Anabella Srebrow

The connection between splicing and cancer

Alternative splicing is a crucial mechanism for generating protein diversity. Different splice variants of a given protein can display different and even antagonistic biological functions. Therefore, appropriate control of their synthesis is required to assure the complex orchestration of cellular processes within multicellular organisms. Mutations in cis-acting splicing elements or changes in the activity of constitutive or alternative splicing could have a profound regulatory proteins that compromise the accuracy of either impact on human pathogenesis, in particular in tumor development and progression. Mutations in splicing elements, for example, have been found in genes such as LKB1, KIT, CDH17, KLF6 and BRCA1, and changes in trans-acting regulators can affect the expression of genes such as Ron, RAC1 and CD44.

Concerted regulation of nuclear and cytoplasmic activities of SR proteins by AKT.

Serine/arginine-rich (SR) proteins are important regulators of mRNA splicing. Several postsplicing activities have been described for a subset of shuttling SR proteins, including regulation of mRNA export and translation. Using the fibronectin gene to study the links between signal-transduction pathways and SR protein activity, we show that growth factors not only modify the alternative splicing pattern of the fibronectin gene but also alter translation of reporter messenger RNAs in an SR protein–dependent fashion, providing two coregulated levels of isoform-specific amplification. These effects are inhibited by specific small interfering RNAs against SR proteins and are mediated by the AKT kinase, which elicits opposite effects to those evoked by overexpressing SR protein kinases Clk and SRPK. These results show how SR protein activity is modified in response to extracellular stimulation, leading to a concerted regulation of splicing and translation.

Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing.

Here we investigate the promoter control of alternative splicing by studying two transcriptional activators on templates under replicating conditions. SV40 large T‐antigen (T‐Ag) activates template replication only 2‐fold but transcription 25‐fold. T‐Ag‐mediated replication, reported to inhibit RNA polymerase II elongation, provokes a 10‐ to 30‐fold increase in the inclusion of the fibronectin EDI exon into mature mRNA. The T‐Ag effect is exon specific, occurs in cis and depends strictly on DNA replication and not on cell transformation. VP16, an activator of transcriptional initiation and elongation, has a similar effect on transcription but the opposite effect on splicing: EDI inclusion is inhibited by 35‐fold. VP16 completely reverts the T‐Ag effect, but a VP16 mutant with reduced elongation ability provokes only partial reversion. Both T‐Ag and VP16 promote conspicuous co‐localization of mRNA with nuclear speckles that contain the SR protein SF2/ASF, a positive regulator of EDI inclusion. Therefore, we conclude that co‐localization of transcripts and speckles is not sufficient to stimulate EDI inclusion.

Coordination between transcription and pre-mRNA processing

A large body of work has proved that transcription by RNA polymerase II and pre‐mRNA processing are coordinated events within the cell nucleus. Capping, splicing and polyadenylation occur while transcription proceeds, suggesting that RNA polymerase II plays a role in the regulation of these events. The presence and degree of phosphorylation of the carboxy‐terminal domain of RNA polymerase II large subunit is important for functioning of the capping enzymes, the assembly of spliceosomes and the binding of the cleavage/polyadenylation complex. Nuclear architecture and gene promoter structure have also been shown to play key roles in coupling between transcription and splicing.

Akt/PKB: one kinase, many modifications.

Akt/PKB, a serine/threonine kinase member of the AGC family of proteins, is involved in the regulation of a plethora of cellular processes triggered by a wide diversity of extracellular signals and is thus considered a key signalling molecule in higher eukaryotes. Deregulation of Akt signalling is associated with a variety of human diseases, revealing Akt-dependent pathways as an attractive target for therapeutic intervention. Since its discovery in the early 1990s, a large body of work has focused on Akt phosphorylation of two residues, Thr308 and Ser473, and modification of these two sites has been established as being equivalent to Akt activation. More recently, Akt has been identified as a substrate for many different post-translational modifications, including not only phosphorylation of other residues, but also acetylation, glycosylation, oxidation, ubiquitination and SUMOylation. These modifications could provide additional regulatory steps for fine-tuning Akt function, Akt trafficking within the cell and/or for determining the substrate specificity of this signalling molecule. In the present review, we provide an overview of these different post-translational modifications identified for Akt, focusing on their consequences for this kinase activity.

DNA Damage-Induced Heterogeneous Nuclear Ribonucleoprotein K SUMOylation Regulates p53 Transcriptional Activation

Background: hnRNP K acts as a p53 cofactor upon DNA damage. Results: DNA damage stimulates hnRNP K sumoylation, and this modification is required for p53 target gene expression. Conclusion: hnRNP K sumoylation links DNA damage-induced signaling to p53 transcriptional activation. Significance: The discovery of how different players within the p53 pathway are regulated will provide important insights into the study of chemotherapeutic drugs. Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a nucleocytoplasmic shuttling protein that is a key player in the p53-triggered DNA damage response, acting as a cofactor for p53 in response to DNA damage. hnRNP K is a substrate of the ubiquitin E3 ligase MDM2 and, upon DNA damage, is de-ubiquitylated. In sharp contrast with the role and consequences of the other post-translational modifications, nothing is known about the role of SUMO conjugation to hnRNP K in p53 transcriptional co-activation. In the present work, we show that hnRNP K is modified by SUMO in lysine 422 within its KH3 domain, and sumoylation is regulated by the E3 ligase Pc2/CBX4. Most interestingly, DNA damage stimulates hnRNP K sumoylation through Pc2 E3 activity, and this modification is required for p53 transcriptional activation. Abrogation of hnRNP K sumoylation leads to an aberrant regulation of the p53 target gene p21. Our findings link the DNA damage-induced Pc2 activation to the p53 transcriptional co-activation through hnRNP K sumoylation.

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.

Control of alternative pre-mRNA splicing by RNA pol II elongation: Faster is not always better

The realization that the mammalian proteomic complexity is achieved with a limited number of genes demands a better understanding of alternative splicing regulation. Promoter control of alternative splicing was originally described by our group in studies performed on the fibronectin gene. Recently, other labs extended our findings to the cystic fibrosis, CD44 and CGRP genes strongly supporting a coupling between transcription and pre-mRNA splicing. A possible mechanism that would fit in these results is that the promoter itself is responsible for recruiting splicing factors, such as SR proteins, to the site of transcription, possibly through transcription factors that bind the promoter or the transcriptional enhancers. An alternative model, discussed more extensively in this review, involves modulation of RNA pol II (pol II) elongation rate. The model is supported by findings that cis- and trans- acting factors that modulate pol II elongation on a particular template also provoke changes in the alternative splicing balance of the encoded mRNAs.

Involvement of hnRNP A1 in the matrix metalloprotease‐3‐dependent regulation of Rac1 pre‐mRNA splicing

Rac1b is an alternatively spliced isoform of the small GTPase Rac1 that includes the 57‐nucleotide exon 3b. Rac1b was originally identified through its over‐expression in breast and colorectal cancer cells, and has subsequently been implicated as a key player in a number of different oncogenic signaling pathways, including tumorigenic transformation of mammary epithelial cells exposed to matrix metalloproteinase‐3 (MMP‐3). Although many of the cellular consequences of Rac1b activity have been recently described, the molecular mechanism by which MMP‐3 treatment leads to Rac1b induction has not been defined. Here we use proteomic methods to identify heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a factor involved in Rac1 splicing regulation. We find that hnRNP A1 binds to Rac1 exon 3b in mouse mammary epithelial cells, repressing its inclusion into mature mRNA. We also find that exposure of cells to MMP‐3 leads to release of hnRNP A1 from exon 3b and the consequent generation of Rac1b. Finally, we analyze normal breast tissue and breast cancer biopsies, and identify an inverse correlation between expression of hnRNP A1 and Rac1b, suggesting the existence of this regulatory axis in vivo. These results provide new insights on how extracellular signals regulate alternative splicing, contributing to cellular transformation and development of breast cancer. J. Cell. Biochem. 113: 2319–2329, 2012.

The CRE-binding factor ATF-2 facilitates the occupation of the CCAAT box in the fibronectin gene promoter

The cAMP response element (CRE) and the CCAAT box of the fibronectin gene promoter are separated by only twenty base pairs. A specific factor that binds the CRE interacts cooperatively with the protein which binds to the adjacent CCAAT box, stimulating transcription [1992, J. Biol. Chem. 267, 12767‐12774]. Here we show that the CRE factor is an heterodimer between a 43 kDa and the ‘73 kDa’ CRE‐binding proteins and we identify the latter as ATF‐2 (also named CRE‐BPI), a protein implicated in recruiting transcriptional activators to promoters, able to form heterodimers with Jun and for which a sequence‐deduced MW of 55 kDa had been previously reported.