Targeting alternative splicing by RNAi: from the differential impact on splice variants to triggering artificial pre-mRNA splicing.

Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsis thaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.

Linking transcription, RNA polymerase II elongation and alternative splicing.

Gene expression is an intricately regulated process that is at the basis of cell differentiation, the maintenance of cell identity and the cellular responses to environmental changes. Alternative splicing, the process by which multiple functionally distinct transcripts are generated from a single gene, is one of the main mechanisms that contribute to expand the coding capacity of genomes and help explain the level of complexity achieved by higher organisms. Eukaryotic transcription is subject to multiple layers of regulation both intrinsic - such as promoter structure - and dynamic, allowing the cell to respond to internal and external signals. Similarly, alternative splicing choices are affected by all of these aspects, mainly through the regulation of transcription elongation, making it a regulatory knob on a par with the regulation of gene expression levels. This review aims to recapitulate some of the history and stepping-stones that led to the paradigms held today about transcription and splicing regulation, with major focus on transcription elongation and its effect on alternative splicing.

The Histone Methyltransferase G9a Controls Axon Growth by Targeting the RhoA Signaling Pathway.

The generation of axonal and dendritic domains is critical for brain circuitry assembly and physiology. Negative players, such as the RhoA-Rho coiled-coil-associated protein kinase (ROCK) signaling pathway, restrain axon development and polarization. Surprisingly, the genetic control of neuronal polarity has remained largely unexplored. Here, we report that, in primary cultured neurons, expression of the histone methyltransferase G9a and nuclear translocation of its major splicing isoform (G9a/E10+) peak at the time of axon formation. RNAi suppression of G9a/E10+ or pharmacological blockade of G9a constrains neuronal migration, axon initiation, and the establishment of neuronal polarity in situ and in vitro. Inhibition of G9a function upregulates RhoA-ROCK activity by increasing the expression of Lfc, a guanine nucleotide exchange factor (GEF) for RhoA. Together, these results identify G9a as a player in neuronal polarization.

GSK-3 is an RNA polymerase II phospho-CTD kinase.

We have previously found that UV-induced DNA damage causes hyperphosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), inhibition of transcriptional elongation and changes in alternative splicing (AS) due to kinetic coupling between transcription and splicing. In an unbiased search for protein kinases involved in the AS response to DNA damage, we have identified glycogen synthase kinase 3 (GSK-3) as an unforeseen participant. Unlike Cdk9 inhibition, GSK-3 inhibition only prevents CTD hyperphosphorylation triggered by UV but not basal phosphorylation. This effect is not due to differential degradation of the phospho-CTD isoforms and can be reproduced, at the AS level, by overexpression of a kinase-dead GSK-3 dominant negative mutant. GSK-3 inhibition abrogates both the reduction in RNAPII elongation and changes in AS elicited by UV. We show that GSK-3 phosphorylates the CTD in vitro, but preferentially when the substrate is previously phosphorylated, consistently with the requirement of a priming phosphorylation reported for GSK-3 efficacy. In line with a role for GSK-3 in the response to DNA damage, GSK-3 inhibition prevents UV-induced apoptosis. In summary, we uncover a novel role for a widely studied kinase in key steps of eukaryotic transcription and pre-mRNA processing.

Reciprocal regulation between alternative splicing and the DNA damage response.

Splicing, the process that catalyzes intron removal and flanking exon ligation, can occur in different ways (alternative splicing) in immature RNAs transcribed from a single gene. In order to adapt to a particular context, cells modulate not only the quantity but also the quality (alternative isoforms) of their transcriptome. Since 95% of the human coding genome is subjected to alternative splicing regulation, it is expected that many cellular pathways are modulated by alternative splicing, as is the case for the DNA damage response. Moreover, recent evidence demonstrates that upon a genotoxic insult, classical DNA damage response kinases such as ATM, ATR and DNA-PK orchestrate the gene expression response therefore modulating alternative splicing which, in a reciprocal way, shapes the response to a damaging agent.

Target RNAs Strike Back on MicroRNAs.

MicroRNAs are extensively studied regulatory non-coding small RNAs that silence animal genes throughout most biological processes, typically doing so by binding to partially complementary sequences within target RNAs. A plethora of studies has described detailed mechanisms for microRNA biogenesis and function, as well as their temporal and spatial regulation during development. By inducing translational repression and/or degradation of their target RNAs, microRNAs can contribute to achieve highly specific cell- or tissue-specific gene expression, while their aberrant expression can lead to disease. Yet an unresolved aspect of microRNA biology is how such small RNA molecules are themselves cleared from the cell, especially under circumstances where fast microRNA turnover or specific degradation of individual microRNAs is required. In recent years, it was unexpectedly found that binding of specific target RNAs to microRNAs with extensive complementarity can reverse the outcome, triggering degradation of the bound microRNAs. This emerging pathway, named TDMD for Target RNA-Directed MicroRNA Degradation, leads to microRNA 3'-end tailing by the addition of A/U non-templated nucleotides, trimming or shortening from the 3' end, and highly specific microRNA loss, providing a new layer of microRNA regulation. Originally described in flies and known to be triggered by viral RNAs, novel endogenous instances of TDMD have been uncovered and are now starting to be understood. Here, we review our current knowledge of this pathway and its potential role in the control and diversification of microRNA expression patterns.

When SUMO met splicing.

Spliceosomal proteins have been revealed as SUMO conjugation targets. Moreover, we have reported that many of these are in a SUMO-conjugated form when bound to a pre-mRNA substrate during a splicing reaction. We demonstrated that SUMOylation of Prp3 (PRPF3), a component of the U4/U6 di-snRNP, is required for U4/U6•U5 tri-snRNP formation and/or recruitment to active spliceosomes. Expanding upon our previous results, we have shown that the splicing factor SRSF1 stimulates SUMO conjugation to several spliceosomal proteins. Given the relevance of the splicing process, as well as the complex and dynamic nature of its governing machinery, the spliceosome, the molecular mechanisms that modulate its function represent an attractive topic of research. We posit that SUMO conjugation could represent a way of modulating spliceosome assembly and thus, splicing efficiency. How cycles of SUMOylation/de-SUMOylation of spliceosomal proteins become integrated throughout the highly choreographed spliceosomal cycle awaits further investigation.

[CRISPR/Cas9 and gene therapy]. / CRISPR/Cas9 y la terapia génica.

The development of techniques that allow the precise and efficient edition of the genome of living cells is one of the main goals of biomedical research. Over the last few decades, a number of genome editing tools have been developed, the most prominent being the CRISPR/Cas9 system, a bacterial defense mechanism that has been redesigned for its use in other cellular systems. The accessibility, both technical and economical, and the enormous potential of CRISPR/Cas9 have contributed to an almost unprecedented revolution in the biomedical sciences and represent an important step forward in the field of gene therapy that needs, however, to be taken cautiously.

CRISPR/Cas9 y la terapia génica / CRISPR/Cas9 and gene therapy

El desarrollo de técnicas que permitan editar o corregir con precisión y eficiencia el genoma de células vivas es uno de los objetivos principales de la investigación biomédica. En las últimas décadas se han investigado e implementado distintas herramientas de edición genómica entre las cuales se destaca el sistema CRISPR/Cas9, un mecanismo de defensa bacteriano que ha sido adaptado y rediseñado para su utilización en otros modelos celulares. La accesibilidad, técnica y económica, y el enorme potencial de CRISPR/Cas9 han dado lugar a una revolución casi sin precedentes en las ciencias biomédicas y representan un gran avance en el campo de la terapia génica que requiere, sin embargo, la cautela apropiada.

The development of techniques that allow the precise and efficient edition of the genome of living cells is one of the main goals of biomedical research. Over the last few decades, a number of genome editing tools have been developed, the most prominent being the CRISPR/Cas9 system, a bacterial defense mechanism that has been redesigned for its use in other cellular systems. The accessibility, both technical and economical, and the enormous potential of CRISPR/Cas9 have contributed to an almost unprecedented revolution in the biomedical sciences and represent an important step forward in the field of gene therapy that needs, however, to be taken cautiously.

Major Roles for Pyrimidine Dimers, Nucleotide Excision Repair, and ATR in the Alternative Splicing Response to UV Irradiation.

We have previously found that UV irradiation promotes RNA polymerase II (RNAPII) hyperphosphorylation and subsequent changes in alternative splicing (AS). We show now that UV-induced DNA damage is not only necessary but sufficient to trigger the AS response and that photolyase-mediated removal of the most abundant class of pyrimidine dimers (PDs) abrogates the global response to UV. We demonstrate that, in keratinocytes, RNAPII is the target, but not a sensor, of the signaling cascade initiated by PDs. The UV effect is enhanced by inhibition of gap-filling DNA synthesis, the last step in the nucleotide excision repair pathway (NER), and reduced by the absence of XPE, the main NER sensor of PDs. The mechanism involves activation of the protein kinase ATR that mediates the UV-induced RNAPII hyperphosphorylation. Our results define the sequence UV-PDs-NER-ATR-RNAPII-AS as a pathway linking DNA damage repair to the control of both RNAPII phosphorylation and AS regulation.

Alternative Splicing of G9a Regulates Neuronal Differentiation.

Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

The RNA Response to DNA Damage.

Multicellular organisms must ensure genome integrity to prevent accumulation of mutations, cell death, and cancer. The DNA damage response (DDR) is a complex network that senses, signals, and executes multiple programs including DNA repair, cell cycle arrest, senescence, and apoptosis. This entails regulation of a variety of cellular processes: DNA replication and transcription, RNA processing, mRNA translation and turnover, and post-translational modification, degradation, and relocalization of proteins. Accumulated evidence over the past decades has shown that RNAs and RNA metabolism are both regulators and regulated actors of the DDR. This review aims to present a comprehensive overview of the current knowledge on the many interactions between the DNA damage and RNA fields.

Chromatin, DNA structure and alternative splicing.

Coupling of transcription and alternative splicing via regulation of the transcriptional elongation rate is a well-studied phenomenon. Template features that act as roadblocks for the progression of RNA polymerase II comprise histone modifications and variants, DNA-interacting proteins and chromatin compaction. These may affect alternative splicing decisions by inducing pauses or decreasing elongation rate that change the time-window for splicing regulatory sequences to be recognized. Herein we discuss the evidence supporting the influence of template structural modifications on transcription and splicing, and provide insights about possible roles of non-B DNA conformations on the regulation of alternative splicing.

E2F1 induces p19INK4d, a protein involved in the DNA damage response, following UV irradiation.

Central to the maintenance of genomic integrity is the cellular DNA damage response. Depending on the type of genotoxic stress and through the activation of multiple signaling cascades, it can lead to cell cycle arrest, DNA repair, senescence, and apoptosis. p19INK4d, a member of the INK4 family of CDK inhibitors, plays a dual role in the DNA damage response, inhibiting cell proliferation and promoting DNA repair. Consistently, p19INK4d has been reported to become upregulated in response to UV irradiation and a great variety of genotoxic agents. Here, this induction is shown to result from a transcriptional stimulatory mechanism that can occur at every phase of the cell cycle except during mitosis. Moreover, evidence is presented that demonstrates that E2F1 is involved in the induction of p19INK4d following UV treatment, as it is prevented by E2F1 protein ablation and DNA-binding inhibition. Specific inhibition of this regulation using triplex-forming oligonucleotides that target the E2F response elements present in the p19INK4d promoter also block p19INK4d upregulation and sensitize cells to DNA damage. These results constitute the first description of a mechanism for the induction of p19INK4d in response to UV irradiation and demonstrate the physiological relevance of this regulation following DNA damage.

E2F1-mediated upregulation of p19INK4d determines its periodic expression during cell cycle and regulates cellular proliferation.

BACKGROUND: A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. CONCLUSIONS/SIGNIFICANCE: The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity.

Mdm2 plays a positive role as an effector of p53-dependent responses.

The p53 tumor suppressor is negatively regulated in cells by the Mdm2 protein. Mdm2 has therefore been the focus of intensive research aiming at using it as a target for cancer therapy with the ultimate goal of restoring p53 activity. Several studies have attempted to ablate Mdm2 expression or disrupt its interaction with p53 in cancer cells. While the p53-Mdm2 duo has concentrated a lot of attention, multiple new and diverse functions and targets of Mdm2 have been uncovered. Downregulation of Mdm2 using an siRNA approach has recently provided evidence for a new role of Mdm2 in the p53 response, by modulating the inhibition of the cyclin-dependent kinase 2 (cdk2) by p21. Here, this and other recent findings are discussed that support a new role for Mdm2 in the regulation of p53 response.

Mdm2 is required for inhibition of Cdk2 activity by p21, thereby contributing to p53-dependent cell cycle arrest.

p53 is extensively posttranslationally modified in response to various types of cellular stress. Such modifications have been implicated in the regulation of p53 protein levels as well as its DNA binding and transcriptional activities. Treatment of cells with doxorubicin causes phosphorylation and acetylation of p53, transcriptional upregulation of p21 and other target genes, and growth arrest. In contrast, downregulation of Mdm2 by a small interfering RNA (siRNA) approach led to increased levels of p53 lacking phosphorylation at serine 15 and acetylation at lysine 382. Levels of binding of p53 to the p21 promoter were comparable following treatment with doxorubicin or Mdm2 siRNA. Moreover, p53 was transcriptionally active and capable of inducing or repressing a variety of its target genes. Surprisingly, p53 upregulated by Mdm2 siRNA had no effect on cell cycle progression. Although comparable in level to that achieved by treatment with the p53 activators actinomycin D and nutlin-3, the increases in p53 and p21 after downregulation of Mdm2 were not sufficient to trigger cell cycle arrest. This version of p21 was capable of interacting with cyclin-dependent kinase 2 (Cdk2) but failed to inhibit its activity. Taken together, these results argue that Mdm2 is needed for full inhibition of Cdk2 activity by p21, thereby positively contributing to p53-dependent cell cycle arrest.

The p53 tumor suppressor participates in multiple cell cycle checkpoints.

The process of cell division is highly ordered and regulated. Checkpoints exist to delay progression into the next cell cycle phase only when the previous step is fully completed. The ultimate goal is to guarantee that the two daughter cells inherit a complete and faithful copy of the genome. Checkpoints can become activated due to DNA damage, exogenous stress signals, defects during the replication of DNA, or failure of chromosomes to attach to the mitotic spindle. Abrogation of cell cycle checkpoints can result in death for a unicellular organism or uncontrolled proliferation and tumorigenesis in metazoans (Nyberg et al., 2002). The tumor suppressor p53 plays a critical role in each of these cell cycle checkpoints and is reviewed here.

Inhibitory effect of AP-1 complex on 5-aminolevulinate synthase gene expression through sequestration of cAMP-response element protein (CRE)-binding protein (CBP) coactivator.

Activation protein-1 (AP-1) transcription factors are early response genes involved in a diverse set of transcriptional regulatory processes. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is often used to induce AP-1 activity. The purpose of this work was to explore the molecular mechanisms involved in the TPA regulation of ubiquitous 5-aminolevulinate synthase (ALAS) gene expression, the first and rate-controlling step of the heme biosynthesis. Previous analysis of the 5'-flanking sequence of ALAS revealed the existence of two cAMP-response elements (CRE) required for basal and cAMP-stimulated expression. The fragment -833 to +42 in the 5'-flanking region of rat ALAS gene was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector pALAS/CAT produced a significant CAT activity in transiently transfected HepG2 human hepatoma cells, which was repressed by TPA. Sequence and deletion analysis detected a TPA response element (TRE), located between -261 and -255 (TRE-ALAS), that was critical for TPA regulation. We demonstrated that c-Fos, c-Jun, and JunD are involved in TPA inhibitory effect due to their ability to bind TRE-ALAS, evidenced by supershift analysis and their capacity to repress promoter activity in transfection assays. Repression of ALAS promoter activity by TPA treatment or Fos/Jun overexpression was largely relieved when CRE protein-binding protein or p300 was ectopically expressed. When the TRE site was placed in a different context with respect to CRE sites, it appeared to act as a transcriptional enhancer. We propose that the decrease in ALAS basal activity observed in the presence of TPA may reflect a lower ability of this promoter to assemble the productive pre-initiation complex due to CRE protein-binding protein sequestration. We also suggest that the transcriptional properties of this AP-1 site would depend on a spatial-disposition-dependent manner with respect to the CRE sites and to the transcription initiation site.