptic proteins DLG and ARD were unchanged. Consistent with the notion that this regulation occurs via translational control, the effect of cholinergic stimulation was blocked by the ribosomal inhibitor Anisomycin but not by Actinomycin D, an inhibitor of transcription (Ashraf, 2006).
These results and the requirement of the CaMKII 3'UTR for dendritic localization suggest that cholinergic activity may induce the translation of CaMKII mRNA at postsynaptic sites. This was examined by monitoring EYFP3'UTR reporter expression in explant culture. A 5 min nicotine incubation increased EYFP3'UTR expression by 30% and, after 20 min, by 250%. The induced EYFP protein was found in large punctae. Cholinergic stimulation did not increase EYFP3'UTR expression at the presynaptic terminals in the calyx. In contrast, CaMKII::EYFPNUT expression was only slightly increased by nicotine or ACh exposure. Nicotine incubation did not alter the expression of cytoplasmic EYFP, CD8::GFP, or an EGFP construct harboring an a1-tubulin 3'UTR (Hh::EGFP-3'UTRtub). These observations indicate that the localization and rapid induction of CaMKII in dendrites is due to 3'UTR-dependent synaptic protein synthesis (Ashraf, 2006).
Odor-specific induction of synaptic protein synthesis occurs when conditioned and unconditioned stimuli are presented coincidentally and with temporal spacing, the experience that establishes an LTM: In Drosophila, an olfactory LTM is induced by 'spaced training,' a protocol where an odor (CS+) and electric shock (US) are presented coincidentally at temporally spaced intervals. A second odor (CS-) follows the CS+ odor in each interval without coincident shock. An LTM appears after several hours and lasts beyond 24 hr, as assayed by tactic behavior in a T-maze. This protocol was followed and EYFP3'UTR was used to report synaptic protein synthesis in animals that developed an olfactory LTM. The analysis focused on the antennal lobe glomeruli because these structures can be reproducibly identified and display clustered synaptic activity. Furthermore, the first-order antennal lobe synapses might participate in an early stage of memory storage, including the storage of LTM. This analysis revealed an odorant-specific pattern of synaptic protein synthesis associated with the induction of a long-term memory (Ashraf, 2006).
Animals harboring the UAS-EYFP3'UTR reporter driven by the PN-specific GH146-GAL4 were trained and analyzed at times from 4 to 24 hr posttraining. The brains of trained and untrained animals were processed for microscopy in parallel. For each glomerulus, a Z stack of 6-8 confocal microscopic images was recorded and analyzed via a thresholding protocol that isolated pixel groups corresponding to synaptic puncta. An average glomerulus intensity change (ΔF/F) was calculated for 5-8 brains in each experiment. Each experiment was repeated five times. LTM was in all cases verified by T-maze performance (Ashraf, 2006).
The analysis was restricted to a set of glomeruli that included those with a primary response to the odorants octanol (OCT) and methylcyclohexanol (MCH). Only particular glomeruli displayed a training-dependent increase in EYFP3'UTR fluorescence, while others did not; their identities depended on the odorant (CS+) paired with shock. When OCT was the CS+, only glomeruli D and DL3 displayed increased fluorescence, by 115% and 108%, respectively. When MCH was the CS+, fluorescence increased significantly in glomeruli DA1 and VA1 by 95% and 70%, respectively. There were modest but possibly insignificant increases in glomeruli DM6 and VC2. The glomerulus-specific increases were noted as early as 4 hr posttraining and were not observed when odorant and/or electric shock was unpaired or left out or when temporal spacing was not employed ('massed training'). In animals that expressed a cytoplasmic EYFP reporter or CaMKII::EYFPNUT, which lack the CaMKII 3'UTR, there were no significant fluorescence changes. This analysis revealed that an odor-specific induction of synaptic protein synthesis occurred when conditioned and unconditioned stimuli were presented coincidentally and with temporal spacing, the experience that establishes an LTM. This plasticity was evidently maintained for at least 24 hr (Ashraf, 2006).
A coordinated program for synaptic gene expression occurs during the storage of a memory: If CaMKII was synthesized at the synapse, its mRNA would be localized there. To address this question, an mRNA tracking system based on the bacteriophage coat protein MS2 and its RNA binding site were utilized. The fusion protein MS2::GFP::nls is concentrated in the nucleus by nuclear localization signals (nls) but can be diverted elsewhere by binding to an MS2 binding site (MS2-bs) tagged mRNA. Three mRNAs were tagged: the Drosophila CaMKII cDNA, its 3'UTR alone, and the mouse aCaMKII 3'UTR, which mediates dendritic localization and synaptic translation in the mouse (Ashraf, 2006).
GFP fluorescence was examined when MS2::GFP::nls was expressed in projection neurons (PNs) with or without an MS2-bs tagged mRNA. Punctate fluorescence was observed in the dendrites when an MS2-bs-tagged mRNA was coexpressed with MS2::GFP::nls but not with MS2::GFP::nls alone. For example, the tagged Drosophila CaMKII mRNA increased the intensity of glomerular fluorescence by 200%. In dendrites, particular mRNAs, including the mouse aCaMKII, are localized to particles containing the motor protein Kinesin. Consistent with this observation, the GFP-positive dendritic puncta were labeled with an antibody against the major kinesin heavy chain, KHC (Ashraf, 2006).
It was asked whether the synaptic CaMKII expression induced by cholinergic activity might be associated with enhanced dendritic localization of CaMKII mRNA, as has been found for the mouse aCaMKII and Arc mRNAs. When explanted into media with nicotine or ACh, brains harboring MS2::GFP::nls and the tagged Drosophila CaMKII mRNA displayed a striking increase in dendritic GFP fluorescence. The effect with cholinergic stimulation was similar with the tagged mouse aCaMKII 3'UTR: a 70%-73% increase relative to culture without nicotine. The activity-enhanced dendritic mRNA transport was blocked by Anisomycin but not by Actinomycin D. It is supposed that mainly existing mRNA can be translocated during the short period of culture. Thus, in Drosophila, like mammals, neural activity increases the rate of mRNA movement to the synapse by a protein synthesis-dependent mechanism (Ashraf, 2006).
It was then asked whether the induction of an LTM might affect mRNA transport to the synapse. When animals expressing MS2::GFP::nls and Drosophila ms2bs-CaMKII were subjected to the spaced training protocol, the number of GFP-labeled puncta in dendrites was substantially increased. The pattern of dendritic punctae did not display evident glomerular specificity, as observed for synaptic protein synthesis. However, the induced punctae were distributed along the dendritic branches, making a determination of glomerular specificity uncertain. These observations reveal that a coordinated program for synaptic gene expression occurs during the storage of a memory (Ashraf, 2006).
Regulation of mRNA transport and synaptic protein synthesis by the RISC pathway: The RNA interference (RISC) pathway silences gene expression by the targeted degradation of mRNAs or their nondestructive silencing. In Drosophila, RISC-mediated translational silencing controls oskar expression in the developing oocyte. An SDE3-class RNA helicase, Armitage (Armi) acts as part of RISC to control oskar translation and regulate cytoskeletal organization, possibly via control of Kinesin heavy chain (Khc) translation. Both the oskar and Khc 3'UTRs have putative binding sites for the microRNA (miRNA) miR-280. The CaMKII 3'UTR has a remarkably similar miR-280 binding site. This site and a nearby site for miR-289 satisfy the predictive rule that 7 of 8 nucleotides at the 5' end of an miRNA are cognate to a target mRNA. Kinesin is also a component of the RNA-containing dendritic particles that bring mRNA to the synapse. Staufen, likewise a mediator of RNA transport, has binding sites for miR-280 and miR-305 in its mRNA 3'UTR. Thus this study explored the role of RISC in CaMKII, KHC, and Staufen expression (Ashraf, 2006).
Dicer-2 is one of two Drosophila ribonucleases that produce short RNA components of RISC. CaMKII synaptic expression was dramatically increased in a dicer-2 mutant, particularly in the antennal lobe and mushroom body. In contrast, there was no difference in the expression of the cell adhesion protein Fasciclin II in the same animals. In Western analysis, there was a striking ~25-fold increase in CaMKII protein in dicer-2 mutant brains. Synaptic CaMKII expression was also elevated in aubergine and armitage mutant brains. The aubergine locus encodes an Argonaute protein involved in RISC assembly and function. The level of Staufen protein was also increased in the armitage mutant brain (Ashraf, 2006).
Whether the miRNA binding sites in the CaMKII 3'UTR might be involved in RISC-mediated regulation was examined with the EYFP3'UTR transgene. When expressed in the PNs, EYFP fluorescence in glomeruli was 80% greater in armi than in the wild-type. The EYFP3'UTR fluorescence was localized to large dendritic puncta like those found in brains explanted into nicotine-containing media. Indeed, EYFP3'UTR expression in armi brains did not increase further upon explant with nicotine, consistent with the notion that cholinergic activation might act via antagonism of RISC. The expression of CaMKII::EYFPNUT, which lacks the 3'UTR, increased slightly in the armi mutant background, while other control constructs, such as CD8::GFP, were essentially unchanged. In addition, RT-PCR analysis of wild-type and armi mutant brains did not reveal a difference in the levels of transgenic mRNAs. There was also a substantial increase in EYFP3'UTR and CaMKII::EYFP3'UTR synaptic fluorescence in dicer-2 and aubergine mutants. Therefore it is concluded that RISC regulates CaMKII expression by a posttranscriptional mechanism, utilizing sites in the CaMKII 3'UTR (Ashraf, 2006).
Armitage expression was found in multiple neuronal populations in the brain, including the PNs and mushroom-body Kenyon cells. It is distributed in puncta in cell bodies and dendrites and to axon termini. A GFP::Armi fusion protein, when expressed in the PNs, displayed a similar punctate distribution that overlaps synaptic puncta containing CaMKII. The GFP::Armi fusion protein retains armi+ activity such that neurons with high levels of GFP::Armi expression would have increased armi+ activity. Several observations indicate that a posttranscriptional autoregulatory circuit modulates Armi expression. Nonetheless, strong transgenic expression of GFP::Armi reduced the level of CaMKII expression, as revealed by Western blot analysis and immunohistochemistry. Neurons that expressed a high level of GFP::Armi displayed reduced expression of both CaMKII and KHC. A control UAS-CD8-GFP transgene was unaffected by GFP::Armi expression (Ashraf, 2006).
Since Armi regulates KHC and Staufen expression, the possibility was considered that it might also regulate the dendritic transport of CaMKII mRNA. When examined with the MS2::GFP system, armi72.1 homozygotes indeed displayed a 78% increase in fluorescence by dendritic GFP-positive puncta, compared to an armi+ control. Therefore, Armi regulation of synaptic protein synthesis reflects a coordinated program with multiple miRNA targets, affecting both mRNA transport and translation at the synapse, where Armi protein is found (Ashraf, 2006).
Neural activity induces rapid proteasome-mediated degradation of Armitage: If mRNA silencing by RISC plays a role in LTM, this pathway would be expected to be somehow regulated by neural function. Given the inverse relationship between CaMKII expression and armi+ activity, whether Armi might be a regulatory target wad considered. The level of GFP::Armi fluorescence rapidly decreased (by 3.5-fold) in brains explanted into nicotine-containing medium. There was a correlated increase in CaMKII expression (by ~4.5-fold) in the PN dendritic arbors of the antennal lobe. A short incubation with nicotine (5 min) resulted in the complete disappearance of Armi protein in Western analysis. The GFP::Armi protein was also eliminated upon explant with nicotine. In contrast, the CaMKII protein level increased and a1-tubulin was unchanged (Ashraf, 2006).
Two experiments were performed to determine whether the activity-induced elimination of Armi required the proteasome. First, GFP::Armi was expressed along with a transgenic dominant-negative mutant of the proteasome β subunit. When the DTS5 transgene was present, the level of GFP::Armi fluorescence was elevated by 3.2-fold. In contrast, the DTS5 transgene did not alter the level of CD8::GFP. Second, incubation with the proteasome inhibitor lactacystin blocked the nicotine-induced loss of GFP::Armi and degradation of endogenous Armi protein. Preincubation with lactacystin also blocked nicotine-induced synaptic CaMKII synthesis, as determined by both Western analysis and by immunohistochemistry. Thus, cholinergic activity evidently acts via the proteasome to induce the degradation of Armitage and synaptic synthesis of CaMKII (Ashraf, 2006).
A degradative pathway for LTM: A key question is whether this degradative pathway has a role in synaptic protein synthesis associated with LTM. Animals expressing the GFP::Armi protein in projection neurons were subjected to olfactory spaced training and analyzed by the same microscopic methods used to assess LTM-associated EYFP3'UTR expression. The GFP::Armi protein was found concentrated in synaptic puncta in the glomeruli. When examined at either 3 or 24 hr posttraining, GFP::Armi fluorescence was significantly reduced in many glomeruli and most strongly reduced in the glomeruli that had displayed the greatest increase in EYFP3'UTR expression. Fluorescence in glomeruli DA1 and VA1 decreased by ~3.1- and 3.8-fold, respectively, when the odorant MCH was paired with shock. When the odorant OCT was paired with shock, the D and DL3 glomeruli displayed the most significant decreases (~2-fold). More modest losses of GFP fluorescence were observed in other glomeruli. These observations reveal an inverse relationship between synaptic Armi protein and CaMKII synthesis during the establishment of an LTM. Since these changes were still present at 24 hr posttraining, the change was evidently maintained long-term, perhaps for the term of the memory (Ashraf, 2006).
Given the role of Armi in the synaptic synthesis of CaMKII, it was wondered whether either of these genes might be required to form an olfactory LTM. Several armi hypomorphic alleles display normal adult viability and behavior, including normal odor and shock sensitivity. Given their normal performance in these tests, armi animals were examined for STM and LTM. The animals (armi72.1/armi72.1 or armi72.1/Df(3L)E1) displayed normal memory in the short-term paradigm but were profoundly deficient in LTM. Expression of the GFP::Armi transgene rescued the armi72.1/armi72.1 LTM deficiency to a normal value. A nearly complete and tissue-specific loss of CaMKII was achieved by use of a construct that generates a CaMKII hairpin RNA. Animals expressing UAS-CaMKIIhpin in all CaMKII-positive neurons (with the CaMKII-GAL4 driver) retained normal short-term memory, but displayed a near-complete loss of LTM. Thus, both CaMKII and Armitage are required for LTM but not for STM (Ashraf, 2006).
It is concluded that memory-specific patterns of synaptic protein synthesis occur with the induction of a long-term memory in Drosophila. These patterns appear to be controlled by the proteasome-mediated degradation of a RISC pathway component, Armitage, to regulate the transport of mRNA to synapses and its translation once there (Ashraf, 2006).
To visualize synaptic protein synthesis, fluorescent reporters were used based on the Drosophila CaMKII gene, which has well-described roles in synaptic plasticity and memory. The 3'UTR of CaMKII shares regulatory motifs with the mammalian aCaMKII mRNA, which mediate dendritic mRNA localization and neural activity-dependent translation. The 3'UTR of Drosophila CaMKII was also necessary and sufficient for mRNA localization to dendrites and synaptic translation. This 3'UTR sufficed for the enhanced dendritic mRNA transport and translation induced by cholinergic stimulation. Hence a simple parallel was found between the synaptic regulation of CaMKII in Drosophila and mammals (Ashraf, 2006).
When these fluorescent reporters were utilized in vivo, the induction of synaptic protein synthesis was observed in several Drosophila brain centers following the spaced training paradigm of repetitive odor paired with electric shock that establishes a long-term memory. There were local patterns of memory specificity identifiable in glomeruli of the antennal lobe where synapses of similar function are clustered. When the odorant OCT was paired with electric shock, protein synthesis was induced selectively in the D and DL3 glomeruli. When the odorant MCH was paired with shock, the DA1 and VA1 glomeruli displayed the most robust enhancement of synaptic protein synthesis. Notably, the animals were exposed to both odorants during training; the pattern of synthesis depended on coincidence with shock. There was no significant induction of protein synthesis when exposure to odor and shock was nonoverlapping, with either stimulus presented alone, or in the absence of temporal spacing ('massed training'). Thus, an odor-specific pattern of synaptic protein synthesis was induced under conditions that produce an LTM (Ashraf, 2006).
Experiments in the honeybee suggest that the antennal lobe is a 'way station' for memory where stimuli are integrated to yield plasticity more labile than a short-term memory. A long-term memory can be formed in the honeybee antennal lobe in a spaced training paradigm. Experiments have revealed plasticity in the Drosophila antennal lobe, where particular glomeruli acquired enhanced synaptic activity after a single episode of paired odor and shock. Remarkably, the enhanced synaptic protein synthesis observed with spaced training occurred in essentially the same glomeruli that displayed enhanced synaptic activity in the STM protocol. These glomeruli are distinct from those that display the greatest odor or electric shock-evoked synaptic activity. Therefore, it is supposed that the mechanism that integrates a single paired odor and shock to produce new synaptic activity might also generate the trigger for synaptic protein synthesis when the paired stimuli are repeated with temporal spacing. It is believed this trigger includes the proteasome-mediated degradation of the RISC factor Armitage (Ashraf, 2006).
Though these 'memory traces' have been recorded in the antennal lobe, there is still no evidence for their role in memory. The mushroom body, in contrast, is required for LTM. The current methods cannot resolve patterns of synaptic protein synthesis in the mushroom body because it lacks the stereotyped synaptic architecture of the antennal lobe. When determined, a global brain map of synaptic protein synthesis will provide significant insights into the mechanisms of memory storage (Ashraf, 2006).
Synaptic protein synthesis and dendritic mRNA transport are well studied for the mammalian aCaMKII gene, which bears recognition motifs in its 3'UTR for CPEB and other proteins with transport and translation control functions. The presence of potential recognition motifs for the CPEB, Pumilio, and Staufen proteins in the Drosophila CaMKII 3'UTR suggests that these mechanisms are conserved in Drosophila. Indeed, Staufen, orb (a CPEB family member), and pumilio have been identified as LTM-deficient mutants. The roles of these genes remain to be fully explored (Ashraf, 2006).
Focus was placed instead on the RISC pathway because of apparent binding motifs for microRNAs miR-280 and miR-289 in the CaMKII 3'UTR. These sites are similar to those in the 3'UTRs of oskar and Kinesin heavy chain (Khc), which are targets for translational silencing by Armitage and other RISC components in the oocyte. Armitage was found in synaptic puncta on dendrites, colocalized with CaMKII. When the level of Armitage was decreased or increased by mutation or transgenic expression, CaMKII synaptic expression was modulated in a reciprocal and cell-autonomous fashion. This regulation could be recapitulated by an EYFP reporter bearing the CaMKII 3'UTR. Mutants for the RISC components Aubergine and Dicer-2 displayed similar phenotypes. It therefore seems likely that multiple tiers of control regulate CaMKII like oskar, where two systems (Bruno/Cup and RISC) act on distinct sites in its 3'UTR (Ashraf, 2006).
A second avenue for RISC control of CaMKII synthesis is via mRNA transport. By tagging CaMKII mRNA with a GFP reporter, dendritic punctae were observed whose frequency and intensity increased under the same conditions that induced synaptic protein synthesis: cholinergic activation and olfactory spaced training. The induction of mRNA transport required new protein synthesis but not transcription. Armitage was also found to regulate the frequency and intensity of the GFP-tagged dendritic puncta. Two proteins that play a role in mRNA transport, Kinesin heavy chain (KHC) and Staufen, recapitulate this pattern of regulation by cholinergic stimulation and Armitage. Both of their mRNAs bear targets for miRNA regulation in the 3'UTR. These studies leave open the possibility that the enhanced synaptic localization of CaMKII mRNA underlies the induction of its synaptic translation. However, the presence of miRNA binding sites in the CaMKII 3'UTR, the localization of Armitage with CaMKII in synaptic punctae, and the rapid induction of CaMKII synthesis by cholinergic activity all suggest that RISC acts at the synapse. Furthermore, local translational control may be required to impose the specificity that was not evident in the pattern of mRNA transport associated with the induction of an LTM (Ashraf, 2006).
A link between the induction of memory and synaptic protein synthesis is the proteasome-mediated degradation of Armitage. In explant culture, cholinergic induction of CaMKII synthesis was accompanied by the rapid degradation of Armi; both events were blocked by inhibition of the proteasome. The relationship between Armi degradation and CaMKII synaptic translation was recapitulated in the brain as animals formed and maintained an LTM. The same glomeruli that displayed the greatest increase in CaMKII synthesis displayed the largest decline in synaptic Armi. This reciprocal relationship between the Armi and CaMKII proteins was detected as early as 3 hr after training and maintained for at least 24 hr posttraining. The training-induced change of synaptic Armi was therefore 'locked in,' possibly for the term of the memory, consistent with a role in maintaining an alteration of synaptic function (Ashraf, 2006).
Therefore a new mechanism is proposed for stable memory in which an integrated sensory trigger induces the proteasome-mediated degradation of a RISC factor, releasing synaptic protein synthesis and mRNA transport from microRNA suppression. It is supposed that this mechanism is triggered with neuronal specificity in order to produce memory-specific patterns of protein synthesis. Whether this specificity is required for memory or extends to the level of a single synapse are questions that remain to be addressed (Ashraf, 2006).
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