Regulating expression levels – the role of the 3’UTR

Another addition to the canon of regulatory mechanisms controlling Ubx expression is the notion that the length of the Ubx 3’ Untranslated Region (3’UTR) can be alternatively chosen during development during the post-transcriptional process of alternative poly-adenylation (APA).

In most cases the termination of the mRNA transcript involves the recognition of specific AU rich elements within the transcript by protein complexes that consequently cleave the mRNA transcript from the RNA Pol II transcriptional machinery and promote the poly-adenylation of the transcript (Fig.1.6A). The addition of these poly-A nucleotides to the gene transcript is important for the stability and translatability of each mRNA transcript within the cell. It is now apparent that the process of cleavage and poly-adenylation occurs co-transcriptionally(Bentley, 2014; Millevoi and Vagner, 2010). Indeed evidence exists that the CTD domain of the RNA Pol II complex directly interacts with and recruits cleavage/poly-A factors to the transcript (Fig.1.6B).

Alternative poly-adenylation occurs when there is more than one set of sequences present within a gene to trigger cleavage and poly-adenylation. The result is the formation of mRNAs that have alternative endings meaning, that each transcript could contain alternative exons and/or 3’UTRs (Proudfoot, 2011).

What determines which site is chosen? The main deciding factor in the decision to which site is chosen, is the relative strength of each site. Sites with high similarity to the consensus poly-adenylation sequences (including upstream and downstream sequences) will have a kinetic advantage in being able to form the cleavage complex and thus initiate transcript termination and activate poly-adenylation.

Recent evidence is emerging that suggests many factors including the relative concentration of required proteins, and the speed of RNA Pol II elongation can also affect site choice (Proudfoot, 2011). The Ubx locus has two possible poly-adenylation sites within its 3’UTR region, a proximal site (closest the 3’ exon) and a more distal site separated by approximately 1100 nucleotides. The choice of site can lead to Ubx transcripts that will have either a short or long (extended) 3’UTR. It has been documented that over the course of Drosophila embryogenesis, the relative abundance of short and long Ubx 3’UTR isoforms changes so that by the time Ubx expression is confined to the CNS during late embryogenesis, the long 3’UTR is the dominant isoform (Kornfeld et al., 1989; O’Connor et al., 1988).

Fig.1.6 The process of alternative poly-adenylation occurs co-transcriptionally

(AB) Schematic showing the common sequence elements associated with the process of transcript cleavage and poly-adenylation site choice (B) Schematic highlighting the relationship between transcription and poly-adenylation. Three complexes/factors – CstF, CPSF and PABP are shown associated with the CTD region of the RNA Pol II machinery. These factors are heavily involved in the poly-adenylation site choice for transcript cleavage and poly-adenylation. Specific site-choice is governed by the recognition of specific sequence elements (coloured bars within transcript) by these factors. Alternative poly-adenylation occurs when more than one suitable site is present within the transcript. Alternative site choice can be affected by many factors including the speed of transcription and the relative concentration of appropriate factors required for poly-adenylation. See (Bentley, 2014; Millevoi and Vagner, 2010; Proudfoot, 2011) for detailed descriptions of these processes.

A

5’

3’

U-Rich

AAUAAA

CA

U/G-rich

0-20 nt

15-30 nt

0-20 nt

B

PolII

The biological relevance of this APA phenomenon has become more apparent since the discovery of microRNAs (miRNAs) - small non-coding RNAs that regulate gene expression along with a greater understanding of the role RNA-Binding Proteins (RBPs) can play in influencing gene expression during animal development. Both of these potential regulators predominantly bind to the 3’UTR of their target genes to exert their function. Thus the extension of the 3’UTRs, now seen as a common phenomenon during development can have real regulatory importance (Hilgers et al., 2011; Smibert et al., 2012).

It has been documented that the Ubx 3’UTR is under regulatory pressure from the miRNAs iab-4/iab-8 during embryogenesis (Bender, 2008; Ronshaugen et al., 2005; Stark et al., 2008; Tyler et al., 2008). In a study by Thomsen et al, a correlation was made with the onset of this miRNA regulation and the transition to the extended long

Ubx 3’UTR, showing that the extended 3’UTR isoform was required for correct Ubx

expression patterns during late embryogenesis (Thomsen et al., 2010). The biological importance of iab-4/iab-8 regulation of Ubx during CNS development is still an unresolved question.

As yet, there is no evidence linking the regulation of Ubx expression through RBP activity but many studies have shown the potential regulatory potential of these proteins. A well characterised example is the RBP Pumilio. This protein has been shown to regulate translation by binding to the 3’UTR of its target genes in more than one developmental context. The interaction of Pumilio and another RBP, Nanos with the 3’UTR of hb mRNA is essential for the posterior patterning in the embryo (Murata and Wharton, 1995; Wreden et al., 1997). Furthermore, both Pumilio and Nanos have also been implicated in the control of translation within developing neurons, affecting their morphogenesis and plasticity (Ye et al., 2004).

An intriguing relationship between RBPs and miRNAs may exist, in which the former can control the accessibility of the latter, affecting the regulatory potential of the miRNAs (Alonso, 2012). This regulatory relationship, shown by Kedde and colleagues, demonstrated that the binding of the vertebrate Pumilio homolog Pumilio-1 (PUM1) to a target 3’UTR, altered its structure, allowing miRNAs to target this gene more efficiently (Kedde et al., 2010). Other studies have implicated the regulation by Pumilio/miRNAs in controlling the expression of potent oncogenes (Miles et al., 2012).

Evidence suggests that the regulation of Hox gene 3’UTRs is a conserved method of fine-tuning expression and function in both vertebrates and invertebrates. An early study focusing on the transcriptional regulation of vertebrate Hox gene expression

alone was unable to reproduce the correct domains of expression. The authors reasoned that destabilisation of the Hox gene transcripts through the 3’UTR was required to maintain the correct posterior domains of expression (Brend et al., 2003). A number of studies have shown that that miR-196 (interestingly, the ortholog of

iab4/8) targets HoxB8 during development, controlling the posterior domains of

expression along the main body axis (Hornstein et al., 2005; McGlinn et al., 2009). A further study was able to show that this miRNA regulates HoxB8 within the neural tube and demonstrated that disruption to this regulation results in incorrect motoneuron formation (Asli and Kessel, 2010).

Regulation of gene expression through the 3’UTR is emerging as a potentially powerful method in fine-tuning the expression and consequently, the functionality of any given gene. In the case of developmental regulators like the Hox genes, transcription factors which have the potential to alter cell-states and developmental pathways, this mechanism of regulation may be very important.