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W4382788274 abstract "Full text Figures and data Side by side Abstract eLife assessment Introduction Results Discussion Materials and methods Appendix 1 Data availability References Peer review Author response Article and author information Metrics Abstract Aurora Kinase A (AURKA) is an oncogenic kinase with major roles in mitosis, but also exerts cell cycle- and kinase-independent functions linked to cancer. Therefore, control of its expression, as well as its activity, is crucial. A short and a long 3′UTR isoform exist for AURKA mRNA, resulting from alternative polyadenylation (APA). We initially observed that in triple-negative breast cancer, where AURKA is typically overexpressed, the short isoform is predominant and this correlates with faster relapse times of patients. The short isoform is characterized by higher translational efficiency since translation and decay rate of the long isoform are targeted by hsa-let-7a tumor-suppressor miRNA. Additionally, hsa-let-7a regulates the cell cycle periodicity of translation of the long isoform, whereas the short isoform is translated highly and constantly throughout interphase. Finally, disrupted production of the long isoform led to an increase in proliferation and migration rates of cells. In summary, we uncovered a new mechanism dependent on the cooperation between APA and miRNA targeting likely to be a route of oncogenic activation of human AURKA. eLife assessment In this important study, the authors provide compelling evidence that the interplay between alternative polyadenylation (APA) of mRNA encoding Aurora Kinase A (AURKA) and hsa-let-7a miRNA governs AURKA protein levels. The authors show that short 3'UTR isoform of mRNA encoding AURKA is efficiently translated throughout the cell cycle, while the long 3'UTR isoform is suppressed by hsa-let-7a miRNA in a cell cycle-dependent manner. These findings delineate post-transcriptional mechanisms regulating AURKA expression that may be implicated in increase in AURKA protein that is frequently observed across a variety of cancers. https://doi.org/10.7554/eLife.87253.2.sa0 About eLife assessments Introduction Aurora Kinase A (AURKA) is a critical positive regulator of the mitotic phase of the cell cycle (Willems et al., 2018). AURKA also plays additional cancer-promoting roles in cell proliferation, survival, migration, and cancer stem cell phenotypes, some of which in interphase and in a kinase-independent manner (Naso et al., 2021). AURKA expression follows a strict cell cycle-dependent pattern, with both protein and mRNA levels extremely low in G1 phase, increasing in S phase, and peaking at G2 phase until mitosis (Cacioppo and Lindon, 2022). High expression of AURKA is strongly associated with cancer progression, drug resistance, and poor prognosis, justifying why oncogenic AURKA represents a renowned target of anticancer drugs (Du et al., 2021), and making evident that oncogenic roles of AURKA are prompted by its highly sustained levels of expression. AURKA overexpression in human cancers is known to be caused by elevated gene copy number, enhanced transcription, or increased protein stability. Dysregulation of translation is also linked to disease and contributions of dysregulated translation to cancer phenotypes are increasingly reported (Kovalski et al., 2022; Modelska et al., 2015). Despite some modest evidence suggesting that modulation of AURKA translation is relevant in disease (Dobson et al., 2013; Lai et al., 2017), control of AURKA expression at the level of translation is widely understudied compared with control of its transcription and mRNA processing (Cacioppo and Lindon, 2022). For example, it is not clear whether AURKA mRNA undergoes translational activation and/or inhibition during the cell cycle, and the precise timing, extent, or regulators of these processes remain unexplored. The process of cleavage of the 3′end of precursor mRNAs (pre-mRNAs) and concomitant addition of a poly(A) tail represents one key event aiding the maturation of mRNAs, termed cleavage, and polyadenylation (C/P) (Gruber and Zavolan, 2019). The cleavage site is typically preceded by a polyadenylation signal (PAS), located 10–30 nucleotides (nt) upstream, and by UGUA and U-rich motifs, whereas it is typically followed by U- and GU-rich motifs. Altogether, these elements constitute the C/P site (Gruber et al., 2016). Most human pre-mRNAs contain multiple C/P sites (Derti et al., 2012), enabling alternative cleavage and polyadenylation (APA) and, thus, distinct expression of transcript isoforms for the same gene. A search using PolyA_DB (Wang et al., 2018) indicates the presence of two C/P sites with canonical PASs (AATAAA) on AURKA 3′ untranslated region (3′UTR) (Figure 1A). This fostered our hypothesis that AURKA mRNA could be subjected to tandem 3′UTR APA, resulting in two 3′UTR isoforms that differ in length. It is currently unknown which AURKA PAS is preferentially used in which cellular context or whether a 3′UTR isoform switch can be modulatable. Figure 1 Download asset Open asset Increased short/long ratio (SLR) of Aurora Kinase A (AURKA) alternative polyadenylation (APA) isoforms in triple-negative breast cancer (TNBC). (A) AURKA transcript isoforms (USCS Genome Browser). AURKA gene is located on (-) strand. (B) Median and range of SLR values for AURKA 3′UTR obtained using APADetect. Mann–Whitney test; ****p<0.0001. (C), (D) RT-qPCR analysis of SLR of AURKA 3′UTR in TNBC cell lines (C) and patient samples (D). SDHA used as reference gene. TN, tissue number. (E) Relapse-free survival rates of TNBC patients with high (highest 25%) or low (lowest 25%) AURKA SLRs. p-value determined by log-rank test. Figure 1—source data 1 Numerical data for graphs. https://cdn.elifesciences.org/articles/87253/elife-87253-fig1-data1-v1.xlsx Download elife-87253-fig1-data1-v1.xlsx APA is involved in most cellular processes and is often altered in cancer (Gruber and Zavolan, 2019). Clinically, human cancers are characterized by unique profiles of alternative 3′UTRs that can be exploited for classification of distinct cancer subtypes (Singh et al., 2009; Wang et al., 2020), and associations between 3′UTR shortening and poor prognosis (Lembo et al., 2012) or drug sensitivity Xiang et al., 2018 have been detected. At the molecular level, a strong positive association between expression of short 3′UTRs, increased protein levels, and proliferative states has been frequently reported (Sandberg et al., 2008; Mayr and Bartel, 2009; Masamha et al., 2014; Xia et al., 2014; Pieraccioli et al., 2022). Such genome-wide 3′UTR shortening sustains cancer cell behavior by removing repressor sequence elements from the 3′UTR of oncogenic mRNAs, for example, microRNA (miRNA) binding sites (Sandberg et al., 2008; Mayr and Bartel, 2009; Masamha et al., 2014), or alternatively by inactivating tumor suppressors through suppression of their expression (Lee et al., 2018; Park et al., 2018). The role of miRNAs in regulating cell cycle genes and the relevance of this regulation in cancer are well understood (Bueno and Malumbres, 2011; Ghafouri-Fard et al., 2020). Few miRNAs have been pointed to as regulators of AURKA mRNA but, importantly, reported cases of miRNA targeting of AURKA occur in those cancers where AURKA overexpression is a promoting factor or a marker of poor prognosis (Fadaka et al., 2020; Zhang et al., 2020; Yuan et al., 2019; Ma et al., 2015). Regardless, none of these studies consider the existence of distinct AURKA 3′UTR isoforms in their experimental design of targeting assessment. The hsa-let-7 miRNA family comprises 11 closely related genes that map in chromosomal regions that are typically deleted in human tumors and, given their pathogenic downregulation in cancer, they are classified as tumor suppressors (Bueno and Malumbres, 2011; Johnson et al., 2007). Roles for hsa-let-7a in breast tumor growth and metastasis have been proposed (Thammaiah and Jayaram, 2016; Shi et al., 2020) and a correlation between hsa-let-7a expression and clinical variables has been detected in triple-negative breast cancer (TNBC) (Avery-Kiejda et al., 2014; Turashvili et al., 2018). AURKA was classified within the TNBC subtype with the highest median index of 3′UTR shortening events (Wang et al., 2020; Akman et al., 2015), and also undergoes 3′UTR shortening in poor-prognosis patients of breast and lung cancer (Lembo et al., 2012). Importantly, AURKA overexpression in TNBC represents a marker of early recurrence, poor prognosis, and shorter overall survival (Xu et al., 2013; Jalalirad et al., 2021). However, the correlation between AURKA PAS usage, protein expression, and pathological cell behavior has not been explored for this or other biological contexts, nor at the molecular level. In this study, we uncover a molecular mechanism leveraging the cellular ratio of APA isoforms and their different translational program during the cell cycle to control acquisition of AURKA oncogenic potential. Results Increased short/long ratio of AURKA APA isoforms in TNBC In a preliminary study using the APADetect in silico tool, we analyzed publicly available microarray data to identify changes in AURKA 3′UTR isoform abundance in tissues (Akman et al., 2015). 520 comparable datasets for TNBC samples came from GSE31519 (Rody et al., 2011) and 65 histologically normal epithelium and cancer-free prophylactic mastectomy patients were used: 32 from GSE20437 (Graham et al., 2010), 12 from GSE9574 (Tripathi et al., 2008), 7 from GSE3744 (Richardson et al., 2006), 6 from GSE6883 (Liu et al., 2007), 5 from GSE26910 (Planche et al., 2011), and 3 from GSE21422 (Kretschmer et al., 2011). The analysis revealed increased short/long ratio (SLR) of AURKA 3′UTR isoforms in TNBC compared to normal breast tissues (Figure 1B). Higher SLR was confirmed by RT-qPCR in multiple TNBC cell lines (Figure 1C). Furthermore, RT-qPCR analysis of normal and TNBC patient cDNAs from Origene Breast Cancer cDNA array IV (BCRT504) also showed higher AURKA SLR in TNBC samples compared to normal (Figure 1D). In addition, the shortening of AURKA 3′UTR correlated with faster relapse times in TNBC patients (clinical data from Rody et al., 2011; Figure 1E). These results therefore suggest a potential oncogenic role of AURKA APA in breast cancer worth further investigations. AURKA shows 3′UTR isoform-dependent protein expression To probe AURKA APA isoform-dependent protein expression, we developed a single-cell expression sensor suitable for experiments in live cell. The construct independently expresses Venus and mCherry fluorescent proteins via a constitutive bi-directional promoter (Figure 2A). The coding sequence (CDS) of Venus is flanked by AURKA UTRs, whereas that of mCherry lacks regulatory regions and is therefore used to normalize for transfection efficiency. To test for APA-sensitive expression, we alternatively mutated the distal (d) or proximal (p) PAS on the reporter 3′UTR, to generate different 3′UTR isoforms (SHORT and LONG, respectively). Constructs lacking AURKA UTRs (Δ) and expressing AURKA wild-type UTRs (WT) were used as controls. Figure 2 with 1 supplement see all Download asset Open asset Aurora Kinase A (AURKA) shows 3′UTR isoform-dependent protein expression. (A) Top: UTR-dependent protein expression reporters. Venus coding sequence (CDS) is flanked by AURKA 5′UTR and 3′UTR, WT or polyadenylation signal (PAS)-mutated. Bottom: representative snapshots of transfected U2OS cells. Scale bar 50 µm. (B) Mean and SEM of median Venus/mCherry mean fluorescence intensity (MFI) ratios from transfected U2OS cells from three biological replicates. n ≥ 129 cells per condition. Ordinary one-way ANOVA with Tukeys multiple-comparisons test. (C) 3′RACE of endogenous AURKA APA isoforms. (D) RT-qPCR of endogenous AURKA short/long ratio (SLR) in U2OS cells. Long isoform abundance plotted as fold change over total AURKA mRNA. 18S rRNA used as reference target. (E) Same as (B) but in MCF10A (left) and RPE1 (right) cells. n ≥ 55 cells per condition. Unpaired t-test. ns, not significant; *p<0.05; **p<0.01; ***p<0.001. Figure 2—source data 1 Raw gel/blot images. https://cdn.elifesciences.org/articles/87253/elife-87253-fig2-data1-v1.zip Download elife-87253-fig2-data1-v1.zip Figure 2—source data 2 Numerical data for graphs. https://cdn.elifesciences.org/articles/87253/elife-87253-fig2-data2-v1.xlsx Download elife-87253-fig2-data2-v1.xlsx We initially assessed the efficiency of the promoter bidirectionality. Correlation between Venus and mCherry expression was strongly maintained at the level of both fluorescence intensity (Figure 2—figure supplement 1A) and mRNA abundance (Figure 2—figure supplement 1B). Promoter strength was, however, not equal in both directions since fewer copies of mCherry mRNA were transcribed compared to Venus mRNA (Figure 2—figure supplement 1B), despite mCherry fluorescence intensity being generally higher than that of Venus (Figure 2A and B, see Δ). We further assessed that mRNA of both mCherry and Venus was stable over time (Figure 3B, see Δ), and fluorescence of both proteins was stable over time and over different cell cycle stages (Figure 2—figure supplement 1C and D). Considering the short maturation time and long half-life of both Venus and mCherry proteins (Shaner et al., 2004; Nagai et al., 2002), the assay allows to reliably measure effects of UTRs on reporter protein levels at any given time and regardless of cell cycle phases. As positive control of our assay, we recapitulated the higher protein expression from the short 3′UTR APA isoform of CDC6 mRNA, which has previously been observed (Akman et al., 2012; Figure 2—figure supplement 1E). Figure 3 Download asset Open asset Aurora Kinase A (AURKA) alternative polyadenylation (APA) isoforms are translated with different efficiency. (A, B) RT-qPCR of reporter mRNAs abundance (A) and decay rate (B) from transfected U2OS cells. mCherry mRNA used as reference target. Ordinary one-way ANOVA with Tukey’s multiple-comparisons test; ns, not significant. (C) Decay rate of endogenous AURKA mRNA as in (B). 18S rRNA used as reference target. Abundance of long isoform plotted as fold change over total AURKA mRNA. (D) Design of the nascent chain immunoprecipitation (NC IP) reporters and assay. (E) Mean and SEM of median FlagVenus/mCherry mean fluorescence intensity (MFI) ratios from transfected U2OS cells from two biological replicates. n ≥ 160 cells per condition. Unpaired t-test; **p<0.005. (F), (G) Immunoblots of NC IP fractions using Δ (F) or Flag-Δ (G) reporter. mCherry used as negative control. (H) RT-qPCR of eluted reporter mRNAs. Results representative of three biological replicates. Figure 3—source data 1 Numerical data for graphs. https://cdn.elifesciences.org/articles/87253/elife-87253-fig3-data1-v1.xlsx Download elife-87253-fig3-data1-v1.xlsx Figure 3—source data 2 Raw gel/blot images. https://cdn.elifesciences.org/articles/87253/elife-87253-fig3-data2-v1.zip Download elife-87253-fig3-data2-v1.zip Addition of AURKA UTRs to Venus CDS significantly increased protein expression (Figure 2A and B), likely due to the role of 5′UTR in facilitating translation (Hinnebusch et al., 2016). We found that the SHORT reporter generates significantly more protein compared to the LONG (Figure 2A and B). Moreover, similar protein expression levels from the WT and LONG reporters suggest that AURKA WT 3′UTR is processed with a preference for dPAS in U2OS cells. Accordingly, we could detect both endogenous AURKA APA isoforms in U2OS cells by 3′RACE (Figure 2C) and confirmed by RT-qPCR that AURKA long isoform is prevalent (~60% of total AURKA mRNA) (Figure 2D). The different SLR observed between U2OS cells, normal breast tissues, and TNBC cell lines and tissues (Figure 1B–D) indicates that AURKA 3′UTR isoform prevalence is dependent on cell type. In addition, the quantitative difference in reporter protein expression by the isoforms also varied among cell types, suggesting cell-specific regulation (Figure 2B and E, SHORT vs. LONG). In sum, these results provide evidence for the first time of a role for APA in controlling AURKA protein expression. AURKA APA isoforms are translated with different efficiency We next investigated the basis of the different protein expression between AURKA APA isoforms. Following transfection of U2OS cells with the constructs in Figure 2A, we first quantified the abundance of reporter mRNA isoforms (Figure 3A). We then assessed the isoforms decay rate by quantifying reporter mRNAs at multiple time points following arrest of transcription by actinomycin D (ActD) (Figure 3B). We observed that while Venus mRNA lacking UTRs was highly stable, reporter mRNA levels decreased at faster rate when carrying UTRs (Figure 3B), indicating that the assay reports on UTR-dependent effects on mRNA stability. Both the abundance and stability of the SHORT and LONG reporter isoforms were similar (Figure 3A and B). We additionally found that the two endogenous AURKA 3′UTR isoforms also have similar decay rates, albeit decaying at a higher rate compared to the reporter mRNAs (Figure 3C), suggesting that features present in AURKA CDS might influence mRNA stability (Narula et al., 2019). Because the reporter APA isoforms share similar abundance and stability, we wondered whether they undergo different translational regulation instead. To this aim, we adapted a biochemical translation efficiency (TE) assay from Williams et al., 2022, which required addition of a 3XFlag tag to the N-terminus of Venus (FlagVenus) in our reporter constructs, and called this nascent chain immunoprecipitation (NC IP) assay (Figure 3D). We first assessed that addition of the 3XFlag tag did not alter Venus expression (Figure 3E). In our NC IP assay, anti-Flag beads were used to immunoprecipitate nascent FlagVenus chains from ribosomes stalled by treatment with cycloheximide (CHX). Ribosome-mRNA complexes were eluted from the IP-immobilized nascent chains using puromycin, which causes release of nascent chains from ribosomes (Aviner, 2020); RNA was then purified from the elution fraction and quantified by RT-qPCR to provide a measure of the amount of reporter mRNA undergoing translation. All fractions were then blotted for FlagVenus and mCherry (negative control) proteins to monitor their presence at different steps of the experiment. As expected, elution with puromycin retained FlagVenus on the beads, whereas mCherry, as well as untagged Venus, are lost in the flow-through (Figure 3G, left, and F). No reporter mRNA could be detected in the elution fraction when untagged Venus was used (data not shown). Alternatively, purified 3XFlag peptide was used to elute the nascent chain-ribosome-mRNA complexes from the beads following IP (Figure 3G, right). RT-qPCR quantification of the FlagVenus nascent chain-cognate reporter mRNAs revealed about twice more copies of Flag-S mRNA compared to Flag-L mRNA, regardless of the elution method (Figure 3H). This indicates that Flag-S mRNA is translated with higher efficiency than Flag-L mRNA. These results show that APA controls AURKA protein expression mainly via differential translational regulation of the 3′UTR isoforms. Translation rate of AURKA APA isoforms follows different cell cycle periodicity Given the known cell cycle-dependent expression of AURKA (Cacioppo and Lindon, 2022), we tested whether differential translational efficiency of AURKA mRNA isoforms might contribute to this regulation. To avoid perturbation of translation provoked by classical cell cycle synchronization methods (Anda and Grallert, 2019), we used a live-cell fluorescence-based translation rate measurement assay in conjunction with a CDK2 activity sensor (Spencer et al., 2013) for in silico cell cycle synchronization. We developed our assay of ‘translation rate imaging by rate of protein stabilization’ (TRIPS) based on a previously introduced reporter system (Han et al., 2014; Tanenbaum et al., 2015). Our bidirectional promoter construct was modified to express superfolder GFP (sfGFP) fused to a mutated Echerichia coli dihydrofolate reductase (DHFR-Y100I) destabilizer domain (DHFR-sfGFP), which is continuously degraded unless the stabilizer molecule trimethoprim (TMP) is added. Addition of TMP leads to an increase of sfGFP signal over time and, given the sfGFP short maturation time (Pédelacq et al., 2006), the accumulation rate of sfGFP reflects DHFR-sfGFP protein synthesis rate (Figure 4A, left, Figure 4—figure supplement 1A). The ratio of the median of single-cell mCherry-normalized sfGFP signals at 2 hr to that at 0 hr of TMP treatment was therefore used as read-out for bulk translation rate. In accordance with our assay being designed to measure translation rate, sfGFP signal could not increase under TMP treatment in the presence of translation inhibitor CHX (Figure 4B). We also ensured that the increase in sfGFP signal was TMP-dependent (Figure 4—figure supplement 1B) and that TMP treatment affected neither mCherry expression (Figure 4—figure supplement 1C) nor DHFR-sfGFP mRNA abundance (Figure 4C). To probe translation rate at different cell cycle phases, we used the CDK2 activity sensor (Spencer et al., 2013) stably expressed in our U2OS cell line (U2OSCDK2) (Figure 4—figure supplement 1A) and called this assay ‘cell cycle-dependent TRIPS’ (C-TRIPS) (Figure 4A, right). Figure 4 with 1 supplement see all Download asset Open asset Translation rate of Aurora Kinase A (AURKA) alternative polyadenylation (APA) isoforms follows different cell cycle periodicity. (A) Design of the translation rate imaging by rate of protein stabilization (TRIPS) reporters and assay (left) and CDK2 activity sensor (right). (B) Mean and SEM of median sfGFP/mCherry mean fluorescence intensity (MFI) ratios from U2OS cells transfected with TRIPS-Δ and imaged at 0 hr and 2 hr of 50 µM trimethoprim (TMP) treatment, with or without 0.1 mg/ml cycloheximide (CHX), from three biological replicates. Baseline at 0 hr. n ≥ 55 cells per condition. (C) RT-qPCR of sfGFP mRNA from U2OS cells transfected with TRIPS-Δ, at 0 hr and 2 hr of 50 µM TMP treatment. mCherry mRNA used as reference target. RNA extracts at 0 hr of treatment used as reference sample. (D) TRIPS (left) and C-TRIPS (right) assays in transfected U2OSCDK2 cells. n ≥ 200 cells per condition. Left: mean and SEM from three biological replicates. Right: median and 95% CI of pooled data from left. Kruskal–Wallis with Dunnett’s multiple-comparisons test. (E) RT-qPCR of endogenous AURKA mRNA in U2OS cells. 18S rRNA used as reference target. Baseline at G1/S. (F) Endogenous AURKA long isoform as in (E) plotted as percentage of total AURKA mRNA. (D) Left, (E, F) Ordinary one-way ANOVA with Tukey’s multiple-comparisons test. ns, not significant; *p<0.05; **p<0.01; ***p<0.0005; ****p<0.0001. Figure 4—source data 1 Numerical data for graphs. https://cdn.elifesciences.org/articles/87253/elife-87253-fig4-data1-v1.xlsx Download elife-87253-fig4-data1-v1.xlsx To test the translation rate of the individual AURKA APA isoforms, we flanked DHFR-sfGFP CDS with AURKA PAS-mutated UTRs (TRIPS-L, TRIPS-S) (Figure 4A, left). We found that our TRIPS assay could recapitulate the difference in translation efficiency of the isoforms previously observed (Figures 3H and 4D, left). Importantly, expression of the CDK2 activity sensor did not affect cellular translation as the different translation rate of AURKA 3′UTR isoforms could be reproduced in U2OS cells lacking the sensor (Figure 4—figure supplement 1D). Following measurements of bulk translation rates (Figure 4D, left), we then binned single-cell translation rate values into three intervals of CDK2 activity (Figure 4D, right). Results of our C-TRIPS assay revealed that, while TRIPS-Δ is translated constantly during the cell cycle, translation rate of TRIPS-L is regulated in the cell cycle. This isoform showed lower translation rate in G1 and S and an enhanced rate at G2, consistent with the increase in both AURKA mRNA and protein levels that occurs in preparation for mitosis (Cacioppo and Lindon, 2022). By contrast, TRIPS-S was translated constantly through the cell cycle and at a maximal rate already in G1 (Figure 4D, right), indicating that this isoform is insensitive to cell cycle regulation of AURKA translation rate. Furthermore, we quantified abundance of endogenous AURKA APA isoforms at different stages of the cell cycle by performing RT-qPCR following synchronization in G1/S, G2, or M phases (Figure 4E). The expected changes in AURKA mRNA abundance following each treatment represent a positive control for the synchronization. However, abundance of the long isoform changed quite concomitantly with changes in total AURKA mRNA levels (Figure 4F). This suggests that the same ratio of 3′UTR isoforms is rather maintained throughout the cell cycle and that AURKA APA is not cell cycle regulated. These results not only provide strong, independent validation of our finding that elements present in 5′ and 3′ UTR of AURKA enable translational activation but additionally indicate that elements present on the long 3′UTR might account for its different pattern of translation during interphase as lack of these on the short 3′UTR allow escape from cell cycle phase-dependent translation. Translational periodicity of long 3′UTR isoform is regulated by hsa-let-7a miRNA Among known post-transcriptional regulators, miRNAs are widely recognized as molecular regulators of both mRNA stability and translation (Jonas and Izaurralde, 2015). We interrogated miRDB (https://mirdb.org/) (Chen and Wang, 2020) to search for miRNAs that could be involved in the differential regulation of the two AURKA mRNA isoforms and selected hsa-let-7a miRNA (Figure 5A) given its widely established tumor-suppressor role of in TNBC. We assessed the hsa-let-7a targeting of AURKA 3′UTR by co-transfecting our AURKA UTR-dependent protein expression reporters (Figure 2A) with hsa-let-7a or a negative control miRNA that does not have any target in the human genome. As positive control of the assay, we cloned Myeloid Zinc Finger1 (MZF1) 3′UTR downstream Venus CDS in our Δ reporter and could reproduce the previously reported targeting of MZF1 3′UTR by hsa-let-7a (Tvingsholm et al., 2018; Figure 5B). Protein expression from the LONG reporter mRNA was reduced by hsa-let-7a, whereas that from the SHORT mRNA and from a LONG mRNA that lacks the hsa-let-7a binding site (Δlet7a) was not (Figure 5B). Also, the loss of hsa-let-7a targeting was sufficient to increase protein expression from the LONG reporter mRNA (compare LONG + NC vs. Δlet7a + NC). To confirm that altered expression was due to the lack of hsa-let-7a targeting and not an effect of the mutation itself, we also observed an increase in protein expression when we co-transfected our LONG reporter and an inhibitor of hsa-let-7a (anti-let7a) (Figure 5C). Figure 5 Download asset Open asset Translational periodicity of long 3′UTR isoform is regulated by hsa-let-7a miRNA. (A) Complementarity of hsa-let-7a binding to Aurora Kinase A (AURKA) 3′UTR. (B) Mean and SEM of median Venus/mCherry mean fluorescence intensity (MFI) ratios from U2OS cells co-transfected with 250 nM hsa-let-7a or a negative control (NC) miRNA from three biological replicates. n ≥ 182 cells per condition. Unpaired t-test. (C) Same as (B) but co-transfecting 300 nM anti-let-7a or NC. n ≥ 94 cells per condition. Unpaired t-test. (D) RT-qPCR of reporter mRNAs abundance from U2OS cells transfected as (B), at 8 hr of 10 μg/ml ActD. mCherry mRNA used as reference target. Ordinary one-way ANOVA with Tukey’s multiple-comparisons test. (E) Translation rate imaging by rate of protein stabilization (TRIPS) (left) and C-TRIPS (right) assays in transfected U2OSCDK2 cells. n ≥ 162 cells per condition Left: mean and SEM from three biological replicates. Ordinary one-way ANOVA with Dunnett’s multiple-comparisons test vs. NC. Right: median and 95% CI of pooled data from left. Kruskal–Wallis with Dunnett’s multiple-comparisons test vs. NC of the respective phase. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Figure 5—source data 1 Numerical data for graphs. https://cdn.elifesciences.org/articles/87253/elife-87253-fig5-data1-v1.xlsx Download elife-87253-fig5-data1-v1.xlsx In order to assess the role of hsa-let-7a in controlling decay rate of the target mRNA, we next co-transfected our Venus reporters and either hsa-let-7a or negative control miRNA and quantified reporter mRNA abundance after 8 hr of ActD treatment. We found that stability of the LONG reporter mRNA was significantly reduced by hsa-let-7a, whereas that of the SHORT reporter mRNA was unaltered. Additionally, mutation of the hsa-let-7a binding site slightly increased reporter mRNA stability (compare LONG + NC vs. Δlet7a + NC) (Figure 5D). We then performed our C-TRIPS assay co-transfecting the TRIPS-L reporter and hsa-let-7a or control miRNA and found that hsa-let-7a reduced both bulk translation rate (Figure 5E, left) and translation rate at all interphase stages (Figure 5E, right) of the long 3′UTR. Furthermore, we asked whether loss of hsa-let-7a targeting is sufficient to cause loss of translational regulation of the long isoform during the cell cycle. For this, we performed the C-TRIPS assay using a TRIPS-L reporter with mutations in the hsa-let-7a binding site (TRIPS-Δlet7a) or, alternatively, co-transfecting the TRIPS-L reporter and the hsa-let-7a inhibitor anti-let7a. Interestingly, in both cases, loss of hsa-let-7a targeting only increased translation rate in G1 and S, but not G2 (Figure 5E, right), suggesting that the targeting in G2 is" @default.
W4382788274 created "2023-07-02" @default.
W4382788274 date "2023-06-29" @default.
W4382788274 modified "2023-09-23" @default.
W4382788274 title "Reviewer #2 (Public Review):: Differential translation of mRNA isoforms underlies oncogenic activation of cell cycle kinase Aurora A" @default.
W4382788274 doi "https://doi.org/10.7554/elife.87253.2.sa2" @default.
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