Meet the authors: Mei-Sheng Xiao, Arun Prasath Damodaran, and Thomas Gonatopoulos-Pournatzis.

We talk to corresponding author Thomas Gonatopoulos-Pournatzis and co-first authors Arun Prasath Damodaran and Mei-Sheng Xiao about their paper "Genome-scale exon perturbation screens uncover exons critical for cell fitness" (in this issue of Molecular Cell) and get insights into their findings, career trajectories, and future directions in the pre-mRNA processing field.

Genome-scale exon perturbation screens uncover exons critical for cell fitness.

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.

Alteration of SC35 localization by transfection reagents.

Transfection is a powerful tool that enables introducing foreign nucleic acids into living cells in order to study the function of a gene product. Ever since the discovery of transfection many side effects or artifacts caused by transfection reagents have been reported. Here, we show that the transfection reagent, JetPRIME alters the localization of the splicing protein SC35 widely used as a nuclear speckle marker. We demonstrate that transfection of plasmids with JetPRIME leads to enlarged SC35 speckles and SC35 cytoplasmic granules. By contrast, transfection of the same plasmid with Lipofectamine 3000 does not have any effect on SC35 localization. The formation of SC35 cytoplasmic granules by JetPRIME-mediated transfection is independent of exogenous expression by plasmid and although similar in morphology they are distinct from P-bodies and stress granules. This method of transfection affected only SC35 and phosphorylated SR proteins but not the nuclear speckles. The JetPRIME-mediated transfection also showed compromised transcription in cells with enlarged SC35 speckles. Our work indicates that the use of JetPRIME alters SC35 localization and can affect gene expression and alternative splicing. Therefore, caution should be exercised when interpreting results after the use of a transient transfection system, particularly when the subject of the study is the function of a protein in the control of gene expression or mRNA splicing.

Abrogation of FBW7α-dependent p53 degradation enhances p53's function as a tumor suppressor.

The gene encoding the tumor suppressor p53 is mutated in most cancers. p53 expression is known to be tightly controlled by several E3 ligases. Here, we show that F-box and WD repeat domain-containing 7α (FBW7α), the substrate-recognition component of the SCFFBW7 multiprotein E3 ligase complex, targets both WT and tumor-derived mutants of p53 for proteasomal degradation in multiple human cancer cell lines (HCT116 and U2OS). We found that lack of FBW7α stabilizes p53 levels, thereby increasing its half-life. p53 ubiquitylation and subsequent degradation require the F-box and the C-terminal WD40 repeats in FBW7α. The polyubiquitylation of p53 occurred via Lys-48 linkage and involved phosphorylation on p53 at Ser-33 and Ser-37 by glycogen synthase kinase 3ß (GSK3ß) and DNA-dependent protein kinase (DNA-PK), respectively. These phosphorylation events created a phosphodegron that enhanced p53 binding to FBW7α, allowing for the attachment of polyubiquitin moieties at Lys-132 in p53. FBW7α-dependent p53 polyubiquitylation apparently occurred during and immediately after DNA double-strand breaks induced by either doxorubicin or ionizing radiation. Accordingly, in cells lacking FBW7α, p53 induction was enhanced after DNA damage. Phosphodegron-mediated polyubiquitylation of p53 on Lys-132 had functional consequences, with cells in which FBW7α-mediated p53 degradation was abrogated exhibiting enhancement of their tumorigenic potential. We conclude that p53, which previously has been reported to transactivate FBW7, is also targeted by the same E3 ligase for degradation, suggesting the presence of a regulatory feedback loop that controls p53 levels and functions during DNA damage.

Aurora A kinase activity is required to maintain an active spindle assembly checkpoint during prometaphase.

During the prometaphase stage of mitosis, the cell builds a bipolar spindle of microtubules that mechanically segregates sister chromatids between two daughter cells in anaphase. The spindle assembly checkpoint (SAC) is a quality control mechanism that monitors proper attachment of microtubules to chromosome kinetochores during prometaphase. Segregation occurs only when each chromosome is bi-oriented with each kinetochore pair attached to microtubules emanating from opposite spindle poles. Overexpression of the protein kinase Aurora A is a feature of various cancers and is thought to enable tumour cells to bypass the SAC, leading to aneuploidy. Here, we took advantage of a chemical and chemical-genetic approach to specifically inhibit Aurora A kinase activity in late prometaphase. We observed that a loss of Aurora A activity directly affects SAC function, that Aurora A is essential for maintaining the checkpoint protein Mad2 on unattached kinetochores and that inhibition of Aurora A leads to loss of the SAC, even in the presence of nocodazole or Taxol. This is a new finding that should affect the way Aurora A inhibitors are used in cancer treatments.This article has an associated First Person interview with the first authors of the paper.

Aurora A Kinase Is a Priority Pharmaceutical Target for the Treatment of Cancers.

Aurora kinases control multiple events during cell cycle progression and are essential for mitotic and meiotic bipolar spindle assembly and function. There are three Aurora kinases in mammals, some of which have oncogenic properties and all of which are overexpressed in multiple cancers. Pharmaceutical companies quickly made these kinases priority targets for the development of inhibitors to be used as cancer treatments. In this review, we focus on Aurora A, against which several inhibiting compounds have been discovered and made available; however, even though some of these compounds underwent clinical trials, none have yet gone beyond Phase III trials. The varying efficiencies and particularities of these drugs raise several questions that are explored in this review: is Aurora A even a good target? What biomarkers can we use to measure its activity in vivo? How can we improve the Aurora A-inhibiting drugs?