No abstract

The generation of new ribosomes is a coordinated process essential to sustain cell growth. As such, it is tightly regulated according to cell needs. As cancer cells require intense protein translation to ensure their enhanced growth rate, they exploit various mechanisms to boost ribosome biogenesis. In this review, we will summarize how oncogenes and tumor suppressors modulate the biosynthesis of the RNA component of ribosomes, starting from the description of well-characterized pathways that converge on ribosomal RNA transcription while including novel insights that reveal unexpected regulatory networks hacked by cancer cells to unleash ribosome production.

Focal deletions occur frequently in the cancer genome. However, the putative tumor‐suppressive genes residing within these regions have been difficult to pinpoint. To robustly identify these genes, we implemented a computational approach based on non‐negative matrix factorization, NMF, and interrogated the TCGA dataset. This analysis revealed a metagene signature including a small subset of genes showing pervasive hemizygous deletions, reduced expression in cancer patient samples, and nucleolar function. Amid the genes belonging to this signature, we have identified PNRC1, a nuclear receptor coactivator. We found that PNRC1 interacts with the cytoplasmic DCP1α/DCP2 decapping machinery and hauls it inside the nucleolus. PNRC1‐dependent nucleolar translocation of the decapping complex is associated with a decrease in the 5′‐capped U3 and U8 snoRNA fractions, hampering ribosomal RNA maturation. As a result, PNRC1 ablates the enhanced proliferation triggered by established oncogenes such as RAS and MYC. These observations uncover a previously undescribed mechanism of tumor suppression, whereby the cytoplasmic decapping machinery is hauled within nucleoli, tightly regulating ribosomal RNA maturation.

The regulation of pre-mRNA processing has important consequences for cell division and the control of cancer cell proliferation but the underlying molecular mechanisms remain poorly understood. We report that three splicing factors, SPF45, SR140 and CHERP form a tight physical and functionally coherent complex that regulates a variety of alternative splicing events, frequently by repressing short exons flanked by suboptimal 3' splice sites. These comprise alternative exons embedded in genes with important functions in cell cycle progression, including the G2/M key regulator FOXM1 and the spindle regulator SPDL1. Knockdown of either of the three factors leads to G2/M arrest and to enhanced apoptosis in HeLa cells. Promoting the changes in FOXM1 or SPDL1 splicing induced by SPF45/SR140/CHERP knockdown partially recapitulate the effects on cell growth, arguing that the complex orchestrates a program of alternative splicing necessary for efficient cell proliferation.

Summary Alternative splicing is a prevalent mechanism of gene regulation that is modulated in response to a wide range of extracellular stimuli. Stress-activated protein kinases (SAPKs) play a key role in controlling several steps of mRNA biogenesis. Here, we show that osmostress has an impact on the regulation of alternative splicing (AS), which is partly mediated through the action of p38 SAPK. Splicing network analysis revealed a functional connection between p38 and the spliceosome component SKIIP, whose depletion abolished a significant fraction of p38-mediated AS changes. Importantly, p38 interacted with and directly phosphorylated SKIIP, thereby altering its activity. SKIIP phosphorylation regulated AS of GADD45α, the upstream activator of the p38 pathway, uncovering a negative feedback loop involving AS regulation. Our data reveal mechanisms and targets of SAPK function in stress adaptation through the regulation of AS.

Background Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts. Results We observe a significant overlap between alternative splicing changes detected in the two reprogramming systems, which are generally uncoupled from changes in transcriptional levels. Correlation between gene expression of potential regulators and specific clusters of alternative splicing changes enables the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of mouse embryonic fibroblasts reprogramming. We further find that these RNA-binding proteins control partially overlapping programs of splicing regulation, involving genes relevant for developmental and morphogenetic processes. Conclusions Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process and their targets.

N6-methyladenosine (m6A) is a widely studied and abundant RNA modification. The m6A mark regulates the fate of RNAs in various ways, which in turn, drives changes in cell physiology, development, and disease pathology. Over the last decade, numerous methods have been developed to map and quantify m6A sites genome-wide through deep sequencing. Alternatively, m6A levels can be quantified from a population of RNAs using techniques such as liquid chromatography-mass spectrometry or thin layer chromatography. However, many methods for quantifying m6A levels involve extensive protocols and specialized data analysis, and often only a few samples can be handled in a single experiment. Here, we developed a simple method for determining relative m6A levels in mRNA populations from various sources based on enzyme-linked immunosorbent-based assay (m6A-ELISA). We have optimized various steps of m6A-ELISA such as sample preparation and the background signal resulting from the primary antibody. We validated the method using mRNA populations from budding yeast and mouse embryonic stem cells. The full protocol takes less than a day, requiring only 25 ng of mRNA. The m6A-ELISA protocol is quick, cost-effective, and scalable, making it a valuable tool for determining relative m6A levels in samples from various sources that could be adapted to detect other mRNA modifications.

The directionality of gene promoters - the ratio of protein-coding over divergent noncoding transcription - is highly variable and regulated. How promoter directionality is controlled remains poorly understood. We show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) dictate promoter directionality by attenuating divergent transcription. Depletion of RSC increased divergent noncoding transcription and decreased protein-coding transcription at promoters with strong directionality. Consistent with RSCs role in regulating chromatin, RSC depletion impacts nucleosome occupancy upstream of the nucleosome depleted region where divergent transcription initiates, suggesting that nucleosome positioning at the 5 prime border of gene promoters physically blocks the recruitment of the transcription machinery and inhibits initiation of divergent transcription. Highly directional promoters were also enriched for the binding of GRFs such as Reb1 and Abf1. Furthermore, ectopic targeting of divergent transcription initiation sites with GRFs or the dCas9 protein can suppress divergent transcription. Our data suggest that RSC-mediated nucleosome positioning and GRFs play a pervasive role in repressing divergent transcription. We propose that any DNA binding factor, when stably associated with cryptic transcription start sites, can form a barrier for repressing divergent transcription. Our study provides an explanation as to why certain promoters are more directional than others.