RNA Polymerase III Subunit POLR3G Regulates Specific Subsets of PolyA(+) and SmallRNA Transcriptomes and Splicing in Human Pluripotent Stem Cells (original) (raw)
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STEM CELLS, 2011
The pluripotency of human embryonic stem cells (hESC) could have great potential for the development of cell replacement therapies. Previous studies have converged on the finding that OCT4, SOX2, and NANOG serve as key regulators in the maintenance of hESC. However, other signals that regulate hESC maintenance remain poorly studied. Here we describe a novel role of an RNA polymerase III (Pol III) subunit, POLR3G, in the maintenance of pluripotency in hESC. We demonstrate the presence of POLR3G in undifferentiated hESC, human induced pluripotent stem cells (hiPSC), and early mouse blastocysts. Downregulation of POLR3G is observed on differentiation of hESC and hiPSC, suggesting that POLR3G can be used as a molecular marker to readily identify undifferentiated pluripotent stem cells from their differentiated derivatives. Using an inducible shRNA lentiviral system, we found evidence that decreased levels of POLR3G result in loss of pluripotency and promote differentiation of hESC to all three germ layers but have no effect on cell apoptosis. On the other hand, overexpression of POLR3G has no effect on pluripotency and apoptosis in undifferentiated hESC. Interestingly, hESC expressing elevated levels of POLR3G are more resistant to differentiation. Furthermore, our experimental results show that POLR3G is a downstream target of OCT4 and NANOG, and our pharmacological study indicated that POLR3G expression can be readily regulated by the Erk1/2 signaling pathway. This study is the first to show an important role of POLR3G in the maintenance of hESC, suggesting a potential role of Pol III transcription in regulating hESC pluripotency. STEM CELLS
Stem Cells International, 2019
The eukaryotic nucleus is a highly complex structure that carries out multiple functions primarily needed for gene expression, and among them, transcription seems to be the most fundamental. Diverse approaches have demonstrated that transcription takes place at discrete sites known as transcription factories, wherein RNA polymerase II (RNAP II) is attached to the factory and immobilized while transcribing DNA. It has been proposed that transcription factories promote chromatin loop formation, creating long-range interactions in which relatively distant genes can be transcribed simultaneously. In this study, we examined long-range interactions between thePOU5F1gene and genes previously identified as beingPOU5F1enhancer-interacting, namely,CDYL,TLE2,RARG, andMSX1(all involved in transcriptional regulation), in human pluripotent stem cells (hPSCs) and their early differentiated counterparts. As a control gene,RUNX1was used, which is expressed during hematopoietic differentiation and no...
Regulatory Non-Coding RNAs in Pluripotent Stem Cells
International Journal of Molecular Sciences, 2013
The most part of our genome encodes for RNA transcripts are never translated into proteins. These include families of RNA molecules with a regulatory function, which can be arbitrarily subdivided in short (less than 200 nucleotides) and long non-coding RNAs (ncRNAs). MicroRNAs, which act post-transcriptionally to repress the function of target mRNAs, belong to the first group. Included in the second group are multi-exonic and polyadenylated long ncRNAs (lncRNAs), localized either in the nucleus, where they can associate with chromatin remodeling complexes to regulate transcription, or in the cytoplasm, acting as post-transcriptional regulators. Pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), represent useful systems for modeling normal development and human diseases, as well as promising tools for regenerative medicine. To fully explore their potential, however, a deep understanding of the molecular basis of stemness is crucial. In recent years, increasing evidence of the importance of regulation by ncRNAs in pluripotent cells is accumulating. In this review, we will discuss recent findings pointing to multiple roles played by regulatory ncRNAs in ESC and iPSCs, where they act in concert with signaling pathways, transcriptional regulatory circuitries and epigenetic factors to modulate the balance between pluripotency and differentiation.
Newly synthesized RNA Sequencing Characterizes Transcription Dynamics in Three Pluripotent States
2021
Unique transcriptomes define naïve, primed and paused pluripotent states in mouse embryonic stem cells. Here we perform transient transcriptome sequencing (TT-seq) to de novo define and quantify coding and non-coding transcription units (TUs) in different pluripotent states. We observe a global reduction of RNA synthesis, total RNA amount and turnover rates in ground state naïve cells (2i) and paused pluripotency (mTORi). We demonstrate that elongation velocity can be reliably estimated from TT-seq nascent RNA and RNA polymerase II occupancy and observe a transcriptome-wide attenuation of elongation velocity in the two inhibitor-induced states. We also discover a relationship between elongation velocity and termination read-through distance. Our analysis suggests that steady-state transcriptomes in mouse ES cells are controlled predominantly on the level of RNA synthesis, and that signaling pathways governing different pluripotent states immediately control key parameters of transcr...
Nucleic Acids Research, 2013
The transcriptional landscape in embryonic stem cells (ESCs) and during ESC differentiation has received considerable attention, albeit mostly confined to the polyadenylated fraction of RNA, whereas the non-polyadenylated (NPA) fraction remained largely unexplored. Notwithstanding, the NPA RNA super-family has every potential to participate in the regulation of pluripotency and stem cell fate. We conducted a comprehensive analysis of NPA RNA in ESCs using a combination of wholegenome tiling arrays and deep sequencing technologies. In addition to identifying previously characterized and new non-coding RNA members, we describe a group of novel conserved RNAs (snacRNAs: small NPA conserved), some of which are differentially expressed between ESC and neuronal progenitor cells, providing the first evidence of a novel group of potentially functional NPA RNA involved in the regulation of pluripotency and stem cell fate. We further show that minor spliceosomal small nuclear RNAs, which are NPA, are almost completely absent in ESCs and are upregulated in differentiation. Finally, we show differential processing of the minor intron of the polycomb group gene Eed. Our data suggest that NPA RNA, both known and novel, play important roles in ESCs.
Long Noncoding RNA Regulation of Pluripotency
Stem Cells International, 2016
Pluripotent stem cells (PSCs) represent a unique kind of stem cell, as they are able to indefinitely self-renew and hold the potential to differentiate into any derivative of the three germ layers. As such, human Embryonic Stem Cells (hESCs) and human induced Pluripotent Stem Cells (hiPSCs) provide a unique opportunity for studying the earliest steps of human embryogenesis and, at the same time, are of great therapeutic interest. The molecular mechanisms underlying pluripotency represent a major field of research. Recent evidence suggests that a complex network of transcription factors, chromatin regulators, and noncoding RNAs exist in pluripotent cells to regulate the balance between self-renewal and multilineage differentiation. Regulatory noncoding RNAs come in two flavors: short and long. The first class includes microRNAs (miRNAs), which are involved in the posttranscriptional regulation of cell cycle and differentiation in PSCs. Instead, long noncoding RNAs (lncRNAs) represent a heterogeneous group of long transcripts that regulate gene expression at transcriptional and posttranscriptional levels. In this review, we focus on the role played by lncRNAs in the maintenance of pluripotency, emphasizing the interplay between lncRNAs and other pivotal regulators in PSCs.
Small RNA changes en route to distinct cellular states of induced pluripotency
Nature Communications, 2014
MicroRNAs (miRNAs) are critical to somatic cell reprogramming into induced pluripotent stem cells (iPSCs), however, exactly how miRNA expression changes support the transition to pluripotency requires further investigation. Here we use a murine secondary reprogramming system to sample cellular trajectories towards iPSCs or a novel pluripotent 'F-class' state and perform small RNA sequencing. We detect sweeping changes in an early and a late wave, revealing that distinct miRNA milieus characterize alternate states of pluripotency. miRNA isoform expression is common but surprisingly varies little between cell states. Referencing other omic data sets generated in parallel, we find that miRNA expression is changed through transcriptional and post-transcriptional mechanisms. miRNA transcription is commonly regulated by dynamic histone modification, while DNA methylation/demethylation consolidates these changes at multiple loci. Importantly, our results suggest that a novel subset of distinctly expressed miRNAs supports pluripotency in the F-class state, substituting for miRNAs that serve such roles in iPSCs.
Transcriptomic Analysis of Human Naïve and Primed Pluripotent Stem Cells
Methods in Molecular Biology
Over the last decade, RNA-Sequencing (RNA-Seq) has revolutionized the field of transcriptomics due to its sheer advantage over previous technologies for studying gene expression. Even the domain of stem cell bioinformatics has benefited from these advancements. It has helped look deeper into how the process of pluripotency is maintained by stem cells and how it may be exploited for application in regenerative medicine. However, as it is still an evolving technology, there is no single accepted protocol for RNA-Seq data analysis. From a wide array of tools and/or algorithms available for the purpose, researchers tend to develop a pipeline that is best suited for their sample, experimental design, and computational power. In this tutorial, we describe a pipeline based on open-source tools to analyze RNA-Seq data from naïve and primed state human pluripotent stem cell samples. Precisely, we show how RNA-Seq data can be downloaded from databases, processed, and used to identify differentially expressed genes and construct a co-expression network. Further, we also show how the list of interesting genes obtained from differential expression testing or co-expression network be analyzed to gain biological insights.
Insight into the key genes of pluripotency in human and their interrelationships is necessary for understanding the underlying mechanism of pluripotency and hence their successful application in regenerative medicine. The recent advances in transcriptomics technologies have created new opportunities to decipher the genes involved in pluripotency, genetic network that governs the unique properties of embryonic stem cells and lineage differentiation mechanisms in a deeper scale. There are a large number of experimental studies on human embryonic stem cells (hESCs) being routinely conducted for unfolding the underlying biology of embryogenesis and their clinical prospects. However, the outcome of these studies often lacks consensus due to differences in samples, experimental techniques and/or analysis protocols. A universal stemness gene list is still lacking. In this quest, we compared transcriptomic profiles of pluripotent and non-pluripotent samples from diverse cell lines/types gen...