scholarly journals Dissecting splicing decisions and cell-to-cell variability with designed sequence libraries

2018 ◽  
Author(s):  
Martin Mikl ◽  
Amit Hamburg ◽  
Yitzhak Pilpel ◽  
Eran Segal
Keyword(s):  
Secondary Structure ◽  
Single Cell ◽  
Splicing Regulation ◽  
Splice Isoforms ◽  
Human Genes ◽  
Large Expansion ◽  
Alternatively Spliced ◽  
Protein Splice ◽  
Cell To Cell Variability

AbstractMost human genes are alternatively spliced, allowing for a large expansion of the proteome.The multitude of regulatory inputs to splicing limits the potential to infer general principles from investigating native sequences. Here, we created a rationally designed library of >32,000 splicing events to dissect the complexity of splicing regulation through systematicsequence alterations. Measuring RNA and protein splice isoforms allowed us to investigate bothcause and effect of splicing decisions, quantify diverse regulatory inputs and accurately predict (R2=0.75–0.85) isoform ratios from sequence and secondary structure. By profiling individual cells, we measure the cell-to-cell variability of splicing decisions and show that it can be encoded in the DNA and influenced by regulatory inputs, opening the door for a novel,single-cell perspective on splicing regulation.

scholarly journals Dissecting splicing decisions and cell-to-cell variability with designed sequence libraries

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Martin Mikl ◽  
Amit Hamburg ◽  
Yitzhak Pilpel ◽  
Eran Segal
Keyword(s):  
Secondary Structure ◽  
Single Cell ◽  
Splicing Regulation ◽  
Splice Isoforms ◽  
Cause And Effect ◽  
Human Genes ◽  
Large Expansion ◽  
Alternatively Spliced ◽  
Protein Splice ◽  
Cell To Cell Variability

Abstract Most human genes are alternatively spliced, allowing for a large expansion of the proteome. The multitude of regulatory inputs to splicing limits the potential to infer general principles from investigating native sequences. Here, we create a rationally designed library of >32,000 splicing events to dissect the complexity of splicing regulation through systematic sequence alterations. Measuring RNA and protein splice isoforms allows us to investigate both cause and effect of splicing decisions, quantify diverse regulatory inputs and accurately predict (R2 = 0.73–0.85) isoform ratios from sequence and secondary structure. By profiling individual cells, we measure the cell-to-cell variability of splicing decisions and show that it can be encoded in the DNA and influenced by regulatory inputs, opening the door for a novel, single-cell perspective on splicing regulation.

scholarly journals Splicing Regulation: A Molecular Device to Enhance Cancer Cell Adaptation

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Vittoria Pagliarini ◽  
Chiara Naro ◽  
Claudio Sette
Keyword(s):  
Cancer Cells ◽  
Splice Variants ◽  
Growth Conditions ◽  
Splicing Regulation ◽  
Molecular Device ◽  
Splice Isoforms ◽  
Regulate Gene Expression ◽  
Human Genes ◽  
Harsh Conditions ◽  
Coding Potential

Alternative splicing (AS) represents a major resource for eukaryotic cells to expand the coding potential of their genomes and to finely regulate gene expression in response to both intra- and extracellular cues. Cancer cells exploit the flexible nature of the mechanisms controlling AS in order to increase the functional diversity of their proteome. By altering the balance of splice isoforms encoded by human genes or by promoting the expression of aberrant oncogenic splice variants, cancer cells enhance their ability to adapt to the adverse growth conditions of the tumoral microenvironment. Herein, we will review the most relevant cancer-related splicing events and the underlying regulatory mechanisms allowing tumour cells to rapidly adapt to the harsh conditions they may face during the occurrence and development of cancer.

scholarly journals Aberrant Alternative Splicing Is Another Hallmark of Cancer

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Michael Ladomery
Keyword(s):  
Alternative Splicing ◽  
Splice Factor ◽  
Splice Isoforms ◽  
Cancer Hallmarks ◽  
Abnormal Expression ◽  
Human Genes ◽  
Alternatively Spliced

The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

scholarly journals Positive Selection in Alternatively Spliced Exons of Human Genes

2008 ◽  
Vol 83 (1) ◽  
pp. 94-98 ◽  
Author(s):  
Vasily E. Ramensky ◽  
Ramil N. Nurtdinov ◽  
Alexei D. Neverov ◽  
Andrei A. Mironov ◽  
Mikhail S. Gelfand
Keyword(s):  
Positive Selection ◽  
Human Genes ◽  
Alternatively Spliced ◽  
Alternatively Spliced Exons

scholarly journals Human Genes Escaping X-inactivation Revealed by Single Cell Expression Data

2018 ◽  
Author(s):  
Kerem Wainer Katsir ◽  
Michal Linial
Keyword(s):  
Single Cell ◽  
X Chromosome ◽  
Noncoding Rna ◽  
Phenotypic Variability ◽  
Cell Types ◽  
X Inactivation ◽  
Specific Expression ◽  
X Chromosomes ◽  
Human Genes ◽  
Cumulative Knowledge

AbstractBackgroundIn mammals, sex chromosomes pose an inherent imbalance of gene expression between sexes. In each female somatic cell, random inactivation of one of the X-chromosomes restores this balance. While most genes from the inactivated X-chromosome are silenced, 15-25% are known to escape X-inactivation (termed escapees). The expression levels of these genes are attributed to sex-dependent phenotypic variability.ResultsWe used single-cell RNA-Seq to detect escapees in somatic cells. As only one X-chromosome is inactivated in each cell, the origin of expression from the active or inactive chromosome can be determined from the variation of sequenced RNAs. We analyzed primary, healthy fibroblasts (n=104), and clonal lymphoblasts with sequenced parental genomes (n=25) by measuring the degree of allelic-specific expression (ASE) from heterozygous sites. We identified 24 and 49 candidate escapees, at varying degree of confidence, from the fibroblast and lymphoblast transcriptomes, respectively. We critically test the validity of escapee annotations by comparing our findings with a large collection of independent studies. We find that most genes (66%) from the unified set were previously reported as escapees. Furthermore, out of the overlooked escapees, 11 are long noncoding RNA (lncRNAs).ConclusionsX-chromosome inactivation and escaping from it are robust, permanent phenomena that are best studies at a single-cell resolution. The cumulative information from individual cells increases the potential of identifying escapees. Moreover, despite the use of a limited number of cells, clonal cells (i.e., same X-chromosomes are coordinately inhibited) with genomic phasing are valuable for detecting escapees at high confidence. Generalizing the method to uncharacterized genomic loci resulted in lncRNAs escapees which account for 20% of the listed candidates. By confirming genes as escapees and propose others as candidates from two different cell types, we contribute to the cumulative knowledge and reliability of human escapees.

scholarly journals Digital signaling decouples activation probability and population heterogeneity

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Ryan A Kellogg ◽  
Chengzhe Tian ◽  
Tomasz Lipniacki ◽  
Stephen R Quake ◽  
Savaş Tay
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Activation ◽  
Population Heterogeneity ◽  
Cellular Phenotype ◽  
Noisy Environments ◽  
Gene Expression Phenotype ◽  
Weak Stimulation ◽  
Cell To Cell Variability ◽  
Activation Probability

Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration × duration) controls the fraction of cells that activate NF-κB in the population. However, stimulus temporal profile determined NF-κB dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity.

Dual-coding Regions in Alternatively Spliced Human Genes

2008 ◽  
Author(s):  
Han Liang ◽  
Laura F Landweber
Keyword(s):  
Dual Coding ◽  
Coding Regions ◽  
Human Genes ◽  
Alternatively Spliced

scholarly journals The Role of Alternative RNA Splicing in the Regulation of hTERT, Telomerase, and Telomeres: Implications for Cancer Therapeutics

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1514 ◽  
Author(s):  
Aaron L. Slusher ◽  
Jeongjin JJ Kim ◽  
Andrew T. Ludlow
Keyword(s):  
Cancer Cell ◽  
Rna Splicing ◽  
Cell Biology ◽  
Science Studies ◽  
De Novo ◽  
Treatment Strategies ◽  
Cancer Therapeutics ◽  
Telomerase Activity ◽  
Splice Isoforms ◽  
Alternatively Spliced

Alternative RNA splicing impacts the majority (>90%) of eukaryotic multi-exon genes, expanding the coding capacity and regulating the abundance of gene isoforms. Telomerase (hTERT) is a key example of a gene that is alternatively spliced during human fetal development and becomes dysregulated in nearly all cancers. Approximately 90% of human tumors use telomerase to synthesize de novo telomere repeats and obtain telomere-dependent cellular immortality. Paradigm shifting data indicates that hTERT alternative splicing, in addition to transcription, plays an important role in the regulation of active telomerase in cells. Our group and others are pursuing the basic science studies to progress this emerging area of telomerase biology. Recent evidence demonstrates that switching splicing of hTERT from the telomerase activity producing full-length hTERT isoform to alternatively spliced, non-coding isoforms may be a novel telomerase inhibition strategy to prevent cancer growth and survival. Thus, the goals of this review are to detail the general roles of telomerase in cancer development, explore the emerging regulatory mechanisms of alternative RNA splicing of the hTERT gene in various somatic and cancer cell types, define the known and potential roles of hTERT splice isoforms in cancer cell biology, and provide insight into new treatment strategies targeting hTERT in telomerase-positive cancers.

Identification of MET genomic amplification, protein expression and alternative splice isoforms in neuroblastomas

2013 ◽  
Vol 66 (11) ◽  
pp. 985-991 ◽  
Author(s):  
Benedict Yan ◽  
Malcolm Lim ◽  
Lihan Zhou ◽  
Chik Hong Kuick ◽  
May Ying Leong ◽  
...  
Keyword(s):  
Protein Expression ◽  
Kinase Inhibitor ◽  
In Situ Hybridisation ◽  
Genomic Amplification ◽  
Splice Isoforms ◽  
Anaplastic Lymphoma ◽  
Met Amplification ◽  
Alternatively Spliced ◽  
Expression Status

BackgroundCrizotinib, a dual anaplastic lymphoma kinase (ALK) and mesenchymal-epithelial transition (MET) tyrosine kinase inhibitor, is currently being evaluated for the treatment of neuroblastoma. Its effects are thought to be mediated mainly via its activity against ALK. Although MET genomic/protein expression status might conceivably affect crizotinib efficacy, this issue has hitherto not received attention in neuroblastomas.Aims/MethodsMET genomic and protein expression status was characterised by silver in situ hybridisation and immunohistochemistry (IHC) respectively, in a cohort of 54 neuroblastoma samples. MET splice isoforms were characterised in 15 of these samples by quantitative PCR.ResultsOne case (1/54; prevalence 1.85%) displayed MET genomic amplification, while another case (1/54; prevalence 1.85%) displayed strong membranous MET protein expression (IHC score 3+). Alternative exon 10-deleted and exon 14-deleted MET splice isoforms were identified.ConclusionsMET amplification and protein expression, although low in prevalence, are present in neuroblastomas. This has implications when crizotinib is employed as a therapeutic agent in neuroblastomas. Additionally, the existence of alternatively spliced MET isoforms may have clinical and biological implications in neuroblastomas.

Intron retention: a human DKC1 gene common splicing event

2013 ◽  
Vol 91 (6) ◽  
pp. 506-512 ◽  
Author(s):  
Mimmo Turano ◽  
Alberto Angrisani ◽  
Nunzia Di Maio ◽  
Maria Furia
Keyword(s):  
Intron Retention ◽  
Cell Types ◽  
Dyskeratosis Congenita ◽  
Telomere Maintenance ◽  
Splice Isoforms ◽  
Nonsense Mediated Decay ◽  
Molecular Approaches ◽  
Additional Layer ◽  
Alternatively Spliced

Identification of alternatively spliced transcripts produced by a gene is a crucial step in deciphering the bulk of its biological roles and the overall processes that regulate its activity. By using a combination of bioinformatic and molecular approaches we identified, cloned, and characterized 3 novel alternative splice isoforms derived from human dyskeratosis congenita 1 (hDKC1), an essential human gene causative of the X-linked dyskeratosis congenita disease and involved in multiple functions related to cell growth, proliferation, and telomere maintenance. Expression of the new isoforms, all characterized by intron retention, was confirmed by RT-PCR in a panel of diverse cell lines and normal human tissues, and despite the presence of premature termination codons, was not down-regulated by the mechanism of nonsense-mediated decay. Accumulation of these transcripts fluctuated distinctly in the diverse tissues and during in vitro differentiation of Caco2 cells, suggesting that their ratio may contribute to the gene functional diversity across different cell types. Intriguingly, the structure of one isoform leads to exonize an intronically encoded small nucleolar RNA (snoRNA), highlighting an additional layer of complexity that can contribute to overall gene regulation.

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