The "raison d'être" of the iab-8 ncRNA seems to be to allow the breaking of posterior prevalence in the CNS, while keeping abd-A repressed in PS13. There seems to be an extremely high evolutionary pressure to prevent ectopic abd-A expression in neurons of this parasegment. The iab-8 ncRNA ensures this repression by an extremely effective microRNA, mir-iab-8, combined with transcriptional interference, this being the only known case in which a noncoding RNA completely shuts down the expression of a Hox gene in an entire parasegment. The importance of this repression becomes evident in the light of the complete sterility exhibited by males and females in which the iab-8 ncRNA is absent. I will not go into detail on the possible causes of this sterility, as its study has not been the case of this thesis, but I would like to draw attention to its neurological basis.
ABD-A as a neuropeptidergic determinant
The reasons for the breaking of posterior prevalence in the CNS were explained before, and probably relate to the role of Hox gene expression in neurons, which ultimately determine their neuropeptidergic identity (which kind of neuropeptides those neurons will release). The ectopic expression of ABD-A in PS13, therefore, could lead to a change of identity in this parasegment in neurons that only should express ABD-B.
It has been shown, for example, that UBX and ABD-A are co-expressed in the abdominal leucokinergic (leucokinin producing) neurons of the ventral nerve cord (ABLKs), being both genes necessary for the specification of their identity (Estacio-Gomez et al. 2013).
In the ventral-abdominal (Va) neurons, a maximum of four Hox proteins have been found co-expressed in the same cell (ANTP, UBX, ABD-A and ABD-B), being all of them involved in the differentiation process of the Capa-expressing neurons (Suska et al. 2011).
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Further more, in Capaergic (Capa-producing) neurons, there is not only an absence of posterior prevalence in the expression of anterior Hox genes by posterior ones, but it has been described that UBX is able to interfere with the normal function exerted by ABD-B under ectopic expression, resulting in a particular case of "anterior phenotypic prevalence" (Gabilondo et al. 2018).
The role of Hox genes in the specification of neuronal identity, however, is in early phases of study. It would be interesting to study the neurons present in PS13 of Fab-864 mutants, to try to identify potential changes in neuronal identity that could happen when ABD-A is expressed in this parasegment.
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Conclusions
In this thesis I had set the objective of providing further insight into the regulation of abdominal-A by the iab-8 ncRNA, via a mechanism that was believed to be transcriptional interference. I also aimed to propose an explanation to the fact that this repressive interaction only seems to take place in the embryonic central nervous system, where the iab-8 ncRNA takes over the role of Abd-B in the repression of abd-A.
The iab-8 ncRNA exerts transcriptional interference over abd-A
The study of the expression of abd-A and the iab-8 ncRNA in the embryonic central nervous system allowed me to describe a CNS-specific splice isoform of the iab-8 transcript. This isoform turned out to be a hybrid RNA composed by the 5' exons of the iab-8 ncRNA and the 3' exons of abd-A, via a process of exon skipping of the terminal exon of iab-8 and a subsequent failure of transcriptional termination, which causes the intergenic splicing of iab-8 into abd-A. This hybrid RNA does not have coding potential, as it lacks the first exons of abd-A. However, its discovery provides for an interesting possibility to explain how transcriptional interference could work.
During the course of my work, I have proven that the transcription of the iab-8 ncRNA is sufficient to repress any given gene placed downstream from its terminal exon. I proved this by placing a GFP gene downstream from the abd-A promoter. This gene, despite lacking any target site for the mir-iab-8 microRNA, is repressed in PS13 of the embryonic CNS by the iab-8 ncRNA expression. This repression is only able to act in cis-, which supports the already proposed model of transcriptional interference.
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The transcription of the abdA:GFP transgene upstream from the endogenous abd-A promoter, however, is not able to repress abd-A (Figure 42B), and it is not able either to rescue the absence of transcriptional interference happening in Fab-864 embryos. This appears as a contradiction, because the transcription of the iab-8 ncRNA, which takes place in roughly the same area of the BX-C, is able to repress abd-A. In fact, the abdA:GFP gene has its transcriptional end closer to abd-A than the iab-8 ncRNA in the unmodified BX-C. This raises the question: what is the main difference between both genes, which allows transcriptional interference in one case, but not in the other?
In addition, the insertion of the abdA:GFP gene between exon 8 of the iab-8 ncRNA and the transcriptional start site of abd-A (an insertion of approximately 2Kb plus an additional terminator sequence) does not have any effect over abd-A expression in PS13 (Figure 42B), which indicates that the iab-8 ncRNA is indeed repressing abd-A by transcriptional interference even over this increased distance.
I tried reproducing the repression exerted by the iab-8 ncRNA by forcing transcription of its terminal exon using the GAL-4/UAS system. This forced transcription, however, was not enough to cause ectopic repression of abd-A. However, this experiment showed that transcriptional interference exerted by the transcript coming from the iab-8 promoter is able to disrupt the interaction between the yeast transcription factor GAL-4 and its target UAS sites (Figure 42C). In this case, transcriptional interference is not exclusive to the CNS, taking place also in the ectoderm of early embryos.
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Figure 42 Schematic representation of the genetic interactions happening between the iab-8 ncRNA, abdA:GFP, UAShsp70ex8GFP and abd-A
The red flat arrows represent transcriptional interference happening (or the absence of it) in each case.
Both of these facts could be explained by hypothesizing that the repression of abd-A in PS13 of the CNS is mediated by the transcription over a potential enhancer, located upstream from exon 8 of the iab-8 ncRNA. This enhancer would not be transcribed over by the abdA:GFP transgene, nor by the ectopic expression of the UAShsp70ex8GFP construct.
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This hypothesis, however, is in direct conflict with the results showed by R. Maeda using CRISPR-mediated deletions of the BX-C. Both of the large deletions which include or exclude exon 8 of the iab-8 ncRNA show de-repression of abd-A in PS13 of the CNS (caused by the absence of the microRNA), which indicates that the enhancer of abd-A that drives its expression in PS13 is still present in those lines, and therefore must lay downstream from exon 8 of the iab-8 ncRNA.
The splicing of the iab-8 ncRNA may be necessary for transcriptional interference There is another difference between the iab-8 ncRNA and the transcripts generated by the abdA:GFP and UAShsp70ex8GFP constructs. The iab-8 ncRNA is spliced, but none of the two transgenes are, being composed of a single exon.
It has been shown that splicing to the terminal exon of a gene occurs cotranscriptionally in yeast (Dye and Proudfoot 1999). Even more, a study made in vitro in HeLa cells proposed that splicing of the last intron does not only promote termination, but it is necessary for the recruitment of the termination machinery to the poly(A) site (Rigo and Martinson 2008).
The structure of the iab-8 ncRNA shows that, out of its 7 introns, 6 of them have an extreme length (more than 10Kb per intron). The insertion of terminator sequences in the BX-C, which stop the transcription of the initiator RNAs, have never been able to prematurely terminate the iab-8 ncRNA (A. Muttero, unpublished). This supports the idea of the need of the terminator being present on an exon in order to efficiently terminate transcription.
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However, the last intron (necessary for the definition of the terminal exon) is small in comparison, comprising less than 2Kb. Outside of the CNS this intron would be normally efficiently spliced, defining exon 8 as the terminal exon and, therefore, an efficient termination of the iab-8 ncRNA (M. Soller, personal communication). In the CNS, however, exon 8 could be skipped, and therefore the terminal exon would not be defined, causing the RNA polymerase to continue transcribing until finding a new exon with a splice acceptor site compatible with the donor site present in exon 7 of the iab-8 ncRNA.
This would be enhanced by the action of the CNS specific factor ELAV (Figure 43):
Figure 43 Involvement of ELAV in the generation of the iab8/abdA chimeric RNA ELAV probably binds to the splice acceptor of exon 8 of the iab-8 ncRNA, preventing splicing between exons 7 and 8. It could additionally bind to the terminator sequence present at the end of exon 8.
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ELAV is necessary for transcriptional interference
The generation of the iab-8 ncRNA transcriptional readthrough is a process that depends on the presence of the RNA-binding protein ELAV (Figure 43). This observation led me to prove that ELAV is also necessary for the repression by transcriptional interference exerted by the iab-8 ncRNA. This was confirmed by the observation of the de-repression of the abdA:GFP transgene in elav mutant embryos.
The initial role described for ELAV as an RNA binding protein was its implication in the splicing of the neural isoform of the gene Nrg (Figure 44, also Figure 25). This splicing is a very similar phenomenon to the one happening in the case of the iab-8 ncRNA, in which the terminal exon of Nrg is skipped in the CNS, allowing for the elongation of its transcriptional unit and for its splicing into a new exon.
Figure 44 Representation of the alternative splicing of the terminal exon of the gene Neuroglian. Modified from (Koushika et al. 1996).
In this case, ELAV has been proposed to bind to sites present in the Nrg167 terminal exon, occluding the splice acceptor and therefore preventing its processing as an exon (Koushika et al. 1996). Interestingly, whole genome ChIP-seq against ELAV has shown that ELAV binds to exon 8 of the iab-8 ncRNA (Oktaba et al. 2014), so it would seem likely that it is exerting a similar function in the generation of the iab8/abdA chimeric RNA.
The implication of ELAV in transcriptional interference mediated by the iab-8 ncRNA would explain its neural specificity.
127 Conservation of transcriptional interference
The conservation of the iab-8 ncRNA transcriptional unit had already been shown in (Gummalla et al. 2012), at least in two other species of Drosophila. In this thesis I have added to this the evidence that, in D. virilis, the iab-8 ncRNA does not efficiently terminate its transcription in the CNS, generating a similar readthrough than the one observed in D. melanogaster. This readthrough is detected transcribing as far as the 3'UTR of abd-A, which may suggest the existence of a chimeric iab8-abdA transcript in this species.
This result, in addition with the detection of such a chimeric transcript by RT-PCR in Bombyx mori (Wang et al. 2019), suggests that intergenic splicing between the iab-8 ncRNA and abd-A is a conserved phenomenon. If this splicing is the cause for transcriptional interference over abd-A, as it seems to be, this would add a strong reinforcement to the proposition made in (Gummalla et al. 2012) which proposed transcriptional interference as the most ancient role of the iab-8 ncRNA.
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