Alternative polyadenylation (APA) modulates mRNA processing in the 3'-untranslated regions (3' UTR), affecting mRNA stability and translation efficiency. Research into genetically regulated APA has the potential to provide insights into cancer risk. In this study, we conducted large APA-wide association studies to investigate associations between APA levels and cancer risk. Genetic models were built to predict APA levels in multiple tissues using genotype and RNA sequencing data from 1,337 samples from the Genotype-Tissue Expression project. Associations of genetically predicted APA levels with cancer risk were assessed by applying the prediction models to data from large genome-wide association studies of six common cancers among European ancestry populations: breast, ovarian, prostate, colorectal, lung, and pancreatic cancers. A total of 58 risk genes (corresponding to 76 APA sites) were associated with at least one type of cancer, including 25 genes previously not linked to cancer susceptibility. Of the identified risk APAs, 97.4% and 26.3% were supported by 3'-UTR APA quantitative trait loci and colocalization analyses, respectively. Luciferase reporter assays for four selected putative regulatory 3'-UTR variants demonstrated that the risk alleles of 3'-UTR variants, rs324015 (STAT6), rs2280503 (DIP2B), rs1128450 (FBXO38), and rs145220637 (LDHA), significantly increased the posttranscriptional activities of their target genes compared with reference alleles. Furthermore, knockdown of the target genes confirmed their ability to promote proliferation and migration. Overall, this study provides insights into the role of APA in the genetic susceptibility to common cancers. Significance: Systematic evaluation of associations of alternative polyadenylation with cancer risk reveals 58 putative susceptibility genes, highlighting the contribution of genetically regulated alternative polyadenylation of 3'UTRs to genetic susceptibility to cancer.

• MeSH
• 3' Untranslated Regions * genetics   MeSH
• Genome-Wide Association Study * MeSH
• Genetic Predisposition to Disease * MeSH
• Polymorphism, Single Nucleotide MeSH
• Humans MeSH
• Quantitative Trait Loci MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Neoplasms * genetics   MeSH
• Polyadenylation * MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Mammalian oocyte development depends on the temporally controlled translation of maternal transcripts, particularly in the coordination of meiotic and early embryonic development when transcription has ceased. The translation of mRNA is regulated by various RNA-binding proteins. We show that the absence of cytoplasmic polyadenylation element-binding protein 3 (CPEB3) negatively affects female reproductive fitness. CPEB3-depleted oocytes undergo meiosis normally but experience early embryonic arrest due to a disrupted transcriptome, leading to aberrant protein expression and the subsequent failure of embryonic transcription initiation. We found that CPEB3 stabilizes a subset of mRNAs with a significantly longer 3'UTR that is enriched in its distal region with cytoplasmic polyadenylation elements. Overall, our results suggest that CPEB3 is an important maternal factor that regulates the stability and translation of a subclass of mRNAs that are essential for the initiation of embryonic transcription and thus for embryonic development.

• MeSH
• 3' Untranslated Regions genetics   MeSH
• Embryonic Development genetics   MeSH
• Meiosis genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mice MeSH
• Oocytes * metabolism   MeSH
• Polyadenylation MeSH
• RNA-Binding Proteins * metabolism   genetics   MeSH
• RNA Stability genetics   MeSH
• Gene Expression Regulation, Developmental MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Osteoblasts orchestrate bone formation through the secretion of type I collagen and other constituents of the matrix on which hydroxyapatite crystals mineralize. Here, we show that TENT5A, whose mutations were found in congenital bone disease osteogenesis imperfecta patients, is a cytoplasmic poly(A) polymerase playing a crucial role in regulating bone mineralization. Direct RNA sequencing revealed that TENT5A is induced during osteoblast differentiation and polyadenylates mRNAs encoding Col1α1, Col1α2, and other secreted proteins involved in osteogenesis, increasing their expression. We postulate that TENT5A, possibly together with its paralog TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization. Importantly, the Tent5a knockout (KO) mouse line displays bone fragility and skeletal hypomineralization phenotype resulting from quantitative and qualitative collagen defects. Thus, we report a biologically relevant posttranscriptional regulator of collagen production and, more generally, bone formation.

• MeSH
• Cell Differentiation MeSH
• Calcification, Physiologic genetics   MeSH
• Collagen Type I, alpha 1 Chain genetics   metabolism   MeSH
• Collagen Type I genetics   metabolism   MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Disease Models, Animal MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Nerve Growth Factors genetics   metabolism   MeSH
• Nucleotidyltransferases genetics   metabolism   MeSH
• Eye Proteins genetics   metabolism   MeSH
• Osteoblasts metabolism   pathology   MeSH
• Osteogenesis Imperfecta genetics   metabolism   pathology   MeSH
• Osteogenesis genetics   MeSH
• Osteonectin genetics   metabolism   MeSH
• Polynucleotide Adenylyltransferase genetics   metabolism   MeSH
• Polyadenylation MeSH
• Protein Isoforms deficiency   genetics   MeSH
• Sequence Analysis, RNA MeSH
• Serpins genetics   metabolism   MeSH
• Signal Transduction MeSH
• Gene Expression Regulation, Developmental MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Tens of thousands of rapidly evolving long non-coding RNA (lncRNA) genes have been identified, but functions were assigned to relatively few of them. The lncRNA contribution to the mouse oocyte physiology remains unknown. We report the evolutionary history and functional analysis of Sirena1, the most expressed lncRNA and the 10th most abundant poly(A) transcript in mouse oocytes. Sirena1 appeared in the common ancestor of mouse and rat and became engaged in two different post-transcriptional regulations. First, antisense oriented Elob pseudogene insertion into Sirena1 exon 1 is a source of small RNAs targeting Elob mRNA via RNA interference. Second, Sirena1 evolved functional cytoplasmic polyadenylation elements, an unexpected feature borrowed from translation control of specific maternal mRNAs. Sirena1 knock-out does not affect fertility, but causes minor dysregulation of the maternal transcriptome. This includes increased levels of Elob and mitochondrial mRNAs. Mitochondria in Sirena1-/- oocytes disperse from the perinuclear compartment, but do not change in number or ultrastructure. Taken together, Sirena1 contributes to RNA interference and mitochondrial aggregation in mouse oocytes. Sirena1 exemplifies how lncRNAs stochastically engage or even repurpose molecular mechanisms during evolution. Simultaneously, Sirena1 expression levels and unique functional features contrast with the lack of functional importance assessed under laboratory conditions.

• MeSH
• Gene Knockout Techniques MeSH
• Rats MeSH
• RNA, Messenger genetics   MeSH
• Mitochondria genetics   ultrastructure   MeSH
• Mice MeSH
• Oocytes growth & development   metabolism   ultrastructure   MeSH
• Polyadenylation genetics   MeSH
• RNA, Long Noncoding genetics   MeSH
• RNA, Mitochondrial genetics   MeSH
• Transcriptome genetics   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The RNA exosome processes a wide variety of RNA and mediates RNA maturation, quality control and decay. In marked contrast to its high processivity in vivo, the purified exosome exhibits only weak activity on RNA substrates in vitro. Its activity is regulated by several auxiliary proteins, and protein complexes. In budding yeast, the activity of exosome is enhanced by the polyadenylation complex referred to as TRAMP. TRAMP oligoadenylates precursors and aberrant forms of RNAs to promote their trimming or complete degradation by exosomes. This chapter provides protocols for the purification of TRAMP and exosome complexes from yeast and the in vitro evaluation of exosome activation by the TRAMP complex. The protocols can be used for different purposes, such as the assessment of the role of individual subunits, protein domains or particular mutations in TRAMP-exosome RNA processing in vitro.

• MeSH
• Cell Nucleus metabolism   MeSH
• Exosome Multienzyme Ribonuclease Complex metabolism   MeSH
• Exosomes metabolism   MeSH
• Polyadenylation physiology   MeSH
• RNA metabolism   MeSH
• Saccharomyces cerevisiae Proteins metabolism   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Serine Endopeptidases metabolism   MeSH
• RNA Stability physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability.

• MeSH
• Active Transport, Cell Nucleus MeSH
• DNA, Neoplasm genetics   metabolism   MeSH
• HeLa Cells MeSH
• Nucleic Acid Heteroduplexes genetics   metabolism   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• RNA, Messenger biosynthesis   genetics   MeSH
• Neoplasms genetics   metabolism   MeSH
• Genomic Instability * MeSH
• Polyadenylation MeSH
• DNA Damage * MeSH
• RNA Precursors biosynthesis   genetics   MeSH
• Cell Cycle Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• DNA Replication * MeSH
• RNA, Neoplasm biosynthesis   genetics   MeSH
• RNA Cleavage * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements (VLEs, also known as linear plasmids) that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5' mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin.

• MeSH
• Cytoplasm MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• Fungal Proteins genetics   MeSH
• Transcription, Genetic * MeSH
• Kluyveromyces genetics   MeSH
• Nucleic Acid Conformation MeSH
• Evolution, Molecular * MeSH
• Polyadenylation MeSH
• Promoter Regions, Genetic * MeSH
• Gene Expression Regulation, Fungal MeSH
• Response Elements * MeSH
• Base Sequence MeSH
• Sequence Homology MeSH
• RNA Stability MeSH
• Viruses genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Alternative polyadenylation is an important and pervasive mechanism that generates heterogeneous 3'-termini of mRNA and is considered an important regulator of gene expression. We performed bioinformatics analyses of ESTs and the 3'-UTRs of the main transcript splice variants of the translational initiation factor eIF4E1 and its family members, eIF4E2 and eIF4E3. This systematic analysis led to the prediction of new polyadenylation signals. All identified polyadenylation sites were subsequently verified by 3'RACE of transcripts isolated from human lymphoblastic cell lines. This led to the observation that multiple simultaneous polyadenylation site utilization occurs in single cell population. Importantly, we described the use of new polyadenylation site in the eIF4E1 mRNA, which lacked any known polyadenylation signal. The proportion of eIF4E1 transcripts derived from the first two polyadenylation sites in eIF4E1 mRNA achieved 15% in a wide range of cell lines. This result demonstrates the ubiquitous presence of ARE-lacking transcripts, which escape HuR/Auf1-mediated control, the main mechanism of eIF4E1 gene expression regulation. We found many EST clones documenting the significant production of transcript variants 2-4 of eIF4E2 gene that encode proteins with C-termini that were distinct from the mainly studied prototypical isoform A. Similarly, eIF4E3 mRNAs are produced as two main variants with the same very long 3'-UTR with potential for heavy post-transcriptional regulation. We identified sparsely documented transcript variant 1 of eIF4E3 gene in human placenta. eIF4E3 truncated transcript variants were found mainly in brain. We propose to elucidate the minor splice variants of eIF4E2 and eIF4E3 in great detail because they might produce proteins with modified features that fulfill different cellular roles from their major counterparts.

• MeSH
• 3' Untranslated Regions MeSH
• Cell Line MeSH
• Eukaryotic Initiation Factor-4E genetics   metabolism   MeSH
• Expressed Sequence Tags MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Brain metabolism   MeSH
• Placenta metabolism   MeSH
• Polyadenylation genetics   MeSH
• RNA Cap-Binding Proteins genetics   MeSH
• Gene Expression Regulation MeSH
• RNA Splicing genetics   MeSH
• Pregnancy MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Although the involvement of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in the regulation of cytostatic factor (CSF) activity; as well as in microtubules organization during meiotic maturation of oocytes; has already been described in detail; rather less attention has been paid to the role of ERK1/2 in the regulation of mRNA translation. However; important data on the role of ERK1/2 in translation during oocyte meiosis have been documented. This review focuses on recent findings regarding the regulation of translation and the role of ERK1/2 in this process in the meiotic cycle of mammalian oocytes. The specific role of ERK1/2 in the regulation of mammalian target of rapamycin (mTOR); eukaryotic translation initiation factor 4E (eIF4E) and cytoplasmic polyadenylation element binding protein 1 (CPEB1) activity is addressed along with additional focus on the other key players involved in protein translation.

• MeSH
• Cytoplasm genetics   metabolism   MeSH
• Eukaryotic Initiation Factor-4E metabolism   MeSH
• mRNA Cleavage and Polyadenylation Factors metabolism   MeSH
• Phosphatidylinositol 3-Kinases metabolism   MeSH
• Humans MeSH
• Meiosis * MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mitogen-Activated Protein Kinase 1 metabolism   MeSH
• Mitogen-Activated Protein Kinase 3 metabolism   MeSH
• Mitogen-Activated Protein Kinases metabolism   MeSH
• Oocytes metabolism   MeSH
• Polyadenylation MeSH
• Protein Biosynthesis * MeSH
• Signal Transduction MeSH
• TOR Serine-Threonine Kinases metabolism   MeSH
• Protein Binding MeSH
• Gene Expression Regulation, Developmental MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

One recently discussed general mechanism affecting gene expression is 3'-untranslated region (3'UTR) length. Events such as shortening, translocation or loss of 3'UTRs are observed within oncogenes and are proposed to associate with increased expression. Thus, increased efforts are being made to understand constitutive and differential transcript 3'end formation. Investigation of AGR2 mRNA revealed a direct impact of its 3'UTR length on AGR2 expression. In silico analyses identified several regulatory sequences within the distal part of AGR2 mRNA that may regulate 3'UTR length and associated protein levels. Short 3'UTRs were observed in a panel of AGR2-positive cancer cell lines and in human breast cancer specimens, in which more extensive 3'UTR shortening correlated with increased AGR2 protein levels. AGR2 is an important member of PI3K/AKT signalling pathway, which along with the proposed involvement of mTOR in the regulation of alternative polyadenylation, prompted us to study the role of mTOR in relation to AGR2 mRNA 3'UTR shortening. A direct impact of mTOR signalling on AGR2 3'UTR shortening associated with increased protein synthesis was found, which led to the identification of a novel molecular mechanism involved in upregulation of AGR2 levels in mTOR-activated cells via modulating the 3'UTR length of AGR2 mRNA.

• MeSH
• 3' Untranslated Regions genetics   MeSH
• A549 Cells MeSH
• HCT116 Cells MeSH
• HEK293 Cells MeSH
• Cloning, Molecular MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• MCF-7 Cells MeSH
• Multiprotein Complexes genetics   MeSH
• Cell Line, Tumor MeSH
• Breast Neoplasms genetics   MeSH
• Polyadenylation genetics   MeSH
• Proteins genetics   metabolism   MeSH
• Protein Biosynthesis genetics   MeSH
• Gene Expression Regulation genetics   MeSH
• Regulatory Sequences, Ribonucleic Acid genetics   MeSH
• Signal Transduction genetics   MeSH
• TOR Serine-Threonine Kinases genetics   metabolism   MeSH
• Up-Regulation genetics   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH