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RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3' UTRs

ABSTRACT: Polyadenylation of mRNA precursors is mediated by a large multisubunit protein complex. Here we show that RBBP6 (retinoblastoma-binding protein 6), identified initially as an Rb- and p53-binding protein, is a component of this complex and functions in 3' processing in vitro and in vivo. RBBP6 associates with other core factors, and this interaction is mediated by an unusual ubiquitin-like domain, DWNN (domain with no name), that is required for 3' processing activity. The DWNN is also expressed, via alternative RNA processing, as a small single-domain protein (isoform 3 [iso3]). Importantly, we show that iso3, known to be down-regulated in several cancers, competes with RBBP6 for binding to the core machinery, thereby inhibiting 3' processing. Genome-wide analyses following RBBP6 knockdown revealed decreased transcript levels, especially of mRNAs with AU-rich 3' untranslated regions (UTRs) such as c-Fos and c-Jun, and increased usage of distal poly(A) sites. Our results implicate RBBP6 and iso3 as novel regulators of 3' processing, especially of RNAs with AU-rich 3' UTRs. RBBP6 siRNA or control siRNA was transfected in MCF7 cells in duplicates and RNA was hybridized to GeneChip Exon 1.0 ST arrays (Affymetrix)

ORGANISM(S): Homo sapiens

SUBMITTER: Di Giammartino DC

PROVIDER: S-ECPF-GEOD-63036 | biostudies-other | 2014 Oct

REPOSITORIES: biostudies-other

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RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3' UTRs.

Project description:Polyadenylation of mRNA precursors is mediated by a large multisubunit protein complex. Here we show that RBBP6 (retinoblastoma-binding protein 6), identified initially as an Rb- and p53-binding protein, is a component of this complex and functions in 3' processing in vitro and in vivo. RBBP6 associates with other core factors, and this interaction is mediated by an unusual ubiquitin-like domain, DWNN ("domain with no name"), that is required for 3' processing activity. The DWNN is also expressed, via alternative RNA processing, as a small single-domain protein (isoform 3 [iso3]). Importantly, we show that iso3, known to be down-regulated in several cancers, competes with RBBP6 for binding to the core machinery, thereby inhibiting 3' processing. Genome-wide analyses following RBBP6 knockdown revealed decreased transcript levels, especially of mRNAs with AU-rich 3' untranslated regions (UTRs) such as c-Fos and c-Jun, and increased usage of distal poly(A) sites. Our results implicate RBBP6 and iso3 as novel regulators of 3' processing, especially of RNAs with AU-rich 3' UTRs.

| S-EPMC4201286 | biostudies-literature

RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3' UTRs

Project description:Polyadenylation of mRNA precursors is mediated by a large multisubunit protein complex. Here we show that RBBP6 (retinoblastoma-binding protein 6), identified initially as an Rb- and p53-binding protein, is a component of this complex and functions in 3' processing in vitro and in vivo. RBBP6 associates with other core factors, and this interaction is mediated by an unusual ubiquitin-like domain, DWNN (domain with no name), that is required for 3' processing activity. The DWNN is also expressed, via alternative RNA processing, as a small single-domain protein (isoform 3 [iso3]). Importantly, we show that iso3, known to be down-regulated in several cancers, competes with RBBP6 for binding to the core machinery, thereby inhibiting 3' processing. Genome-wide analyses following RBBP6 knockdown revealed decreased transcript levels, especially of mRNAs with AU-rich 3' untranslated regions (UTRs) such as c-Fos and c-Jun, and increased usage of distal poly(A) sites. Our results implicate RBBP6 and iso3 as novel regulators of 3' processing, especially of RNAs with AU-rich 3' UTRs. RBBP6 siRNA or control siRNA was transfected in MCF7 cells in duplicates and RNA was hybridized to GeneChip Exon 1.0 ST arrays (Affymetrix)

2014-11-06 | E-GEOD-63036 | biostudies-arrayexpress

RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3' UTRs

Project description:Polyadenylation of mRNA precursors is mediated by a large multisubunit protein complex. Here we show that RBBP6 (retinoblastoma-binding protein 6), identified initially as an Rb- and p53-binding protein, is a component of this complex and functions in 3' processing in vitro and in vivo. RBBP6 associates with other core factors, and this interaction is mediated by an unusual ubiquitin-like domain, DWNN (domain with no name), that is required for 3' processing activity. The DWNN is also expressed, via alternative RNA processing, as a small single-domain protein (isoform 3 [iso3]). Importantly, we show that iso3, known to be down-regulated in several cancers, competes with RBBP6 for binding to the core machinery, thereby inhibiting 3' processing. Genome-wide analyses following RBBP6 knockdown revealed decreased transcript levels, especially of mRNAs with AU-rich 3' untranslated regions (UTRs) such as c-Fos and c-Jun, and increased usage of distal poly(A) sites. Our results implicate RBBP6 and iso3 as novel regulators of 3' processing, especially of RNAs with AU-rich 3' UTRs.

2014-11-06 | GSE63036 | GEO

AU-rich elements regulate Drosophila gene expression.

Project description:In mammals, AU-rich elements (AREs) are critical regulators of mRNA turnover. They recruit ARE-binding proteins that inhibit or stimulate rapid mRNA degradation in response to stress or developmental cues. Using a bioinformatics approach, we have identified AREs in Drosophila melanogaster 3' untranslated regions and validated their cross-species conservation in distant Drosophila genomes. We have generated a Drosophila ARE database (D-ARED) and established that about 16% of D. melanogaster genes contain the mammalian ARE signature, an AUUUA pentamer in an A/U-rich context. Using candidate ARE genes, we show that Drosophila AREs stimulate reporter mRNA decay in cultured cells and in the physiological context of the immune response in D. melanogaster. In addition, we found that the conserved ARE-binding protein Tis11 regulates temporal gene expression through ARE-mediated decay (AMD) in D. melanogaster. Our work reveals that AREs are conserved and functional cis regulators of mRNA decay in Drosophila and highlights this organism as a novel model system to unravel in vivo the contribution of AMD to various processes.

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Oncogenic Ras and transforming growth factor-beta synergistically regulate AU-rich element-containing mRNAs during epithelial to mesenchymal transition.

Project description:Colon cancer progression is characterized by activating mutations in Ras and by the emergence of the tumor-promoting effects of transforming growth factor-beta (TGF-beta) signaling. Ras-inducible rat intestinal epithelial cells (RIE:iRas) undergo a well-described epithelial to mesenchymal transition and invasive phenotype in response to H-RasV12 expression and TGF-beta treatment, modeling tumor progression. We characterized global gene expression profiles accompanying Ras-induced and TGF-beta-induced epithelial to mesenchymal transition in RIE:iRas cells by microarray analysis and found that the regulation of gene expression by the combined activation of Ras and TGF-beta signaling was associated with enrichment of a class of mRNAs containing 3' AU-rich element (ARE) motifs known to regulate mRNA stability. Regulation of ARE-containing mRNA transcripts was validated at the mRNA level, including genes important for tumor progression. Ras and TGF-beta synergistically increased the expression and mRNA stability of vascular endothelial growth factor (VEGF), a key regulator of tumor angiogenesis, in both RIE:iRas cells and an independent cell culture model (young adult mouse colonocyte). Expression profiling of human colorectal cancers (CRC) further revealed that many of these genes, including VEGF and PAI-1, were differentially expressed in stage IV human colon adenocarcinomas compared with adenomas. Furthermore, genes differentially expressed in CRC are also significantly enriched with ARE-containing transcripts. These studies show that oncogenic Ras and TGF-beta synergistically regulate genes containing AREs in cultured rodent intestinal epithelial cells and suggest that posttranscriptional regulation of gene expression is an important mechanism involved in cellular transformation and CRC tumor progression.

| S-EPMC2572152 | biostudies-literature

ARED-Plus: an updated and expanded database of AU-rich element-containing mRNAs and pre-mRNAs.

Project description:Here we present an updated version of the AU-Rich Element Database (ARED-Plus) that is freely available at http://brp.kfshrc.edu.sa/ared. AREs are conserved sequence elements that were first discovered in the 3'UTR of mammalian transcripts. Over the past years, we compiled a series of ARE databases that revealed the extent and wide distribution of ARE-containing genes. For this update, we adopted an optimized search algorithm with improved specificity and sensitivity in ARE selection. The designation of the different ARE clusters was simplified by directly correlating the number of the ARE cluster to the number of overlapping AUUUA pentamers. Additionally, the new database was expanded to include genes with intronic AREs (pre-mRNAs) and their characteristics since recent observations reported their abundance and biological significance. Several enhancements were incorporated such as customized column view, additional search options and live search functionalities. The new version includes links to AREsite and AREScore, two related ARE assessment algorithms for further evaluation of the ARE characteristics. ARED-Plus now contains an updated repertoire of AREs in the human transcriptome that may be useful in several research fields.

| S-EPMC5753209 | biostudies-literature

Selective degradation of AU-rich mRNAs promoted by the p37 AUF1 protein isoform.

Project description:An AU-rich element (ARE) consisting of repeated canonical AUUUA motifs confers rapid degradation to many cytokine mRNAs when present in the 3' untranslated region. Destabilization of mRNAs with AREs (ARE-mRNAs) is consistent with the interaction of ARE-binding proteins such as tristetraprolin and the four AUF1 isoforms. However, the association of the AUF1-mRNA interaction with decreased ARE-mRNA stability is correlative and has not been directly tested. We therefore determined whether overexpression of AUF1 isoforms promotes ARE-mRNA destabilization and whether AUF1 isoforms are limiting components for ARE-mRNA decay. We show that the p37 AUF1 isoform and, to a lesser extent, the p40 isoform possess ARE-mRNA-destabilizing activity when overexpressed. Surprisingly, overexpressed p37 AUF1 also destabilized reporter mRNAs containing a noncanonical but AU-rich 3' untranslated region. Since overexpressed p37 AUF1 could interact in vivo with the AU-rich reporter mRNA, AUF1 may be involved in rapid turnover of mRNAs that lack canonical AREs. Moreover, overexpression of p37 AUF1 restored the ability of cells to rapidly degrade ARE-mRNAs when that ability was saturated and inhibited by overexpression of ARE-mRNAs. Finally, activation of ARE-mRNA decay often involves a translation-dependent step, which was eliminated by overexpression of p37 AUF1. These data indicate that the p37 AUF1 isoform and, to some extent, the p40 isoform are limiting factors that facilitate rapid decay of AU-rich mRNAs.

| S-EPMC193711 | biostudies-literature

Hsp70 is a novel posttranscriptional regulator of gene expression that binds and stabilizes selected mRNAs containing AU-rich elements.

Project description:The AU-rich elements (AREs) encoded within many mRNA 3' untranslated regions (3'UTRs) are targets for factors that control transcript longevity and translational efficiency. Hsp70, best known as a protein chaperone with well-defined peptide-refolding properties, is known to interact with ARE-like RNA substrates in vitro. Here, we show that cofactor-free preparations of Hsp70 form direct, high-affinity complexes with ARE substrates based on specific recognition of U-rich sequences by both the ATP- and peptide-binding domains. Suppressing Hsp70 in HeLa cells destabilized an ARE reporter mRNA, indicating a novel ARE-directed mRNA-stabilizing role for this protein. Hsp70 also bound and stabilized endogenous ARE-containing mRNAs encoding vascular endothelial growth factor (VEGF) and Cox-2, which involved a mechanism that was unaffected by an inhibitor of its protein chaperone function. Hsp70 recognition and stabilization of VEGF mRNA was mediated by an ARE-like sequence in the proximal 3'UTR. Finally, stabilization of VEGF mRNA coincided with the accumulation of Hsp70 protein in HL60 promyelocytic leukemia cells recovering from acute thermal stress. We propose that the binding and stabilization of selected ARE-containing mRNAs may contribute to the cytoprotective effects of Hsp70 following cellular stress but may also provide a novel mechanism linking constitutively elevated Hsp70 expression to the development of aggressive neoplastic phenotypes.

| S-EPMC3536313 | biostudies-literature

Massively parallel analysis of human 3' UTRs reveals that AU-rich element length and registration predict mRNA destabilization.

Project description:AU-rich elements (AREs) are 3' UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 3' UTR sequences that conform to these motifs affect their function. Here we use functional annotation of sequences from 3' UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 3' UTR sequence fragments from 4,653 transcripts on gene expression and mRNA stability in Jurkat and Beas2B cells. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Finally, to investigate the advantages of our general experimental design we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 3' UTRs.

| S-EPMC8728028 | biostudies-literature

NF90 selectively represses the translation of target mRNAs bearing an AU-rich signature motif.

Project description:The RNA-binding protein nuclear factor 90 (NF90) has been implicated in the stabilization, transport and translational control of several target mRNAs. However, a systematic analysis of NF90 target mRNAs has not been performed. Here, we use ribonucleoprotein immunoprecipitation analysis to identify a large subset of NF90-associated mRNAs. Comparison of the 3'-untranslated regions (UTRs) of these mRNAs led to the elucidation of a 25- to 30-nucleotide, RNA signature motif rich in adenines and uracils. Insertion of the AU-rich NF90 motif ('NF90m') in the 3'UTR of an EGFP heterologous reporter did not affect the steady-state level of the chimeric EGFP-NF90m mRNA or its cytosolic abundance. Instead, the translation of EGFP-NF90m mRNA was specifically repressed in an NF90-dependent manner, as determined by analysing nascent EGFP translation, the distribution of chimeric mRNAs on polysome gradients and the steady-state levels of expressed EGFP protein. The interaction of endogenous NF90 with target mRNAs was validated after testing both endogenous mRNAs and recombinant biotinylated transcripts containing NF90 motif hits. Further analysis showed that the stability of endogenous NF90 target mRNAs was not significantly influenced by NF90 abundance, while their translation increased when NF90 levels were reduced. In summary, we have identified an AU-rich RNA motif present in NF90 target mRNAs and have obtained evidence that NF90 represses the translation of this subset of mRNAs.

| S-EPMC2800222 | biostudies-literature

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