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

We wanted to assess to what extent miRNA regulation may be integrated into the Ubx directed transcriptome during haltere development. Many published studies indicate that Ubx directly and indirectly regulates countless numbers of transcripts within the haltere imaginal disc (Hersh et al., 2007; Pavlopoulos and Akam, 2011). This regulation is negative and positive, many transcripts increasing or decreasing expression levels. We were interested in how the miRNA content of the haltere imaginal disc may be integrated into the global regulation of gene expression within the haltere. Do the miRNAs help reduce the expression levels of genes transcriptionally down-regulated by

Ubx or alternatively help fine-tune and buffer the transcriptionally up-regulated genes

within the haltere?

To try and answer these questions we integrated our haltere miRNA expression profiles with available microarray transcriptomic data published by Pavlopoulos and Akam, 2011, which experimentally uncovered transcriptional targets of Ubx. Using this data, we generated three cohorts of genes, containing the top 10% of transcripts significantly down-regulated or up-regulated by Ubx. These were termed ‘Ubx

Downregulated’ and ‘Ubx Upregulated’ respectively. Additionally, we included a ‘Ubx Neutral’ set of transcripts representing 100 genes that show no response to Ubx

transcriptional activity to be used as a control group.

For all genes in each cohort, the 3’UTR sequence was obtained and submitted to the PITA Target Prediction software (Kertesz et al., 2007) along with our experimentally defined set of miRNAs present within the haltere. The resulting target scores for every gene within each cohort was collated. The resulting data was hierarchically clustered using similarities in miRNA targeting scores (Fig.5.8A-C). Comparison of these three target score maps shows that most genes within each cohort have a high potential for

targeting interactions with haltere miRNAs. What is noticeable when comparing the three gene cohorts is that the Ubx Upregulated cohort appears to contain a greater number of genes targeted by each individual miRNA. Additionally, genes within the Ubx Downregulated cohort appear to have a higher prediction score values for each miRNA interaction indicated by the darker orange hues presented within the heat map.

We examined these two points further. Since the number of genes within each Ubx regulated cohort differs, we first calculated the number of genes targeted by each miRNA as a percentage of the total number of genes found within each respective cohort. Analysing the distributions of these Gene Target percentages, comparing each cohort, we revealed a highly significant difference in target percentage when comparing the Ubx Upregulated cohort to both the Ubx Neutral and Ubx Downregulated cohorts respectively (p<0.001 Fig.5.8D). There was no significant difference in score distribution when comparing the Ubx Downregulated to Ubx Neutral cohorts. Together this data indicates that miRNAs found within the haltere are predicted to target a greater percentage of genes transcriptionally up-regulated by Ubx. We furthered this analysis by examining the gene target percentages when each cohort distribution was segregated into the respective miRNA expression groups (Fig.5.8E). There is a general trend amongst expression groups where each tends to target a greater percentage of the Ubx Upregulated cohort. There are no significant changes in percentage score distributions comparing Ubx Upregulated and Ubx Downregulated genes within the Halt Down and Halt Up expression groups, indicating these miRNAs can efficiently target genes in both cohorts.

Fig.5.8 Integration of miRNA regulation into the Ubx directed haltere transcriptome

(A-C) Heatmaps displaying the potential regulatory interactions between transcripts from the Ubx Downregulated (A), Ubx Upregulated (B) and Ubx Neutral (C) gene cohorts miRNAs present in the haltere. (D) Box plots displaying the distributions of the percentage of Ubx regulated genes targeted by individual miRNA within the haltere comparing Ubx Downregulated, Ubx Upregulated and Ubx Neutral gene cohorts (*** p<0.001 Mann-Whitney U-test). (E) The same distribution analysis, now showing changes in target percentages between Ubx regulated cohorts in each miRNA expression group (*** p<0.001, ** p<0.01 Mann-Whitney U-test) (F) Box plots displaying the distributions of the average scores for each haltere miRNA in the Ubx regulated gene cohorts (* p<0.05). (G) The same analysis average score distributions in each miRNA expression group.

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In summary, we see that all expression groups are more likely to target Ubx Upregulated transcripts, however the Halt Up and Halt Down groups are also still likely to have a strong regulatory influence on Ubx Downregulated transcripts. It is intriguing to speculate that perhaps the causative reason for differentially expressed miRNAs within the haltere, is their differing abilities to target Ubx regulated transcripts.

Previously we noted that genes from the Ubx Downregulated cohort appeared to have stronger target predictions when compared with the other cohorts. We explored this further by calculating the Average Score of targeting strength for each miRNA, looking for differences in Average Score distribution (Fig.5.8F). We observe a nominally significant (p<0.05) change when comparing the Ubx Upregulated and Ubx Downregulated cohorts. Transcripts up-regulated by Ubx tend to have more negative average scores implying these genes are under greater targeting pressure by haltere miRNAs. This analysis does not correspond to our initial observations of the data. We further analysed the Average Score distributions broken down by miRNA expression group (Fig.5.8G). Here we see a general trend where the average scores of the Ubx Upregulated genes tend to be more negative, however there are no significant disparities when comparing each miRNA expression group.

Finally, we documented the top predicted miRNA regulators in both Ubx regulated cohorts (Fig.5.9A-B). In each grouping of genes, the major miRNA expression group represented is that of the Average expression group (see pie-chart inserts). Interestingly, each gene cohort is targeted best by the same two miRNAs suggesting that these RNAs could be fundamental regulators required during haltere development. Comparing both lists, we see a 75% similarity in miRNAs within each list. This may indicate that these miRNAs have important roles in regulating the general haltere transcriptome. Interestingly, in both cases there are 5 miRNAs which are specific to each cohort. It is intriguing to think that these miRNAs may have particular roles in helping Ubx regulate haltere development.

In summary, we explored the possibility that the miRNAs were integrated into the Ubx regulated transcriptome of the developing haltere. Analyses of target prediction scores suggest that Ubx up-regulated transcripts are more likely to undergo miRNA targeting and subsequent regulation of gene expression. However, we still observe that transcripts undergoing negative transcriptional regulation by Ubx are also likely to be targeted.

Fig.5.9 Top predicted miRNA regulators of the Ubx instructed transcriptome

Summary bar plots of the top targeting miRNAs detected for the Ubx Downregulated and Ubx Upregulated gene cohorts respectively. miRNAs which are specific to each top 20 list are shaded orange and blue respectively.

In general these results raise the intriguing possibility that a main requirement for miRNA activity within the developing haltere is to help buffer and fine-tune the positive transcriptional regulation induced by Ubx during haltere development. We also identify a number of miRNAs that are prominent in their targeting abilities of both down- regulated and up-regulated genes within the developing haltere. It will be of interest to look further at these miRNAs and their relationship to Ubx activity and function. This analysis may also serve as a starting point in the study of the potential combinatorial regulation of gene targets by multiple miRNAs. Many genes from both cohorts are predicted to be targeted by multiple miRNA. The analysis of these genes, the miRNAs that potentially target them and their role in the development of the haltere are exciting potential avenues of further research.

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