Functional consequences of haltere miRNA expression – The Regulation of

Ultrabithorax

In the Chapter 3, we showed that the fine-tuning of Ubx expression by miRNAs can be an important regulatory step in controlling Ubx functionality. Now, with a better understanding of the miRNA content within the haltere, we re-assessed likely Ubx- miRNA interactions during the development of this appendage. We were interested in first examining the relationship between predicted miRNA targeting strength and miRNA expression group association, and secondly, miRNA targeting strength and overall miRNA expression level within the haltere. Were miRNAs that are preferentially or highly expressed in the haltere, more or less likely to target Ubx transcripts?

We submitted the long isoform of the Ubx 3’UTR to the PITA target prediction algorithm using the most current miRNA annotations within the Drosophila genome (BDGP5.6). We analysed the PITA results, cross-referencing with our documented haltere miRNA

expression profile data. At the time of analysis there were 192 mature miRNAs annotated within the Drosophila genome. Following PITA target prediction, we see that 154 (78%) miRNAs have one or more target sites within the Ubx 3’UTR (Fig.5.6A). How do these 154 Ubx targeting miRNAs distribute amongst the miRNA expression groups defined in our profiling experiments? We observe that just over 70% of all Ubx targeting miRNAs had detectable expression within the haltere, the largest contingent of miRNAs belonging to the Average expression group. This would be expected since the Average expression group contained the most miRNAs and therefore would by chance have more miRNAs capable of targeting Ubx. Interestingly, the Halt Only expression group had the second highest contribution of miRNAs targeting Ubx (Fig.5.6B). We next analysed the relationship between individual miRNA target sites of the Ubx 3’UTR and their associated PITA prediction scores by plotting these two factors against each other (Fig.5.6C). In this scatter plot, each data point represents a specific region of the Ubx 3’UTR along the x-axis and the PITA ΔΔG score associated with each target site along the y-axis. There are 503 predicted target sites within the

Ubx 3’UTR with a negative ΔΔG. Overall, we observe an increased density of target

sites with a ΔΔG score between 0 to -8 (represented by white dashed line). There are noticeably fewer miRNA sites with scores lower than this. This may be of significance when considering which miRNAs could be potent regulators of Ubx (the more negative the ΔΔG score, the more likely a miRNA-target interaction will occur). Using this analysis we do not see obvious differences in the distribution of target sites along the length of the Ubx 3’UTR, suggesting there are no specific regions within the 3’UTR where miRNA targeting elements are enriched.

Fig.5.6 miRNA group associations and potential for Ubx-miRNA regulatory interactions

(A) Summary of the number of miRNAs predicted to target the extended Ubx 3’UTR. (B) Group associations for miRNAs predicted to target the extended Ubx 3’UTR. (C) Scatter plot displaying the relationship of Ubx targets sites along the Ubx 3’UTR and their predicted targeting strength. (D) Analysis of miRNA target score distributions broken down into the respective miRNA expression groups. (E) Scatter plot displaying the association between a miRNAs overall targeting strength against Ubx and the number of target sites predicted within the Ubx 3’UTR. (F) Distribution analysis of the number of target sites detected for each miRNA predicted to target Ubx, broken down into each miRNA group. In panels C & E, white vertical line represents approximate location of the first poly-adenylation site.

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154

192

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Number of miRNAs Targeting Ubx

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miRNA Groups

** p<0.01 ** p<0.01

F

* p<0.05 ** p<0.01 29.22 13.64 9.74 17.53 1.30 28.57

% of miRNAs Targeting Ubx

Average Halt Up Halt Down

We analysed the distribution of overall miRNA targeting strength (the collective score of all individual target sites for each miRNA) for all miRNA expression groups (Fig.5.6D). We observe that each expression group except the Halt Down miRNAs have similar ΔΔG score distributions. The Halt Down expression group had a significant decrease in ΔΔG score distribution corresponding to their associated miRNA target sites (p<0.002 comparing Halt Up x Halt Down and Halt Down x Halt Only). These target scores (only one below -8) suggest that these miRNAs are less likely to target the Ubx 3’UTR within the developing haltere tissue.

We next assessed the association between the number of sites per miRNA and the overall score for each miRNA (Fig.5.6E). Again there is a notable threshold in miRNA density with relatively few miRNAs having more than 10 target sites within the 3’UTR. Of the 12 miRNAs to have more than 10 target sites, only four had detectable expression within the haltere. Analysis of miRNA distributions showed no obvious correlation between the number of target sites and the overall ΔΔG score. This suggests that in general, most miRNAs that potentially target Ubx have a small number of sites which could lead to miRNA-target interactions.

We analysed the distributions of target site number per miRNA within each miRNA expression group (Fig.5.6F). Here, the Halt Up group has a small but significant increase in the target site distribution (p<0.01 comparing Average x Halt Up, p<0.05 comparing Halt Up x Halt Down). Thus miRNAs within the Halt Up cohort tend to have an increased number of miRNA target sites within the Ubx 3’UTR.

Having seen that specific miRNA expression groups have certain associations with either target score distribution or the number of target sites, we next assessed to what extent the expression levels of each miRNA may correlate with the likelihood of targeting the Ubx 3’UTR.

We examined the association between each miRNA target site and its corresponding ΔΔG score. However, we now took into context the expression level of each miRNA within the haltere, defined by our sequencing data and shown by the changing colour of each target site data point (Fig.5.7A). Overall we see that the most highly expressed miRNAs have target sites associated with higher ΔΔG values. This suggests that these miRNAs would be less likely to target the Ubx 3’UTR. However this was not a definitive association, a number of miRNA target sites with very negative ΔΔG scores are associated with highly expressed miRNAs within the haltere (see arrowheads Fig.5.7A).

Haltere Expression Groups – Very High, High, Medium, Low and None dependant on their expression value within the haltere. Each miRNA association with a particular expression group was determined by calculating the quartile ranges of all miRNA expression values within the haltere. miRNAs with no expression in the haltere were automatically grouped into the None expression group.

Having sorted each miRNA into their respective haltere expression groups, we examined the distribution of overall miRNA target scores (Fig.5.7B). Although the distributions of the Very High and High expression groups are greater than the Medium, Low and None, these differences are not statistically significant. This suggests there is no correlation between overall expression level within the haltere and the likelihood of a miRNA targeting Ubx transcripts.

We next investigated the associations between the number of target sites, the ΔΔG score of individual miRNAs and their respective expression values (Fig.5.7C). The most striking observation to be made when analysing this data is that none of the miRNAs with numerous target sites are highly expressed within the haltere tissue. To examine this further, we determined the distribution of target sites amongst the miRNA associated haltere expression groups (Fig.5.7D). Although there appears to be very little change in distribution between groups, there are statistically significant differences. miRNAs present within the Very High expression group tend to have fewer sites per miRNA than the Medium expression group (p<0.05). Additionally, we observe that miRNAs with no expression in the haltere tend to have more target sites per miRNA than the Low expression group (p<0.01).

To summarise, 70% of all potential miRNA regulators targeting Ubx are expressed within haltere tissue. The analysis of miRNA target score distributions shows that there is no particular miRNA expression group more likely to target Ubx than another. However, our data does indicate that miRNAs down-regulated in the haltere (Halt Down group) are less likely to target Ubx transcripts. Analysis of target site number for each miRNA group association shows that miRNAs enriched for haltere expression are more likely to have an increased number of target sites within the Ubx 3’UTR. This may be evidence of the evolution of target sites within the 3’UTR for miRNAs preferentially expressed within this tissue.

Fig.5.7 miRNA expression levels and the effect on potential Ubx-miRNA regulatory interactions

(A) Scatter plot displaying the relationship of Ubx targets sites along the Ubx 3’UTR and their predicted targeting strength. Each miRNA dot colour is representative of the miRNA expression level detected in the haltere. (B) Analysis of miRNA target score distributions broken down into five expression groups based on the miRNA expression level detected in the haltere. (C) Scatter plot displaying the association between a miRNAs overall targeting strength against Ubx and the number of target sites predicted within in the Ubx 3’UTR. Each miRNA dot colour is representative of the miRNA expression level detected in the haltere. (D) Distribution analysis of the number of target sites detected for each miRNA predicted to target Ubx, broken down into the five haltere expression groups. Statistical significance determined using Students’ t-test, p- values given in figure. In panels A & C, white vertical line represents approximate location of the first poly-adenylation site.

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miRNA Target Sites Along the Ubx 3’UTR

Relationship between miRNA Score and Number of Target Sites

Distribution of miRNA Target Scores by Haltere Expression

Distribution of the number of miRNA Sites by Haltere Expression

* p<0.05 ** p<0.01 Fig.5.7 miRNA expression levels and the effect on potential Ubx-miRNA regulatory interactions

likelihood of targeting Ubx. This could be an indication that high expression levels are not required to evolve potential miRNA-target interactions. However, an intriguing observation is that highly expressed miRNAs show a small but significant tendency to have fewer target sites within the Ubx 3’UTR. An interpretation of these observations is that miRNAs with low expression levels are compensated by an increased number of target sites within a 3’UTR to compete with highly expressed miRNAs.

5.6 The integration of miRNA regulation into the Ubx regulated transcriptome of