Ezequiel Petrillo

Alternative splicing: a pivotal step between eukaryotic transcription and translation

Alternative splicing was discovered simultaneously with splicing over three decades ago. Since then, an enormous body of evidence has demonstrated the prevalence of alternative splicing in multicellular eukaryotes, its key roles in determining tissue- and species-specific differentiation patterns, the multiple post- and co-transcriptional regulatory mechanisms that control it, and its causal role in hereditary disease and cancer. The emerging evidence places alternative splicing in a central position in the flow of eukaryotic genetic information, between transcription and translation, in that it can respond not only to various signalling pathways that target the splicing machinery but also to transcription factors and chromatin structure.

Control of alternative splicing through siRNA-mediated transcriptional gene silencing.

When targeting promoter regions, small interfering RNAs (siRNAs) trigger a previously proposed pathway known as transcriptional gene silencing by promoting heterochromatin formation. Here we show that siRNAs targeting intronic or exonic sequences close to an alternative exon regulate the splicing of that exon. The effect occurred in hepatoma and HeLa cells with siRNA antisense strands designed to enter the silencing pathway, suggesting hybridization with nascent pre-mRNA. Unexpectedly, in HeLa cells the sense strands were also effective, suggesting that an endogenous antisense transcript, detectable in HeLa but not in hepatoma cells, acts as a target. The effect depends on Argonaute-1 and is counterbalanced by factors favoring chromatin opening or transcriptional elongation. The increase in heterochromatin marks (dimethylation at Lys9 and trimethylation at Lys27 of histone H3) at the target site, the need for the heterochromatin-associated protein HP1α and the reduction in RNA polymerase II processivity suggest a mechanism involving the kinetic coupling of transcription and alternative splicing.

A methyl transferase links the circadian clock to the regulation of alternative splicing

Circadian rhythms allow organisms to time biological processes to the most appropriate phases of the day–night cycle. Post-transcriptional regulation is emerging as an important component of circadian networks, but the molecular mechanisms linking the circadian clock to the control of RNA processing are largely unknown. Here we show that PROTEIN ARGININE METHYL TRANSFERASE 5 (PRMT5), which transfers methyl groups to arginine residues present in histones and Sm spliceosomal proteins, links the circadian clock to the control of alternative splicing in plants. Mutations in PRMT5 impair several circadian rhythms in Arabidopsis thaliana and this phenotype is caused, at least in part, by a strong alteration in alternative splicing of the core-clock gene PSEUDO RESPONSE REGULATOR 9 (PRR9). Furthermore, genome-wide studies show that PRMT5 contributes to the regulation of many pre-messenger-RNA splicing events, probably by modulating 5′-splice-site recognition. PRMT5 expression shows daily and circadian oscillations, and this contributes to the mediation of the circadian regulation of expression and alternative splicing of a subset of genes. Circadian rhythms in locomotor activity are also disrupted in dart5-1, a mutant affected in the Drosophila melanogaster PRMT5 homologue, and this is associated with alterations in splicing of the core-clock gene period and several clock-associated genes. Our results demonstrate a key role for PRMT5 in the regulation of alternative splicing and indicate that the interplay between the circadian clock and the regulation of alternative splicing by PRMT5 constitutes a common mechanism that helps organisms to synchronize physiological processes with daily changes in environmental conditions.

A chloroplast retrograde signal regulates nuclear alternative splicing

No Light Control Light is the main source of energy for plants and is also used as a signal for growth and development: Indeed, it can modulate up to a fifth of the entire transcriptome in both Arabidopsis thaliana and rice. Petrillo et al. (p. 427, published online 10 April) show that light can affect gene expression through alternative splicing of the serine-arginine rich protein At-RS31, required for proper plant growth. But photoreceptors are not involved; rather, a mobile retrograde signal from the chloroplast controls the alternative splicing of At-RS31. In plants, light-dependent regulation of nuclear alternative splicing involves a signal generated within the chloroplast. Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action, we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions.

Transcriptional elongation and alternative splicing.

Alternative splicing has emerged as a key contributor to proteome diversity, highlighting the importance of understanding its regulation. In recent years it became apparent that splicing is predominantly cotranscriptional, allowing for crosstalk between these two nuclear processes. We discuss some of the links between transcription and splicing, with special emphasis on the role played by transcription elongation in the regulation of alternative splicing events and in particular the kinetic model of alternative splicing regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation

Alternative splicing contributes to cell type‐specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5‐azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation‐induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts.

The serine/arginine-rich protein SF2/ASF regulates protein sumoylation

Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF.

Alternative splicing at the right time.

Alternative splicing (AS) allows the production of multiple mRNA variants from a single gene, which contributes to increase the complexity of the proteome. There is evidence that AS is regulated not only by auxiliary splicing factors, but also by components of the core spliceosomal machinery, as well as through epigenetic modifications. However, to what extent these different mechanisms contribute to the regulation of AS in response to endogenous or environmental stimuli is still unclear. Circadian clocks allow organisms to adjust physiological processes to daily changes in environmental conditions. Here we review recent evidence linking circadian clock and AS, and discuss the role of Protein Arginine Methyltransferase 5 (PRMT5) in these processes. We propose that the interactions between daily oscillations in AS and circadian rhythms in the expression of splicing factors and epigenetic regulators offer a great opportunity to dissect the contribution of these mechanisms to the regulation of AS in a physiologically relevant context.

The control of root growth by reactive oxygen species in Salix nigra Marsh. seedlings

The production of reactive oxygen species (ROS) in specific regions of Salix seedlings roots seems essential for the normal growth of this organ. We examined the role of different ROS in the control of root development in Salix nigra seedlings, and explored possible mechanisms involved in the regulation of ROS generation and action. Root growth was not significantly affected by OH quenchers, while it was either partially or completely inhibited in the presence of H₂O₂ or O₂·⁻ scavengers, respectively. O₂·⁻ production was elevated in the root apex, particularly in the subapical meristem and protodermal zones. Apical O₂·⁻ generation activity was correlated to a high level of either Cu/Zn superoxide dismutase protein as well as carbonylated proteins. While NADPH-oxidase (NOX) was probably the main source of O₂·⁻ generation, the existence of other sources should not be discarded. O₂·⁻ production was also high in root hairs during budding, but it markedly decreased when the hair began to actively elongate. Root hair formation increased in the presence of H₂O₂ scavengers, and was suppressed when H₂O₂ or peroxidase inhibitors were supplied. The negative effect of H₂O₂ was partially counteracted by a MAPKK inhibitor. Possible mechanisms of action of the different ROS in comparison with other plant model systems are discussed.

Alternative splicing adds a new loop to the circadian clock

Circadian clocks allow organisms to adjust multiple physiological and developmental processes in anticipation of daily and seasonal changes in the environment. At the molecular level these clocks consist of interlocked feedback loops, involving transcriptional activation and repression, but also post-translational modifications. In a recently published work we provided evidence that PRMT5, a protein arginine methyl transferase, is part of a novel loop within the circadian clock of the plant Arabidopsis thaliana by regulating alternative splicing of key clock mRNAs. We also found evidence indicating that PRMT5 has a role in the regulation of alternative splicing and the circadian network in Drosophila melanogaster, although the clock connection in the latter is more elusive and seems to be at the output level. We conclude that alternative precursor messenger RNA (pre-mRNA) splicing is part of the circadian program and could be a main actor in the fine-tuning of biological clocks. Here, we embrace the alternative splicing process as part of the circadian program and discuss the possibility that this mechanism is of fundamental relevance for the fine-tuning of biological clocks.