Isabelle Busseau

Oskar allows nanos mRNA translation in Drosophila embryos by preventing its deadenylation by Smaug/CCR4

Anteroposterior patterning of the Drosophila embryo depends on a gradient of Nanos protein arising from the posterior pole. This gradient results from both nanos mRNA translational repression in the bulk of the embryo and translational activation of nanos mRNA localized at the posterior pole. Two mechanisms of nanos translational repression have been described, at the initiation step and after this step. Here we identify a novel level of nanos translational control. We show that the Smaug protein bound to the nanos 3′ UTR recruits the deadenylation complex CCR4-NOT, leading to rapid deadenylation and subsequent decay of nanos mRNA. Inhibition of deadenylation causes stabilization of nanos mRNA, ectopic synthesis of Nanos protein and head defects. Therefore, deadenylation is essential for both translational repression and decay of nanos mRNA. We further propose a mechanism for translational activation at the posterior pole. Translation of nanos mRNA at the posterior pole depends on oskar function. We show that Oskar prevents the rapid deadenylation of nanos mRNA by precluding its binding to Smaug, thus leading to its stabilization and translation. This study provides insights into molecular mechanisms of regulated deadenylation by specific proteins and demonstrates its importance in development.

I factors inDrosophila melanogaster: Transposition under control

I factors are responsible for the I-R system of hybrid dysgenesis inDrosophila melanogaster. They belong to the LINE class of mobile elements, which transpose via reverse transcription of a full-length RNA intermediate. I factors are active members of the I element family, which also contains defective I elements that are immobilized within peri-centromeric heterochromatin and represent very old components of the genome. Active I factors have recently invaded natural populations ofDrosophila melanogaster, giving rise to inducer strains. Reactive strains, devoid of active I factors, derive from old laboratory stocks established before the invasion. Transposition of I factors is activated at very high frequencies in the germline of hybrid females issued from crosses between females from reactive strains and males from inducer strains. It results in the production of high rates of mutations and chromosomal rearrangements as well as in a particular syndrome of sterility. The frequency of transposition of I factors is dependent on the amount of full-length RNA that is synthesized from an internal promoter. This full-length RNA serves both as an intermediate of transposition and presumably as a messenger for protein synthesis. Regulators of transposition apparently affect transcription initiation from the internal promoter. The data presented here lead to the proposal of a tentative model for transposition.

A search for reverse transcriptase-coding sequences reveals new non-LTR retrotransposons in the genome of Drosophila melanogaster

BackgroundNon-long terminal repeat (non-LTR) retrotransposons are eukaryotic mobile genetic elements that transpose by reverse transcription of an RNA intermediate. We have performed a systematic search for sequences matching the characteristic reverse transcriptase domain of non-LTR retrotransposons in the sequenced regions of the Drosophila melanogaster genome.ResultsIn addition to previously characterized BS, Doc, F, G, I and Jockey elements, we have identified new non-LTR retrotransposons: Waldo, You and JuanDm. Waldo elements are related to mosquito RTI elements. You to the Drosophila I factor, and JuanDm to mosquito Juan-A and Juan-C. Interestingly, all JuanDm elements are highly homogeneous in sequence, suggesting that they are recent components of the Drosophila genome.ConclusionsThe genome of D. melanogaster contains at least ten families of non-site-specific non-LTR retrotransposons representing three distinct clades. Many of these families contain potentially active members. Fine evolutionary analyses must await the more accurate sequences that are expected in the next future.

The CCR4 deadenylase acts with Nanos and Pumilio in the fine-tuning of Mei-P26 expression to promote germline stem cell self-renewal.

Summary Translational regulation plays an essential role in Drosophila ovarian germline stem cell (GSC) biology. GSC self-renewal requires two translational repressors, Nanos (Nos) and Pumilio (Pum), which repress the expression of differentiation factors in the stem cells. The molecular mechanisms underlying this translational repression remain unknown. Here, we show that the CCR4 deadenylase is required for GSC self-renewal and that Nos and Pum act through its recruitment onto specific mRNAs. We identify mei-P26 mRNA as a direct and major target of Nos/Pum/CCR4 translational repression in the GSCs. mei-P26 encodes a protein of the Trim-NHL tumor suppressor family that has conserved functions in stem cell lineages. We show that fine-tuning Mei-P26 expression by CCR4 plays a key role in GSC self-renewal. These results identify the molecular mechanism of Nos/Pum function in GSC self-renewal and reveal the role of CCR4-NOT-mediated deadenylation in regulating the balance between GSC self-renewal and differentiation.

A genetically tagged, defective I element can be complemented by actively transposing I factors in the germaine of I-R dysgenic females inDrosophila melanogaster

Non-LTR retrotransposons, also known as LINEs, transpose by reverse transcription of an RNA intermediate. Their mechanism of transposition is apparently different from that of retrotransposons and similar to that of proviruses of retroviruses. The I factor is responsible for the I-R system of hybrid dysgenesis inDrosophila melanogaster. Inducer strains contain several functional I factors whereas reactive strains do not. Transposition of I factors can be experimentally induced: they are stable in inducer strains, but transpose at high frequency in the germline of females, known as SF females, produced by crossing reactive females and inducer males. We have constructed an I element, calledIviP2, marked with thevermilion gene, the coding sequence of which was interrupted by an intron. Splicing of the intron can only occur in the transcript initiated from the I element promoter. Transposed copies expressing a wild-typevermilion phenotype were recovered in the germline of SF females in which I factors were actively transposing. This indicates thattrans-complementation of a defective I element, deficient for the second open reading frame, by functional I factors can occur in the germline of dysgenic females.

Translational Control of Autophagy by Orb in the Drosophila Germline.

Drosophila Orb, the homolog of vertebrate CPEB, is a key translational regulator involved in oocyte polarity and maturation through poly(A) tail elongation of specific mRNAs. orb also has an essential function during early oogenesis that has not been addressed at the molecular level. Here, we show that orb prevents cell death during early oogenesis, thus allowing oogenesis to progress. It does so through the repression of autophagy by directly repressing, together with the CCR4 deadenylase, the translation of Autophagy-specific gene 12 (Atg12) mRNA. Autophagy and cell death observed in orb mutant ovaries are reduced by decreasing Atg12 or other Atg mRNA levels. These results reveal a role of Orb in translational repression and identify autophagy as an essential pathway regulated by Orb during early oogenesis. Importantly, they also establish translational regulation as a major mode of control of autophagy, a key process in cell homeostasis in response to environmental cues.

Trans-complementation of an endonuclease-defective tagged I element as a tool for the study of retrotransposition in Drosophila melanogaster

Abstract. I factors are non-LTR retrotransposons of Drosophila melanogaster that transpose at high frequency in the germline of females resulting from appropriate crosses, allowing in vivo studies of the retrotransposition process. Reverse transcription of a full-length RNA intermediate is thought to occur at the site of integration, using a 3′ hydroxyl group generated by endonucleolytic cleavage of the genomic DNA to prime synthesis of the first cDNA strand. This target-primed reverse transcription (TPRT) process is mediated by endonuclease and reverse transcriptase activities encoded by the element. We have designed a molecularly tagged, endonuclease-defective I element that can be mobilised with high efficiency by constructs that express the product of the I factor ORF2 in trans. This indicates that the endonuclease activity required for retrotransposition of the I factor can be provided in trans. Using this system, we show that the endonuclease domain of the R1Bm retrotransposon from Bombyx mori cannot functionally replace that of the I factor.

Functional lability of RNA-dependent RNA polymerases in animals

RNA interference (RNAi) requires RNA-dependent RNA polymerases (RdRPs) in many eukaryotes, and RNAi amplification constitutes the only known function for eukaryotic RdRPs. Yet in animals, classical model organisms can elicit RNAi without possessing RdRPs, and only nematode RNAi was shown to require RdRPs. Here we show that RdRP genes are much more common in animals than previously thought, even in insects, where they had been assumed not to exist. RdRP genes were present in the ancestors of numerous clades, and they were subsequently lost at a high frequency. In order to probe the function of RdRPs in a deuterostome (the cephalochordate Branchiostoma lanceolatum), we performed high-throughput analyses of small RNAs from various Branchiostoma developmental stages. Our results show that Branchiostoma RdRPs do not appear to participate in RNAi: we did not detect any candidate small RNA population exhibiting classical siRNA length or sequence features. Our results show that RdRPs have been independently lost in dozens of animal clades, and even in a clade where they have been conserved (cephalochordates) their function in RNAi amplification is not preserved. Such a dramatic functional variability reveals an unexpected plasticity in RNA silencing pathways. Author summary RNA interference (RNAi) is a conserved gene regulation system in eukaryotes. In non-animal eukaryotes, it necessitates RNA-dependent RNA polymerases (”RdRPs”). Among animals, only nematodes appear to require RdRPs for RNAi. Yet additional animal clades have RdRPs and it is assumed that they participate in RNAi. Here, we find that RdRPs are much more common in animals than previously thought, but their genes were independently lost in many lineages. Focusing on a species with RdRP genes (a cephalochordate), we found that it does not use them for RNAi. While RNAi is the only known function for eukaryotic RdRPs, our results suggest additional roles. Eukaryotic RdRPs thus have a complex evolutionary history in animals, with frequent independent losses and apparent functional diversification.

Orb prevents autophagy in the Drosophila germline through translational repression of Atg12 mRNA

Drosophila Orb, the homologue of vertebrate CPEB is a key translational regulator involved in oocyte polarity and maturation through poly(A) tail elongation of specific mRNAs. orb has also an essential function during early oogenesis which has not been addressed at the molecular level. Here, we show that orb prevents cell death during early stages of oogenesis, thus allowing oogenesis to progress. It does so through the repression of autophagy, by directly repressing, together with the CCR4 deadenylase, the translation of Autophagy-specific gene 12 (Atg12) mRNA. The uncontrolled autophagy observed in orb mutant ovaries is reduced when Atg12 mRNA levels are decreased. These results reveal a role of Orb in translational repression and identify autophagy as an essential pathway regulated by Orb during early oogenesis. Importantly, they also establish translational regulation as a major mode of control of autophagy, a key process in cell homeostasis in response to environmental cues.