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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.

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.

The removal of introns from mRNA precursors and its regulation by alternative splicing are key for eukaryotic gene expression and cellular function, as evidenced by the numerous pathologies induced or modified by splicing alterations. Major recent advances have been made in understanding the structures and functions of the splicing machinery, in the description and classification of physiological and pathological isoforms and in the development of the first therapies for genetic diseases based on modulation of splicing. Here, we review this progress and discuss important remaining challenges, including predicting splice sites from genomic sequences, understanding the variety of molecular mechanisms and logic of splicing regulation, and harnessing this knowledge for probing gene function and disease aetiology and for the design of novel therapeutic approaches.Alternative splicing of pre-mRNAs is key for cellular function and underpins the aetiology of numerous diseases. Here, we review major advances in understanding the structures and functions of the splicing machinery and its regulation, and in harnessing this knowledge for the design of novel therapies.

Mutations in RNA-binding proteins can lead to pleiotropic phenotypes including craniofacial, skeletal, limb, and neurological symptoms. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in nucleic acid binding, transcription, and splicing through direct binding to DNA and RNA, or through interaction with other proteins in the spliceosome. We show a developmental role for Hnrnpul1 in zebrafish, resulting in reduced body and fin growth and missing bones. Defects in craniofacial tendon growth and adult-onset caudal scoliosis are also seen. We demonstrate a role for Hnrnpul1 in alternative splicing and transcriptional regulation using RNA-sequencing, particularly of genes involved in translation, ubiquitination, and DNA damage. Given its cross-species conservation and role in splicing, it would not be surprising if it had a role in human development. Whole-exome sequencing detected a homozygous frameshift variant in HNRNPUL1 in 2 siblings with congenital limb malformations, which is a candidate gene for their limb malformations. Zebrafish Hnrnpul1 mutants suggest an important developmental role of hnRNPUL1 and provide motivation for exploring the potential conservation of ancient regulatory circuits involving hnRNPUL1 in human development.

The directionality of gene promoters—the ratio of protein-coding over divergent noncoding transcription—is highly variable. How promoter directionality is controlled remains poorly understood. Here, we show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) dictate promoter directionality by attenuating divergent transcription relative to protein-coding transcription. At gene promoters that are highly directional, depletion of RSC leads to a relative increase in divergent noncoding transcription and thus to a decrease in promoter directionality. We find that RSC has a modest effect on nucleosome positioning upstream in promoters at the sites of divergent transcription. These promoters are also enriched for the binding of GRFs such as Reb1 and Abf1. Ectopic targeting of divergent transcription initiation sites with GRFs or the dCas9 DNA-binding protein suppresses divergent transcription. Our data suggest that RSC and GRFs play a pervasive role in limiting divergent transcription relative to coding direction transcription. We propose that any DNA-binding factor, when stably associated with cryptic transcription start sites, forms a barrier which represses divergent transcription, thereby promoting promoter directionality.

Transcription initiation is a highly dynamic and tightly regulated process involving the coordinated action of transcription factors, chromatin remodelers, and RNA polymerase which determine where and when transcription begins. Accurately mapping and quantifying transcription start sites (TSSs) from nascently transcribed RNAs remains a key area of interest, as it provides critical insights into transcription dynamics. Here, we combined transient transcriptome sequencing with transcription start site sequencing (TT-TSS-seq) to accurately map and quantify transcription initiation sites from nascent transcripts. Since transient metabolic labelling yields low-input RNA, we optimized the TSS-seq protocol to enhance sensitivity and accuracy. Specifically, we refined enzymatic reactions for decapping and RNA ligation and incorporated 5’ oligonucleotides containing unique molecular identifiers (UMIs) and barcodes to enable accurate quantification and sample multiplexing. The TT-TSS-seq approach detected transcription initiation of unstable transcripts, such as enhancer RNAs. Moreover, we identified that a large fraction of genes use multiple transcription initiation sites, yet often produce only a single stable transcript. Overall, TT-TSS-seq provides precise mapping and quantification of transcription initiation sites, offering new insights into transcriptional dynamics and expanding the toolkit for studying gene regulation.

Most genes are transcribed from multiple transcription start sites (TSSs), defined as alternative TSSs, which are highly regulated and can lead to various gene regulatory outcomes including changes in translation efficiency and protein isoform expression. Transcription factors and chromatin regulators control alternative TSS selection. DNA supercoiling affects multiple aspects of transcription including transcription initiation; however, its regulatory effect on genes with multiple TSSs is not known. Here, we investigated how DNA supercoiling impacts alternative TSS usage in Saccharomyces cerevisiae. We depleted topoisomerases during early meiosis, where alternative TSS usage is prevalent, and applied an improved TSS sequencing protocol. We show that supercoiling affects alternative TSS usage at almost 600 genes. Increased alternative and aberrant TSS usage was observed near and within open reading frames, likely resulting from transcription-induced supercoiling originating from upstream alternative TSSs. DNA supercoiling had the greatest impact on genes with a dominant alternative TSS, significant spacing between alternative TSSs, and greater overall gene length. Our results establish that DNA supercoiling release during transcription is critical for correct TSS selection.

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. Here, we show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) dictate promoter directionality by attenuating divergent transcription. At gene promoters that are highly directional, depletion of RSC leads to a relative increase in divergent noncoding transcription and thus a decrease in promoter directionality. We find that RSC facilitates nucleosome positioning upstream in promoters at the sites of divergent transcription. These highly directional promoters are also enriched for the binding of GRFs such as Reb1 and Abf1. Ectopic targeting of divergent transcription initiation sites with GRFs or the dCas9 protein suppresses divergent transcription. Our data suggest that RSC and GRFs play a pervasive role in limiting divergent transcription. We propose that any DNA binding factor, when stably associated with cryptic transcription start sites, form barriers for repressing divergent transcription. Our study provides an explanation as to why certain promoters are more directional than others. Competing Interest Statement The authors have declared no competing interest.

Mutations in RNA binding proteins can lead to pleiotropic phenotypes including craniofacial, skeletal, limb and neurological symptoms. Heterogeneous Nuclear Ribonucleoproteins (hnRNPs) are involved in nucleic acid binding, transcription and splicing through direct binding to DNA and RNA, or through interaction with other proteins in the spliceosome. We show a developmental role for Hnrnpul1 in zebrafish, resulting in reduced craniofacial tendon length, severe adult-onset scoliosis and reduced fin size. We demonstrate a role of Hnrnpul1 in alternative splicing and transcriptional regulation using RNA sequencing. Given its cross-species conservation and role in splicing it would not be surprising if it had a role in human development but the developmental role of this gene in humans has not been explored. Whole exome sequencing detected a frameshift variant in HNRNPUL1 in two siblings with congenital limb malformations which remain variants of unknown significance. Zebrafish Hnrnpul1 mutants suggest an important developmental role of hnRNPUL1 and provide motivation for exploring potential conservation of ancient regulatory circuits involving hnRNPUL1 in human development.

Abstract In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for cell reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we have studied the dynamics of alternative splicing (AS) changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells. These changes, generally uncoupled from transcriptional regulation, significantly overlapped with splicing programs reported during reprogramming of mouse embryonic fibroblasts (MEFs). Correlation between gene expression of potential regulators and specific clusters of AS changes enabled the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of MEFs reprogramming. These RNA-binding proteins control partially overlapping programs of splicing regulation affecting genes involved in developmental and morphogenetic processes. Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process. Competing Interest Statement The authors have declared no competing interest.