Cell Cycle Kinase Polo Is Controlled by a Widespread 3′ Untranslated Region Regulatory Sequence in Drosophila melanogaster

Abstract

Alternative polyadenylation generates transcriptomic diversity, although the physiological impact and regulatory mechanisms involved are still poorly understood. The cell cycle kinase Polo is controlled by alternative polyadenylation in the 3= untranslated region (3=UTR), with critical physiological consequences. Here, we characterized the molecular mechanisms required for polo alternative polyadenylation. We identified a conserved upstream sequence element (USE) close to the polo proximal poly(A) signal. Transgenic flies without this sequence show incorrect selection of polo poly(A) signals with consequent downregulation of Polo expression levels and insufficient/defective activation of Polo kinetochore targets Mps1 and Aurora B. Deletion of the USE results in abnormal mitoses in neuroblasts, revealing a role for this sequence in vivo. We found that Hephaestus binds to the USE RNA and that hephaestus mutants display defects in polo alternative polyadenylation concomitant with a striking reduction in Polo protein levels, leading to mitotic errors and aneuploidy. Bioinformatic analyses show that the USE is preferentially localized upstream of noncanonical polyadenylation signals in Drosophila melanogaster genes. Taken together, our results revealed the molecular mechanisms involved in polo alternative polyadenylation, with remarkable physiological functions in Polo expression and activity at the kinetochores, and disclosed a new in vivo function for USEs in Drosophila melanogaster.

On the function and relevance of alternative 3′‐UTRs in gene expression regulation

“…Additionally, U1 also regulates IgM heavy chain APA by preventing the shift from the membrane-bound form to the secreted form during B-cell activation, through binding of U1A to GU-rich sequences downstream of the secretory poly(A) site (Phillips et al, 2004). Polypyrimidine Tract Binding Protein 1 (PTBP1), which belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) subfamily of RBPs, is a protein involved in splicing (Singh et al, 1995;Spellman et al, 2005), polyadenylation (Castelo-Branco et al, 2004;Le Sommer et al, 2005;Lou et al, 1999;Moreira et al, 1998), and APA (Gruber et al, 2018;Oliveira et al, 2019). The function of PTBP1 as a regulator of poly(A) site choice has been recently described in glioblastomas, where PTBP1 masks poly(A) sites preventing the occurrence of cleavage and polyadenylation (Gruber et al, 2018).…”

“…Furthermore, in vivo studies in Drosophila have disclosed a function for the human PTBP1 orthologue, Hephaestus, in the APA of polo , which codes for a key cell cycle kinase. Moreover, Hephaestus mutants revealed a physiological role for this protein in mitosis and aneuploidy (Oliveira et al, 2019).…”

“…This hexanucleotide sequence is the canonical PAS and the strongest in polyadenylation efficiency; however, more than 10 variants with weaker polyadenylation efficiencies have been described (Beaudoing et al, 2000). Cis ‐auxiliary elements flanking the PAS, such as upstream sequence elements (USEs; Danckwardt et al, 2007; Moreira et al, 1998; Natalizio et al, 2002; Oliveira et al, 2019) and downstream sequence elements (DSEs) (Nunes et al, 2010; Zarudnaya et al, 2003) are also critical to assist weak PASs and to help define the poly(A) site.…”

So close, no matter how far: multiple paths connecting transcription to mRNA translation in eukaryotes

“…However, the positive impact of short 3 0 UTRs on translation is not absolute. In Drosophila, for example, the two polo mRNA isoforms exhibit different TE, the shorter isoform being three-fold less translationally productive (Pinto et al, 2011), exerting a regulatory role in mitosis (Oliveira et al, 2019). Interestingly, the regulation of PAS use in the case of polo depends on the levels of Polo protein, supporting the idea that APA may serve the needs of balanced and dynamic protein expression.…”

Alternative polyadenylation of mRNA and its role in cancer

“… 41 , 105 , 106 The difference in translation efficiency enables neuronal activation/differentiation to trigger a rapid accumulation of BDNF protein levels. Similarly, the Drosophila transcript polo , which encodes Polo-like kinase, 90 , 107 was demonstrated to exhibit higher translation efficiency from its longer 3′UTR isoform than for the shorter 3′UTR isoform. Loss of the distal PAS of polo is lethal for flies during development, while proximal PAS deletion has no phenotypic effect.…”