Widespread regulation of the maternal transcriptome by Nanos in Drosophila
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Abstract
The translational repressor Nanos (Nos) regulates a single target, maternal hunchback ( hb) mRNA, to govern abdominal segmentation in the early Drosophila embryo. Nos is recruited specifically to sites in the 3’-UTR of hb mRNA in collaboration with the sequence-specific RNA-binding protein Pumilio (Pum); on its own, Nos has no binding specificity. Nos is expressed at other stages of development, but very few mRNA targets that might mediate its action at these stages have been described. Nor has it been clear whether Nos is targeted to other mRNAs in concert with Pum or via other mechanisms. In this report, we identify mRNAs targeted by Nos via two approaches. In the first method, we identify mRNAs depleted upon expression of a chimera bearing Nos fused to the nonsense mediated decay (NMD) factor Upf1. We find that, in addition to hb , Upf1-Nos depletes ∼2600 mRNAs from the maternal transcriptome in early embryos. Virtually all of these appear to be targeted in a canonical, hb -like manner in concert with Pum. In a second, more conventional approach, we identify mRNAs that are stabilized during the maternal zygotic transition (MZT) in embryos from nos - females. Most (86%) of the 1185 mRNAs regulated by Nos are also targeted by Upf1-Nos, validating use of the chimera. Approximately 60% of mRNAs targeted by Upf1-Nos are not stabilized in the absence of Nos. However, Upf1-Nos mRNA targets are hypo-adenylated and inefficiently translated at the ovary-embryo transition, whether or not they suffer Nos-dependent degradation in the embryo. We suggest that the late ovarian burst of Nos represses a large fraction of the maternal transcriptome, priming it for later degradation by other factors during the MZT in the embryo.
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In this thorough and clever study, the authors identify the diverse targets of the translational repressor Nanos, an RNA binding protein that secondarily promotes transcript degradation. While Nanos is known chiefly for regulating the transcription factor hunchback, its other targets have not been well-identified. The authors took the unconventional approach of identifying Nos targets by enhancing its transcript-degrading abilities through fusion to the Upf protein. Through a combination of morphological analysis, RNA-seq and differential expression, re-analysis of existing datasets, and even yeast hybrid experiments, the authors thoroughly identify thousands other binding targets of Nos.
Throughout the manuscript, the authors conduct a variety of sophisticated and multi-layered experiments that leverage a variety of techniques and …
In this thorough and clever study, the authors identify the diverse targets of the translational repressor Nanos, an RNA binding protein that secondarily promotes transcript degradation. While Nanos is known chiefly for regulating the transcription factor hunchback, its other targets have not been well-identified. The authors took the unconventional approach of identifying Nos targets by enhancing its transcript-degrading abilities through fusion to the Upf protein. Through a combination of morphological analysis, RNA-seq and differential expression, re-analysis of existing datasets, and even yeast hybrid experiments, the authors thoroughly identify thousands other binding targets of Nos.
Throughout the manuscript, the authors conduct a variety of sophisticated and multi-layered experiments that leverage a variety of techniques and existing datasets, allowing them to quite comprehensively examine all of the features of their dataset to understand how Upf fusion affects their results or to identify the Bruno gene as an additional candidate binding partner of Nanos. This work is creative, resourceful, and deep, and it will be exciting to see how others might take similar approaches to more deeply investigate other proteins important for the regulation of maternal gene expression.
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Recruitment is dependent on both the NTD and RBD ofNos, raising the possibility that protein-protein interactions between Bru and Nos as wellas protein-RNA interactions between Nos and the RNA are required for formation of theternary Bru/Nos/RNA complex.
It is very impressive that the authors followed up on the observations that emerged from this control experiment with such rigor.
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same minor extent as does Upf1
Are the authors able to directly compare Upf1-NosL7 / Upf1-Nos / Upf / Nos to each other via differential expression analysis? That might more specifically reveal the differences caused by addition Nos to the Upf protein, etc.
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hb, bcd, and alpha-Importin
Fig. 2A shows the expression of bcd in the data already, but it would be nice to include the other three as well, since they are referenced here.
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As shown in Fig. 2B, the two methods show excellent agreement, with aPearson's correlation R value of 0.98
This is a very clean result!
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revealed a striking bicaudal body plan
It would be interesting to see some images of the bicaudal phenotype generated by this experiment, perhaps as part of Fig. 1.
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Nos
duplication of "Nos"?
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two row
To more clearly demonstrate rescue caused by the L7 mutation in Upf1-NosL7, it might be helpful to have images from a WT (non-transgenic) embryo.
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C. The distribution of bcd mRNA in Upf1-Nosand Upf1-NosL7 embryos at two different stages of early embryogenesis
For ease of comprehension, it might be helpful to indicate with text on top of the corners of each image the precise channel (bcd mRNA, DAPI, etc.)
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B. Embryonicphenotypes resulting from maternal expression of the four regulatory proteins, asindicated.
It might be helpful to include the raw numbers for these figures in the figure legend or the figure itself, rather than having a separate supplementary file.
Also, what are these percentage in WT embryos from the parent strain of these transgenic organisms?
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