U-rich Element Research Articles

Delineating yeast cleavage and polyadenylation signals using deep learning.

3'-end cleavage and polyadenylation is an essential process for eukaryotic mRNA maturation. In yeast species, the polyadenylation signals that recruit the processing machinery are degenerate and remain poorly characterized compared with the well-defined regulatory elements in mammals. Here we address this issue by developing deep learning models to deconvolute degenerate cis-regulatory elements and quantify their positional importance in mediating yeast poly(A) site formation, cleavage heterogeneity, and strength. In S. cerevisiae, cleavage heterogeneity is promoted by the depletion of U-rich elements around poly(A) sites as well as multiple occurrences of upstream UA-rich elements. Sites with high cleavage heterogeneity show overall lower strength. The site strength and tandem site distances modulate alternative polyadenylation (APA) under the diauxic stress. Finally, we develop a deep learning model to reveal the distinct motif configuration of S. pombe poly(A) sites, which show more precise cleavage than S. cerevisiae Altogether, our deep learning models provide unprecedented insights into poly(A) site formation of yeast species, and our results highlight divergent poly(A) signals across distantly related species.

Overexpression of stress granule protein TZF1 enhances salt stress tolerance by targeting ACA11 mRNA for degradation in Arabidopsis.

Tandem CCCH zinc finger (TZF) proteins play diverse roles in plant growth and stress response. Although as many as 11 TZF proteins have been identified in Arabidopsis, little is known about the mechanism by which TZF proteins select and regulate the target mRNAs. Here, we report that Arabidopsis TZF1 is a bona-fide stress granule protein. Ectopic expression of TZF1 (TZF1 OE), but not an mRNA binding-defective mutant (TZF1H186Y OE), enhances salt stress tolerance in Arabidopsis. RNA-seq analyses of NaCl-treated plants revealed that the down-regulated genes in TZF1 OE plants are enriched for functions in salt and oxidative stress responses. Because many of these down-regulated mRNAs contain AU- and/or U-rich elements (AREs and/or UREs) in their 3'-UTRs, we hypothesized that TZF1-ARE/URE interaction might contribute to the observed gene expression changes. Results from RNA immunoprecipitation-quantitative PCR analysis, gel-shift, and mRNA half-life assays indicate that TZF1 binds and triggers degradation of the autoinhibited Ca2+-ATPase 11 (ACA11) mRNA, which encodes a tonoplast-localized calcium pump that extrudes calcium and dampens signal transduction pathways necessary for salt stress tolerance. Furthermore, this salt stress-tolerance phenotype was recapitulated in aca11 null mutants. Collectively, our findings demonstrate that TZF1 binds and initiates degradation of specific mRNAs to enhance salt stress tolerance.

The 3'‑untranslated region of XB130 regulates its mRNA stability and translational efficiency in non‑small cell lung cancer cells.

Silencing XB130 inhibits cell proliferation and epithelial-mesenchymal transition in non-small cell lung cancer (NSCLC), suggesting that downregulating XB130 expression may impede NSCLC progression. However, the molecular mechanism underlying the regulation of XB130 expression remains unclear. In the present study, the role of the 3'-untranslated region (3'-UTR) in the regulation of XB130 expression was investigated. Recombinant psiCHECK-2 vectors with wild-type, truncated, or mutant XB130 3'-UTR were constructed, and the effects of these insertions on reporter gene expression were examined using a dual-luciferase reporter assay and reverse transcription-quantitative PCR. Additionally, candidate proteins that regulated XB130 expression by binding to critical regions of the XB130 3'-UTR were screened for using an RNA pull-down assay, followed by mass spectrometry and western blotting. The results revealed that insertion of the entire XB130 3'-UTR (1,218 bp) enhanced reporter gene expression. Positive regulatory elements were primarily found in nucleotides 113-989 of the 3'-UTR, while negative regulatory elements were found in the 1-112 and 990-1,218 regions of the 3'-UTR. Deletion analyses identified nucleotides 113-230 and 503-660 of the 3'-UTR as two major fragments that likely promote XB130 expression by increasing mRNA stability and translation rate. Additionally, a U-rich element in the 970-1,053 region of the 3'-UTR was identified as a negative regulatory element that inhibited XB130 expression by suppressing translation. Furthermore, seven candidate proteins that potentially regulated XB130 expression by binding to the 113-230, 503-660, and 970-1,053 regions of the 3'-UTR were identified, shedding light on the regulatory mechanism of XB130 expression. Collectively, these results suggested that complex sequence integrations in the mRNA 3'-UTR variably affected XB130 expression in NSCLC cells.

A genetic screen in Arabidopsis reveals the identical roles for RBP45d and PRP39a in 5' cryptic splice site selection.

Cryptic splice sites in eukaryotic genome are generally dormant unless activated by mutation of authentic splice sites or related splicing factors. How cryptic splice sites are used remains unclear in plants. Here, we identified two cryptic splicing regulators, RBP45d and PRP39a that are homologs of yeast U1 auxiliary protein Nam8 and Prp39, respectively, via genetic screening for suppressors of the virescent sot5 mutant, which results from a point mutation at the 5' splice site (5' ss) of SOT5 intron 7. Loss-of-function mutations in RBP45d and PRP39a significantly increase the level of a cryptically spliced variant that encodes a mutated but functional sot5 protein, rescuing sot5 to the WT phenotype. We furtherly demonstrated that RBP45d and PRP39a interact with each other and also with the U1C, a core subunit of U1 snRNP. We found that RBP45d directly binds to the uridine (U)-rich RNA sequence downstream the 5' ss of SOT5 intron 7. However, other RBP45/47 members do not function redundantly with RBP45d, at least in regulation of cryptic splicing. Taken together, RBP45d promotes U1 snRNP to recognize the specific 5' ss via binding to intronic U-rich elements in plants.

Negative feedback regulation by HuR controls TRIM21 expression and function in response to UV radiation

The E3 ubiquitin ligase TRIM21 plays a crucial role as a negative regulator of innate immune responses. Recent evidence has also indicated the involvement of TRIM21 in the genotoxic stress response and suppressing tumorigenesis. Our previous work has demonstrated a new function of TRIM21 in inhibiting p53 protein synthesis by degrading the RNA-binding protein HuR in response to UV radiation. This suggested a pro-oncogenic role of TRIM21. In this study, we have shown that TRIM21 enhances the proliferation of MCF7 breast carcinoma cells and counteracts the decrease in cell proliferation and colony formation caused by UV-induced DNA damage. Further, this pro-oncogenic role of TRIM21 in response to DNA damage is mediated by its degradation of HuR. Conversely, we found that HuR binds to a U-rich element in the 3′UTR of TRIM21 mRNA and activates its translation, thereby constituting a negative feedback loop. We found that dihydrotanshinone-I (DHTS-I), a plant-derived product which prevents HuR binding to specific RNAs, prevented HuR-mediated upregulation of TRIM21, while increasing the HuR-mediated upregulation of p53. Together, these findings demonstrate a negative feedback regulation between TRIM21 and HuR, which may play an important role in regulating the level of p53 in the genotoxic stress response.

Genome-wide analysis identifies cis-acting elements regulating mRNA polyadenylation and translation during vertebrate oocyte maturation.

Most cells change patterns of gene expression through transcriptional regulation. In contrast, oocytes are transcriptionally silent and regulate mRNA poly(A) tail length to control protein production. However, the genome-wide relationship of poly(A) tail changes to mRNA translation during vertebrate oocyte maturation is not known. We used Tail-seq and polyribosome analysis to measure poly(A) tail and translational changes during oocyte maturation in Xenopus laevis. We identified large-scale poly(A) and translational changes during oocyte maturation, with poly(A) tail length changes preceding translational changes. Proteins important for completion of the meiotic divisions and early development exhibited increased polyadenylation and translation during oocyte maturation. A family of U-rich sequence elements was enriched near the polyadenylation signal of polyadenylated and translationally activated mRNAs. We propose that changes in mRNA polyadenylation are a conserved mechanism regulating protein expression during vertebrate oocyte maturation and that these changes are controlled by a spatial code of cis-acting sequence elements.

HuR counteracts miR-330 to promote STAT3 translation during inflammation-induced muscle wasting

Debilitating cancer-induced muscle wasting, a syndrome known as cachexia, is lethal. Here we report a posttranscriptional pathway involving the RNA-binding protein HuR as a key player in the onset of this syndrome. Under these conditions, HuR switches its function from a promoter of muscle fiber formation to become an inducer of muscle loss. HuR binds to the STAT3 (signal transducer and activator of transcription 3) mRNA, which encodes one of the main effectors of this condition, promoting its expression both in vitro and in vivo. While HuR does not affect the stability and the cellular movement of this transcript, HuR promotes the translation of the STAT3 mRNA by preventing miR-330 (microRNA 330)-mediated translation inhibition. To achieve this effect, HuR directly binds to a U-rich element in the STAT3 mRNA-3'untranslated region (UTR) located within the vicinity of the miR-330 seed element. Even though the binding sites of HuR and miR-330 do not overlap, the recruitment of either one of them to the STAT3-3'UTR negatively impacts the binding and the function of the other factor. Therefore, together, our data establish the competitive interplay between HuR and miR-330 as a mechanism via which muscle fibers modulate, in part, STAT3 expression to determine their fate in response to promoters of muscle wasting.

KSRP modulates melanoma growth and efficacy of vemurafenib

The majority of melanomas carry an oncogenic BRAF mutation (BRAFV600E), which results in constitutive kinase activity driving melanoma proliferation. While inhibitors of BRAFV600E (BRAFi) effectively lead to rapid tumor shrinkage, most patients treated with BRAFi develop acquired resistance. Identification of factors as regulators of melanoma growth and as potential sources of resistance is thus crucial for the design of improved therapies to treat advanced melanoma with more durable responses. Here, we show that KH-type splicing regulatory protein (KSRP) is critical for proliferation of melanoma cells without and with acquired resistance to vemurafenib. Silencing KSRP reduces cell proliferation and augments the growth suppressive effects of vemurafenib. We identify killin (KLLN), a p53-regulated DNA replication inhibitor, as a downstream effector of growth inhibition by KSRP silencing and demonstrate that KSRP promotes decay of KLLN mRNA through an RNA-protein interaction. Using heterologous mRNA reporters, we show that a U-rich element within the 3′ untranslated region of KLLN is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of melanoma cell growth in part through controlling KLLN mRNA stability.

Bi-phased regulation of the post-transcriptional inflammatory response by Tristetraprolin levels

ABSTRACTAU-rich elements (AREs) are cis-acting instability and translation inhibition elements that are present in the 3ʹUTR of most inducible inflammatory mRNAs such as TNF and Cxcl2. mRNAs that contain AREs are, by default, repressed and only transiently expressed in response to stimuli. They are targeted by the inducible RNA-binding protein Tristetraprolin (TTP) which blocks their translation and facilitates their decay, thereby contributing to the quick termination of their expression. The exogenous over-expression of TTP in HEK293 cells can unexpectedly lead to the upregulation and extended expression of a nanoLuciferase reporter that contains the ARE of TNF. Here we show that, a moderate downregulation of the highly expressed endogenous TTP after LPS induction by siRNA in macrophages can lead to a reduction in the release of TNF and Cxcl2. We propose that, in contrast to their canonical function, very high levels of induced TTP at the onset of the inflammatory response can enhance the expression of ARE-mRNAs at the post-transcriptional level, independently of phosphorylation status. As the inflammatory response progresses, TTP levels diminish but they continuously regain their ability to reduce the expression of ARE-mRNAs to reach a turning point of ‘optimal TTP level’ with a maximum ability to repress ARE-mRNA expression. Below this level, a further reduction in TTP levels now leads to the loss of canonical-TTP function resulting in increased ARE-mRNA expression. These novel findings should contribute to the understanding of feedback loops that control the kinetics of the inflammatory response.

Negative Regulation of BOK Expression by Recruitment of TRIM28 to Regulatory Elements in Its 3′ Untranslated Region

SummaryBCL-2-related ovarian killer (BOK) is a pro-apoptotic BAX-like member of the BCL-2 family with suggested tumor suppressor activity. The molecular mechanisms regulating BOK expression are poorly understood and fail to explain a frequent lack of concordance between protein and transcript levels. Here, we describe a potent post-transcriptional mechanism that negatively regulates BOK expression mediated by conserved (AU/U)-rich elements within its 3’ UTR. Using proteomics approaches we identified TRIM28 as a key component associating with U-rich elements in the human BOK 3’ UTR, resulting in a dramatic reduction of BOK expression. TRIM28 is overexpressed in several cancers, correlating with poor patient outcome, whereas the BOK locus is frequently deleted or its expression downregulated in human cancers. Data mining indicated that, for certain cancers, high TRIM28 and low BOK expression are significantly correlated in the stratum of patients with the worst survival, suggesting that this mechanism might be of potential therapeutic value.

RBM38 plays a tumor-suppressor role via stabilizing the p53-mdm2 loop function in hepatocellular carcinoma

BackgroundMisregulation of the p53-mdm2 loop function is a major mechanism to promote hepatocellular carcinoma (HCC). RBM38, a member of the RNA recognition motif (RRM) family of RNA binding proteins (RBPs), plays a fundamental role in the posttranscriptional control of gene expression and regulatory functions in human tumors. A novel RBM38-p53-mdm2 autoregulatory feedback loop has been demonstrated. However, its mechanistic role in HCC remains unclear.MethodsIn the present study, we investigated the role and molecular mechanism of misregulation in the p53-mdm2 loop function by RBM38 in HCC. First we investigated the correlation of RBM38 activity and p53-mdm2 loop function in liver cancer cells and HCC tissues by western blot and quantitative RT-PCR. We then conducted functional assays to investigate the molecular roles of RBM38 in inhibiting liver cancer cells aggressiveness in vitro and suppressing tumorigenicity in vivo.ResultsWe observed RBM38 protein expression was commonly silenced coupled with increased mdm2 and decreased wild type (wt) p53 in liver cancer cells and HCC tissues compared to the corresponding normal liver cells and adjacent liver tissues. RBM38 mRNA level was significantly lower in HCC than adjacent liver tissues, whereas mdm2 and wtp53 mRNA levels were similar between HCC and adjacent liver tissues. This implied that deactivation of RBM38 could disrupt the p53-mdm2 loop and promote HCC, even though p53 and mdm2 transcript amounts were stable. Then, we generated stable liver cancer cell lines with overexpressed RBM38 (RBM38-OE) and found that up-regulation of RBM38 could inhibit mdm2 and restore wtp53 expression. Luciferase assay shown that RBM38 destabilized the mdm2 transcript through binding to multiple AU-/U-rich elements in mdm2 3’-UTR. Furthermore, functional assays showed that ectopic expression of RBM38 could induce liver cancer cell apoptosis and senescence, inhibit proliferation and colony growth, and suppress migration and invasion in vitro. Lastly, RBM38 could suppress HCC tumorigenicity in vivo.ConclusionOur findings suggested that RBM38 may be a core contributor in stabilizing the p53-mdm2 loop function to prevent HCC, and a potential novel target to provide a therapeutic strategy for HCC by inhibiting mdm2 and rescuing p53 from inactivation.

The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated Stress Response and Cell Cycle Progression

TIA1 and TIAL1 encode a family of U-rich element mRNA-binding proteins ubiquitously expressed and conserved in metazoans. Using PAR-CLIP, we determined that both proteins bind target sites with identical specificity in 3' UTRs and introns proximal to 5'as well as 3' splice sites. Double knockout (DKO) of TIA1 and TIAL1 increased target mRNA abundance proportional to the number of binding sites and also caused accumulation of aberrantly spliced mRNAs, most of which are subject to nonsense-mediated decay. Loss of PRKRA by mis-splicing triggered the activation of the double-stranded RNA (dsRNA)-activated protein kinase EIF2AK2/PKR andstress granule formation. Ectopic expression of PRKRA cDNA or knockout of EIF2AK2 in DKO cells rescued this phenotype. Perturbation of maturation and/or stability of additional targets further compromised cell cycle progression. Our study reveals the essential contributions of the TIA1 protein family to the fidelity of mRNA maturation, translation, and RNA-stress-sensing pathways in human cells.

Dicer-2 promotes mRNA activation through cytoplasmic polyadenylation.

Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation. In vertebrates, this process requires two sequence elements in target 3′ UTRs: the U-rich cytoplasmic polyadenylation element and the AAUAAA hexanucleotide. In Drosophila melanogaster, cytoplasmic polyadenylation of Toll mRNA occurs independently of these canonical elements and requires a machinery that remains to be characterized. Here we identify Dicer-2 as a component of this machinery. Dicer-2, a factor previously involved in RNA interference (RNAi), interacts with the cytoplasmic poly(A) polymerase Wispy. Depletion of Dicer-2 from polyadenylation-competent embryo extracts and analysis of wispy mutants indicate that both factors are necessary for polyadenylation and translation of Toll mRNA. We further identify r2d2 mRNA, encoding a Dicer-2 partner in RNAi, as a Dicer-2 polyadenylation target. Our results uncover a novel function of Dicer-2 in activation of mRNA translation through cytoplasmic polyadenylation.

Abstract 4491: Modulation of p63 expression by phosphorylation of RBM38 at S195 via miR-203

Abstract p63, a p53 family protein, acts as a sequence-specific DNA-binding transcriptional factor to regulate the expression of target genes involving in various cellular processes, including growth suppression and epithelial development. RBM38, a RNA-binding protein, is a target of p53 family and is known to interact with its target mRNAs and regulate their expression via mRNA stability. Previously, we found that RBM38 negatively regulates p63 mRNA stability through binding to the AU/U-rich element in 3′ untranslated region (3′-UTR). We also found that phosphorylation of RBM38 at Ser195 alters the conformation and function of RBM38 protein. However, it is unclear whether phosphorylation of RBM38 or miR-203 alters the ability of RBM38 to regulate p63 expression. In this study, we found that in both MCF7 and HaCaT cells, wild-type RBM38 and S195A mutant, but not phosphor-mimic S195D mutant, inhibit p63 mRNA stability, suggesting that Ser195 phosphorylation abrogates the ability of RBM38 to repress the expression of p63. Importantly, we found that phosphorylation of RBM38 disrupts the binding of RBM38 to the U-rich element in p63 3′-UTR. Additionally, we found that miR-203 inhibitor increases, whereas miR203 mimic decreases p63 expression through binding to p63 3′-UTR, consistent with a previous report. Interestingly, we found that the ability of RBM38 to repress p63 expression is disrupted by miR-203 inhibitor but enhanced by miR-203 mimic. Furthermore, we found that wild-type RBM38 protein, but not phosphor-mimic S195D mutant, physically interacts with Ago2, indicating that RBM38 may inhibit p63 mRNA stability by modulating Ago2-miR-203 complex to bind to the U-rich element in p63 3′-UTR. Together, we uncovered a novel mechanism by which RBM38 modulates p63 expression via miR-203. Note: This abstract was not presented at the meeting. Citation Format: YANHONG ZHANG, WENSHENG YAN, JIN ZHANG, XINBIN CHEN. Modulation of p63 expression by phosphorylation of RBM38 at S195 via miR-203 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4491. doi:10.1158/1538-7445.AM2017-4491

TAp63γ and ΔNp63γ are regulated by RBM38 via mRNA stability and have an opposing function in growth suppression

The p63 gene is expressed as TAp63 from the P1 promoter and as ΔNp63 from the P2 promoter. Through alternative splicing, five TA and five ΔN isoforms (α-ε) are expressed. Isoforms α-β and δ share an identical 3’ untranslated region (3’UTR) whereas isoform γ has a unique 3’UTR. Recently, we found that RBM38 RNA-binding protein is a target of p63 and RBM38 in turn regulates p63α/β expression via mRNA stability. However, it is uncertain whether p63γ has a unique biological activity and whether p63γ is regulated by RBM38. Here, we found that the levels of ΔNp63γ transcript and protein are induced upon overexpression of RBM38 but decreased by RBM38 knockdown. Conversely, we found that the levels of ΔNp63β transcript and protein are decreased by ectopic expression of RBM38 but increased by RBM38 knockdown, consistent with our previous report. Interestingly, RBM38 increases the half-life of p63γ mRNA by binding to a GU-rich element in p63γ 3’UTR. In contrast, our previous studies showed that RBM38 decreases the half-life of p63α/β mRNAs by binding to AU-/U-rich elements in their 3’UTR. We also found that knockout of p63γ in ME180 and HaCaT cells, in which ΔNp63 isoforms are predominant, inhibits cell proliferation and migration, suggesting that ΔNp63γ has a pro-growth activity. In contrast, we found that knockout of TAp63γ in MIA PaCa-2 cells, in which TAp63 isoforms are predominant, promotes cell proliferation, migration, and inhibits cellular senescence. Taken together, we conclude that ΔNp63γ has an oncogenic potential whereas TAp63γ is a tumor suppressor.

Identification and dynamic changes of RNAs isolated from RALY-containing ribonucleoprotein complexes.

RALY is a member of the heterogeneous nuclear ribonucleoprotein family (hnRNP), a large family of RNA-binding proteins involved in many aspects of RNA metabolism. Although RALY interactome has been recently characterized, a comprehensive global analysis of RALY-associated RNAs is lacking and the biological function of RALY remains elusive. Here, we performed RIP-seq analysis to identify RALY interacting RNAs and assessed the role of RALY in gene expression. We demonstrate that RALY binds specific coding and non-coding RNAs and associates with translating mRNAs of mammalian cells. Among the identified transcripts, we focused on ANXA1 and H1FX mRNAs, encoding for Annexin A1 and for the linker variant of the histone H1X, respectively. Both proteins are differentially expressed by proliferating cells and are considered as markers for tumorigenesis. We demonstrate that cells lacking RALY expression exhibit changes in the levels of H1FX and ANXA1 mRNAs and proteins in an opposite manner. We also provide evidence for a direct binding of RALY to the U-rich elements present within the 3΄UTR of both transcripts. Thus, our results identify RALY as a poly-U binding protein and as a regulator of H1FX and ANXA1 in mammalian cells.

A comprehensive analysis of 3' end sequencing data sets reveals novel polyadenylation signals and the repressive role of heterogeneous ribonucleoprotein C on cleavage and polyadenylation.

Alternative polyadenylation (APA) is a general mechanism of transcript diversification in mammals, which has been recently linked to proliferative states and cancer. Different 3′ untranslated region (3′ UTR) isoforms interact with different RNA-binding proteins (RBPs), which modify the stability, translation, and subcellular localization of the corresponding transcripts. Although the heterogeneity of pre-mRNA 3′ end processing has been established with high-throughput approaches, the mechanisms that underlie systematic changes in 3′ UTR lengths remain to be characterized. Through a uniform analysis of a large number of 3′ end sequencing data sets, we have uncovered 18 signals, six of which are novel, whose positioning with respect to pre-mRNA cleavage sites indicates a role in pre-mRNA 3′ end processing in both mouse and human. With 3′ end sequencing we have demonstrated that the heterogeneous ribonucleoprotein C (HNRNPC), which binds the poly(U) motif whose frequency also peaks in the vicinity of polyadenylation (poly(A)) sites, has a genome-wide effect on poly(A) site usage. HNRNPC-regulated 3′ UTRs are enriched in ELAV-like RBP 1 (ELAVL1) binding sites and include those of the CD47 gene, which participate in the recently discovered mechanism of 3′ UTR–dependent protein localization (UDPL). Our study thus establishes an up-to-date, high-confidence catalog of 3′ end processing sites and poly(A) signals, and it uncovers an important role of HNRNPC in regulating 3′ end processing. It further suggests that U-rich elements mediate interactions with multiple RBPs that regulate different stages in a transcript's life cycle.

Alternative cleavage and polyadenylation in spermatogenesis connects chromatin regulation with post-transcriptional control.

BackgroundMost mammalian genes display alternative cleavage and polyadenylation (APA). Previous studies have indicated preferential expression of APA isoforms with short 3’ untranslated regions (3’UTRs) in testes.ResultsBy deep sequencing of the 3’ end region of poly(A) + transcripts, we report widespread shortening of 3’UTR through APA during the first wave of spermatogenesis in mouse, with 3’UTR size being the shortest in spermatids. Using genes without APA as a control, we show that shortening of 3’UTR eliminates destabilizing elements, such as U-rich elements and transposable elements, which appear highly potent during spermatogenesis. We additionally found widespread regulation of APA events in introns and exons that can affect the coding sequence of transcripts and global activation of antisense transcripts upstream of the transcription start site, suggesting modulation of splicing and initiation of transcription during spermatogenesis. Importantly, genes that display significant 3’UTR shortening tend to have functions critical for further sperm maturation, and testis-specific genes display greater 3’UTR shortening than ubiquitously expressed ones, indicating functional relevance of APA to spermatogenesis. Interestingly, genes with shortened 3’UTRs tend to have higher RNA polymerase II and H3K4me3 levels in spermatids as compared to spermatocytes, features previously known to be associated with open chromatin state.ConclusionsOur data suggest that open chromatin may create a favorable cis environment for 3’ end processing, leading to global shortening of 3’UTR during spermatogenesis. mRNAs with shortened 3’UTRs are relatively stable thanks to evasion of powerful mRNA degradation mechanisms acting on 3’UTR elements. Stable mRNAs generated in spermatids may be important for protein production at later stages of sperm maturation, when transcription is globally halted.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0229-6) contains supplementary material, which is available to authorized users.

AREsite2: an enhanced database for the comprehensive investigation of AU/GU/U-rich elements.

AREsite2 represents an update for AREsite, an on-line resource for the investigation of AU-rich elements (ARE) in human and mouse mRNA 3′UTR sequences. The new updated and enhanced version allows detailed investigation of AU, GU and U-rich elements (ARE, GRE, URE) in the transcriptome of Homo sapiens, Mus musculus, Danio rerio, Caenorhabditis elegans and Drosophila melanogaster. It contains information on genomic location, genic context, RNA secondary structure context and conservation of annotated motifs. Improvements include annotation of motifs not only in 3′UTRs but in the whole gene body including introns, additional genomes, and locally stable secondary structures from genome wide scans. Furthermore, we include data from CLIP-Seq experiments in order to highlight motifs with validated protein interaction. Additionally, we provide a REST interface for experienced users to interact with the database in a semi-automated manner. The database is publicly available at: http://rna.tbi.univie.ac.at/AREsite

Different motif requirements for the localization zipcode element of β-actin mRNA binding by HuD and ZBP1.

Interactions of RNA-binding proteins (RBPs) with their target transcripts are essential for regulating gene expression at the posttranscriptional level including mRNA export/localization, stability, and translation. ZBP1 and HuD are RBPs that play pivotal roles in mRNA transport and local translational control in neuronal processes. While HuD possesses three RNA recognition motifs (RRMs), ZBP1 contains two RRMs and four K homology (KH) domains that either increase target specificity or provide a multi-target binding capability. Here we used isolated cis-element sequences of the target mRNA to examine directly protein-RNA interactions in cell-free systems. We found that both ZBP1 and HuD bind the zipcode element in rat β-actin mRNA's 3′ UTR. Differences between HuD and ZBP1 were observed in their binding preference to the element. HuD showed a binding preference for U-rich sequence. In contrast, ZBP1 binding to the zipcode RNA depended more on the structural level, as it required the proper spatial organization of a stem-loop that is mainly determined by the U-rich element juxtaposed to the 3′ end of a 5′-ACACCC-3′ motif. On the basis of this work, we propose that ZBP1 and HuD bind to overlapping sites in the β-actin zipcode, but they recognize different features of this target sequence.