Emerging roles of alternative cleavage and polyadenylation (APA) in human disease.

In the messenger RNA (mRNA) maturation process, the 3'-end of pre-mRNA is cleaved and a poly(A) sequence is added, this is an important determinant of mRNA stability and its cellular functions. More than 60%-70% of human genes have three or more polyadenylation (APA) sites and can be cleaved at different sites, generating mRNA transcripts of varying lengths. This phenomenon is termed as alternative cleavage and polyadenylation (APA) and it plays role in key biological processes like gene regulation, cell proliferation, senescence, and also in various human diseases. Loss of regulatory microRNA binding sites and interactions with RNA-binding proteins leading to APA are largely investigated in human diseases. However, the functions of the core APA machinery and related factors during disease conditions remain largely unknown. In this review, we discuss the roles of polyadenylation machinery in relation to brain disease, cardiac failure, pulmonary fibrosis, cancer, infectious conditions, and other human diseases. Collectively, we believe this review will be a useful avenue for understanding the emerging role of APA in the pathobiology of various human diseases.

Nuclear mRNA maturation and mRNA export control: from trypanosomes to opisthokonts.

The passage of mRNAs through the nuclear pores into the cytoplasm is essential in all eukaryotes. For regulation, mRNA export is tightly connected to the full machinery of nuclear mRNA processing, starting at transcription. Export competence of pre-mRNAs gradually increases by both transient and permanent interactions with multiple RNA processing and export factors. mRNA export is best understood in opisthokonts, with limited knowledge in plants and protozoa. Here, I review and compare nuclear mRNA processing and export between opisthokonts and Trypanosoma brucei. The parasite has many unusual features in nuclear mRNA processing, such as polycistronic transcription and trans-splicing. It lacks several nuclear complexes and nuclear-pore-associated proteins that in opisthokonts play major roles in mRNA export. As a consequence, trypanosome mRNA export control is not tight and export can even start co-transcriptionally. Whether trypanosomes regulate mRNA export at all, or whether leakage of immature mRNA to the cytoplasm is kept to a low level by a fast kinetics of mRNA processing remains to be investigated. mRNA export had to be present in the last common ancestor of eukaryotes. Trypanosomes are evolutionary very distant from opisthokonts and a comparison helps understanding the evolution of mRNA export.

Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1.

The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling axis is a target of covalent drugs and bioactive native electrophiles. However, much of our understanding of Nrf2 regulation has been focused at the protein level. Here we report a post-transcriptional modality to directly regulate Nrf2-mRNA. Our initial studies focused on the effects of the key mRNA-binding protein (mRBP) HuR on global transcriptomic changes incurred upon oxidant or electrophile stimulation. These RNA-sequencing data and subsequent mechanistic analyses led us to discover a novel role of HuR in regulating Nrf2 activity, and in the process, we further identified the related mRBP AUF1 as an additional novel Nrf2 regulator. Both mRBPs regulate Nrf2 activity by direct interaction with the Nrf2 transcript. Our data showed that HuR enhances Nrf2-mRNA maturation and promotes its nuclear export, whereas AUF1 stabilizes Nrf2-mRNA. Both mRBPs target the 3'-UTR of Nrf2-mRNA. Using a Nrf2 activity-reporter zebrafish strain, we document that this post-transcriptional control of Nrf2 activity is conserved at the whole-vertebrate level.-Poganik, J. R., Long, M. J. C., Disare, M. T., Liu, X., Chang, S.-H., Hla, T., Aye, Y. Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1.

B52 promotes alternative splicing of Dscam in Chinese mitten crab, Eriocheir sinensis.

B52 is a member of the classical serine/arginine (SR)-rich proteins, which are phylogenetically conserved and play significant roles in mRNA maturation, including alternative splicing. In the present study, the docking site, selector sequences and locus control region of the Chinese mitten crab (Eriocheir sinensis) Down syndrome cell adhesion molecule (EsDscam) were identified. Alternative splicing of Dscam is essential to generate different isoforms. We also isolated and characterised the B52 gene from E. sinensis (EsB52). The 876 bp open reading frame of EsB52 encodes a 291 amino acid residue polypeptide, and EsB52 has two RNA recognition motifs (RRMs) at the N-terminus and an arginine/serine-rich domain at the C-terminus. Each RRM contains two degenerate short submotifs, RNP-1 and RNP2. Analysis of tissue distribution revealed that EsB52 mRNA expression was widespread in all tested tissues, and especially high in brain and hemocytes. In hemocytes, EsB52 was upregulated significantly after stimulation with pathogen-associated molecular patterns and bacteria. Furthermore, EsB52 RNAi decreased the number of Ig7 inclusion in mRNA rather than Ig2 or Ig3. Taken together, these findings suggest that EsB52 acts as an alternative splicing activator of EsDscam.

Interactome Analysis of NS1 Protein Encoded by Influenza A H7N9 Virus Reveals an Inhibitory Role of NS1 in Host mRNA Maturation.

Influenza A virus infections can result in severe respiratory diseases. The H7N9 subtype of avian influenza A virus has been transmitted to humans and caused severe disease and death. Nonstructural protein 1 (NS1) of influenza A virus is a virulence determinant during viral infection. To elucidate the functions of the NS1 encoded by influenza A H7N9 virus (H7N9 NS1), interaction partners of H7N9 NS1 in human cells were identified with immunoprecipitation followed by SDS-PAGE coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). We identified 36 cellular proteins as the interacting partners of the H7N9 NS1, and they are involved in RNA processing, mRNA splicing via spliceosome, and the mRNA surveillance pathway. Two of the interacting partners, cleavage and polyadenylation specificity factor subunit 2 (CPSF2) and CPSF7, were confirmed to interact with H7N9 NS1 using coimmunoprecipitation and immunoblotting based on the previous finding that the two proteins are involved in pre-mRNA polyadenylation machinery. Furthermore, we illustrate that overexpression of H7N9 NS1, as well as infection by the influenza A H7N9 virus, interfered with pre-mRNA polyadenylation in host cells. This study comprehensively profiled the interactome of H7N9 NS1 in host cells, and the results demonstrate a novel endotype for H7N9 NS1 in inhibiting host mRNA maturation.

Analysis of psoriasis-relevant gene expression and exon usage alterations after silencing of SR-rich splicing regulators.

In our recent cDNA microarray experiment, three SR-rich splicing factors-SFRS18, PPIG and LUC7L3-were shown to exert altered responsiveness upon T-lymphokine stimulation of psoriatic non-involved and healthy epidermis samples. We have also demonstrated that double silencing LUC7L3 and SFRS18 efficiently decreased production of the psoriasis-associated EDA+ fibronectin isoform. These findings prompted the further investigation of signalling pathways affected by LUC7L3 and SFRS18. To detect gene expression and splicing pattern alterations upon double silencing of LUC7L3 and SFRS18 in an HPV-immortalised keratinocyte cell culture, paired-end RNA sequencing was carried out. Marked changes in exon usage were revealed, in contrast to the modest alterations detected in gene expression, providing a closer delineation of the potential targets of the examined splicing factors. The most prominent gene expression change was detected for IFI6, an interferon-inducible gene highly expressed in psoriasis. Interacting partners of IFI6 and certain psoriasis-associated transcripts also exhibited significantly increased expression upon silencing. In addition to elevated abundance of the EDA+ fibronectin interactor ITGA5, we confirmed decreased EDA domain inclusion, which agrees well with our prior experimental data. Furthermore, differential exon usage was established for the transcription element CREB1, along with HERC6 and CUL1, which are implicated in ubiquitination. Although immortalised keratinocytes express low levels of TINCR, a long non-coding RNA involved in terminal differentiation of keratinocytes, splicing alterations were successfully demonstrated for this RNA as well. We believe that the targeted investigation of mRNA maturation disturbances may help us gain deeper insight into the molecular pathogenesis of psoriasis.

Sororin pre-mRNA splicing is required for proper sister chromatid cohesion in human cells.

Sister chromatid cohesion, which depends on cohesin, is essential for the faithful segregation of replicated chromosomes. Here, we report that splicing complex Prp19 is essential for cohesion in both G2 and mitosis, and consequently for the proper progression of the cell through mitosis. Inactivation of splicing factors SF3a120 and U2AF65 induces similar cohesion defects to Prp19 complex inactivation. Our data indicate that these splicing factors are all required for the accumulation of cohesion factor Sororin, by facilitating the proper splicing of its pre-mRNA. Finally, we show that ectopic expression of Sororin corrects defective cohesion caused by Prp19 complex inactivation. We propose that the Prp19 complex and the splicing machinery contribute to the establishment of cohesion by promoting Sororin accumulation during S phase, and are, therefore, essential to the maintenance of genome stability.

Translation of pre-spliced RNAs in the nuclear compartment generates peptides for the MHC class I pathway.

The scanning of maturing mRNAs by ribosomes plays a key role in the mRNA quality control process. When ribosomes first engage with the newly synthesized mRNA, and if peptides are produced, is unclear, however. Here we show that ribosomal scanning of prespliced mRNAs occurs in the nuclear compartment, and that this event produces peptide substrates for the MHC class I pathway. Inserting antigenic peptide sequences in introns that are spliced out before the mRNAs exit the nuclear compartment results in an equal amount of antigenic peptide products as when the peptides are encoded from the main open reading frame (ORF). Taken together with the detection of intron-encoded nascent peptides and RPS6/RPL7-carrying complexes in the perinucleolar compartment, these results show that peptides are produced by a translation event occurring before mRNA splicing. This suggests that ribosomes occupy and scan mRNAs early in the mRNA maturation process, and suggests a physiological role for nuclear mRNA translation, and also helps explain how the immune system tolerates peptides derived from tissue-specific mRNA splice variants.

Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression.

The expression of eukaryotic mRNAs is achieved though an intricate series of molecular processes that provide many steps for regulating the production of a final gene product. However, the relationships between individual steps in mRNA biosynthesis and the rates at which they occur are poorly understood. By applying RNA-seq to chromatin-associated and soluble nucleoplasmic fractions of RNA from Lipid A-stimulated macrophages, we examined the timing of exon ligation and transcript release from chromatin relative to the induction of transcription. We find that for a subset of genes in the Lipid A response, the ligation of certain exon pairs is delayed relative to the synthesis of the complete transcript. In contrast, 3' end cleavage and polyadenylation occur rapidly once transcription extends through the cleavage site. Our data indicate that these transcripts with delayed splicing are not released from the chromatin fraction until all the introns have been excised. These unusual kinetics result in a chromatin-associated pool of completely transcribed and 3'-processed transcripts that are not yet fully spliced. We also find that long introns containing repressed exons that will be excluded from the final mRNA are excised particularly slowly relative to other introns in a transcript. These results indicate that the kinetics of splicing and transcript release contribute to the timing of expression for multiple genes of the inflammatory response.

eIF4E orchestrates mRNA processing, RNA export and translation to modify specific protein production.

The eukaryotic translation initiation factor eIF4E acts as a multifunctional factor that simultaneously influences mRNA processing, export, and translation in many organisms. Its multifactorial effects are derived from its capacity to bind to the methyl-7-guanosine cap on the 5'end of mRNAs and thus can act as a cap chaperone for transcripts in the nucleus and cytoplasm. In this review, we describe the multifactorial roles of eIF4E in major mRNA-processing events including capping, splicing, cleavage and polyadenylation, nuclear export and translation. We discuss the evidence that eIF4E acts at two levels to generate widescale changes to processing, export and ultimately the protein produced. First, eIF4E alters the production of components of the mRNA processing machinery, supporting a widescale reprogramming of multiple mRNA processing events. In this way, eIF4E can modulate mRNA processing without physically interacting with target transcripts. Second, eIF4E also physically interacts with both capped mRNAs and components of the RNA processing or translation machineries. Further, specific mRNAs are sensitive to eIF4E only in particular mRNA processing events. This selectivity is governed by the presence of cis-acting elements within mRNAs known as USER codes that recruit relevant co-factors engaging the appropriate machinery. In all, we describe the molecular bases for eIF4E's multifactorial function and relevant regulatory pathways, discuss the basis for selectivity, present a compendium of ~80 eIF4E-interacting factors which play roles in these activities and provide an overview of the relevance of its functions to its oncogenic potential. Finally, we summarize early-stage clinical studies targeting eIF4E in cancer.

The Cap-Binding Complex CBC and the Eukaryotic Translation Factor eIF4E: Co-Conspirators in Cap-Dependent RNA Maturation and Translation.

The translation of RNA into protein is a dynamic process which is heavily regulated during normal cell physiology and can be dysregulated in human malignancies. Its dysregulation can impact selected groups of RNAs, modifying protein levels independently of transcription. Integral to their suitability for translation, RNAs undergo a series of maturation steps including the addition of the m7G cap on the 5' end of RNAs, splicing, as well as cleavage and polyadenylation (CPA). Importantly, each of these steps can be coopted to modify the transcript signal. Factors that bind the m7G cap escort these RNAs through different steps of maturation and thus govern the physical nature of the final transcript product presented to the translation machinery. Here, we describe these steps and how the major m7G cap-binding factors in mammalian cells, the cap binding complex (CBC) and the eukaryotic translation initiation factor eIF4E, are positioned to chaperone transcripts through RNA maturation, nuclear export, and translation in a transcript-specific manner. To conceptualize a framework for the flow and integration of this genetic information, we discuss RNA maturation models and how these integrate with translation. Finally, we discuss how these processes can be coopted by cancer cells and means to target these in malignancy.

Dynamic RNA holo-editosomes with subcomplex variants: Insights into the control of trypanosome editing.

RNA editing causes massive remodeling of the mitochondrial mRNA transcriptome in trypanosomes and related kinetoplastid protozoa. This type of editing involves the specific insertion or deletion of uridylates (U) directed by small noncoding guide RNAs (gRNAs). Because U-insertion exceeds U-deletion by a factor of 10, editing increases the nascent mRNA size by up to 55%. In Trypanosoma brucei, the editing apparatus uses ~40 proteins and >1,200 gRNAs to create the functional open reading frame in 12 mRNAs. Thousands of sites are specifically recognized in the pre-edited mRNAs and a myriad of partially edited transcript intermediates accumulates in mitochondria. The control of editing is poorly understood, but past work suggests that it occurs during substrate recognition, the initiation and progression of editing, and during the life-cycle in different hosts. The growing understanding of the editing proteins offers clues about editing control. Most editing proteins reside in the "RNA-free" RNA editing core complex (RECC) and in the accessory RNA editing substrate complex (RESC) that contains gRNA. Two accessory RNA helicases are known, including one in the RNA editing helicase 2 complex (REH2C). Both the RESC and the REH2C associate with mRNA, providing a rationale for the assembly of mRNA or its mRNPs, RESC, and the RECC enzyme. Identified variants of the canonical editing complexes further complicate the model of RNA editing. We examine specific examples of complex variants, differential effects of editing proteins on the mRNAs within and between T. brucei life stages, and possible control points in RNA holo-editosomes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Characterization of the B Cell Transcriptome Bound by RNA-Binding Proteins with iCLIP.

Posttranscriptional regulation of gene expression shapes the B cell transcriptome and controls messenger RNA (mRNA) translation into protein. Recent reports have highlighted the importance of RNA binding proteins (RBPs) for mRNA splicing, subcellular location, stability, and translation during B lymphocyte development, activation, and differentiation. Here we describe individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) in primary lymphocytes, a method that maps RNA-protein interactions in a genome-wide scale allowing mechanistic analysis of RBP function. We discuss the latest improvements in iCLIP technology and provide some examples of how integration of the RNA-protein interactome with other high-throughput mRNA sequencing methodologies uncovers the important role of RBP-mediated RNA regulation in key biological cell processes.