Ubx isoform distribution during post-embryonic development

To be able to understand the regulation of Ubx by miRNAs through targeting of the 3’UTR during haltere development, we first needed to determine which Ubx 3’UTR isoforms were present during the post-embryonic development of Drosophila and specifically, the haltere imaginal discs.

After embryogenesis, Drosophila development continues through three larval developmental stages (L1-L3), a short pre-pupal stage (WPP) and pupal development (P) in which the metamorphosis of the adult occurs (A). Early studies in Ubx transcript processing and expression suggested that APA of the Ubx 3’UTR is actively regulated during embryonic development (Kornfeld et al., 1989; O’Connor et al., 1988). These studies suggested that through the use of a proximal (PAS1) and distal (PAS2) poly- adenylation site, both a short and long 3’UTR isoform was present during larval and pupal development (Fig.3.1A).

We first confirmed these results by determining the relative transcript levels of Ubx- short and Ubx-long 3’UTR isoforms during post-embryonic development.

Fig.3.1 Ubx 3’UTR isoform distribution and functionality during post-embryonic development

(A) Genomic map of Ultrabithorax, the gene spans approximately 75kbs. Exon sequences are identified in grey boxes. The 3’ exon and UTR are shown in expanded sequence. The two active poly-adenylation sites are shown. The extended 3’UTR sequence is approximately 2kb in length. (B) Analysis of Ubx 3’UTR isoforms during post-embryonic development. Amplicons representing all Ubx transcripts are shown in blue, amplicons representing Ubx transcripts containing extended 3’UTR are shown in red. Respective relative expression levels are plotted in blue and red. Error bars represent standard deviation (variation) between biological replicates. Values are calculate by averaging three technical replicates for each biological sample (C) mCherry constructs used in 3’UTR expression analysis, the control mCh-K10 construct carries a viral K10 3’UTR sequence, the experimental mCh-Ubx construct carries an extended Ubx 3’UTR lacking PAS1.(D - E) Sample images of mCh-K10 and mCh-Ubx expression in haltere imaginal disc. Yellow boxes highlight regions measured for expression analysis. (F) Plot profile showing expression analysis of mCh-K10 and mCh-Ubx constructs. (G) Magnification of areas measured for expression intensity of mCh-K10 and mCh-Ubx constructs. Scale bar represents 25µm.

C

Ubx Isoform Expression

Ubx Universal Ubx Distal

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A

Anterior - Posterior Axis

mCherry Construct Expression Levels mCh-K10 mCh-Ubx

F

D

E

G

Fig.3.1 Ubx 3’ UTR isoform distribution and functionality during post-embryonic development

Using a semi-quantitative RT-PCR approach (SQ-RTPCR), we determined the total

Ubx mRNA levels using oligonucleotides that amplify a ~200bp amplicon of the 3’UTR

proximal to the first poly-adenylation site. This was termed the Ubx-universal amplicon. The relative expression levels of Ubx-long isoforms were detected by oligonucleotides that amplified a ~200bp region distal to the first poly-adenylation site and termed the

Ubx-distal amplicon (Fig.3.1B). As a control reaction across all samples and technical

replicates, we designed oligonucleotides to detect the ribosomal protein rp49. Using this experimental strategy we could determine the total levels of Ubx transcript expression and the abundance of Ubx-long isoform specific transcripts across multiple post-embryonic stages and within the haltere imaginal discs.

Our results show that total levels of Ubx vary greatly during post-embryonic development (Fig.3.1B). Ubx expression initially decreases from L1 to L2 stages; a point in which the larvae are growing rapidly, expression of Ubx then increases to its highest level at L3 stage, a time when the imaginal discs are undergoing extensive pre- patterning. From this developmental time point, expression gradually decreases through the WPP stage (where the larvae begins to form the pupae) and pupal stage (where the larvae begins its transformation into the adult form). When the adult emerges, levels of Ubx expression reach their lowest point, likely reflecting the diminished number of cells and tissues still expressing Ubx. Over these developmental stages, the expression levels of Ubx-long isoforms matched total Ubx transcript expression. From this data we can infer that during post-embryonic development the majority of Ubx transcripts carry the Ubx-long 3’UTR. To determine if this distribution is seen in the haltere imaginal discs specifically, we assayed expression of Ubx in haltere discs dissected from WPP stages larvae. Our data indicates that the dominant isoform among all Ubx transcripts during post-embryonic development carry the extended long 3’UTR.

Having determined the dominant Ubx 3’UTR isoforms present within the developing haltere imaginal disc, we next looked for evidence that this 3’UTR was regulated in a manner that could affect expression patterns in the haltere. To achieve this we monitored the expression of a transgenic UAS::mCherry fluorescent reporter protein coupled to either a control viral K10 3’UTR or Ubx-long 3’UTR (Thomsen et al., 2010) (Fig.3.1C). Expression of these transgenic insertions was driven by the UbxGAL4M1 _(De

Navas et al., 2006a)which expresses throughout the haltere imaginal disc.

We determined the relative signal intensity of mCherry expression across the dorsal pouch region of the haltere in both control and experimental UTRs (Fig.3.1D-E). The

axis in the disc were plotted (Fig.3.1F). It is clear that overall mCherry expression is greatly reduced by the presence of the Ubx 3’UTR. The relative intensity of mCherry- K10 expression is on average between 35 and 45 units whereas mCherry-Ubx expression is between 10 and 20 units. There are also distinct peaks of intensity at the far left and far right of the mCherry-K10 plot (see blue shaded areas), these are noticeably absent in the mCherry-Ubx plots. This may indicate the corresponding regions within the imaginal disc are under a high degree of negative regulation. Interestingly, we note that individual measurements of the mCh-K10 expression patterns (light grey lines) are far more varied than the individual measurements seen with the mCh-Ubx samples. This suggests that there is a great deal of transcriptional variation at the Ubx locus, picked up by the UbxGAL4M1 _{insertion, but perhaps masked}

by the presence of regulatory elements present within the Ubx 3’UTR.

Through semi-quantitative RT-PCR analysis, we have seen that Ubx mRNA levels vary during the post-embryonic life cycle of Drosophila, peaking at late larval and pre-pupae stages. This peak of expression correlates with the latter stages of a prolonged phase of growth and differentiation. We show that the dominant 3’UTR isoform among the total Ubx mRNAs is that of the extended long 3’UTR. Specific analysis of the haltere imaginal disc also shows this isoform distribution. The complex transition of different 3’UTR isoforms caused by APA during embryogenesis is not seen post-embryonically.