scholarly journals Characterization of Transcription Termination-Associated RNAs: New Insights into their Biogenesis, Tailing, and Expression in Primary Tumors

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Ilaria Laudadio ◽  
Sara Formichetti ◽  
Silvia Gioiosa ◽  
Filippos Klironomos ◽  
Nikolaus Rajewsky ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Transcription Termination ◽  
Dna Integrity ◽  
Transcription Start ◽  
Cycle Progression ◽  
Primary Tumors ◽  
Transcription Start Sites ◽  
Generation Sequencing ◽  
Comprehensive Study

Next-generation sequencing has uncovered novel classes of small RNAs (sRNAs) in eukaryotes, in addition to the well-known miRNAs, siRNAs, and piRNAs. In particular, sRNA species arise from transcription start sites (TSSs) and the transcription termination sites (TTSs) of genes. However, a detailed characterization of these new classes of sRNAs is still lacking. Here, we present a comprehensive study of sRNAs derived from TTSs of expressed genes (TTSa-RNAs) in human cell lines and primary tissues. Taking advantage of sRNA-sequencing, we show that TTSa-RNAs are present in the nuclei of human cells, are loaded onto both AGO1 and AGO2, and their biogenesis does not require DICER and AGO2 endonucleolytic activity. TTSa-RNAs display a strong bias against a G residue in the first position at 5′ end, a known feature of AGO-bound sRNAs, and a peculiar oligoA tail at 3′ end. AGO-bound TTSa-RNAs derive from genes involved in cell cycle progression regulation and DNA integrity checkpoints. Finally, we provide evidence that TTSa-RNAs can be detected by sRNA-Seq in primary human tissue, and their expression increases in tumor samples as compared to nontumor tissues, suggesting that in the future, TTSa-RNAs might be explored as biomarker for diagnosis or prognosis of human malignancies.

scholarly journals Nep1, a Schizosaccharomyces pombe deneddylating enzyme

2005 ◽  
Vol 389 (2) ◽  
pp. 307-314 ◽  
Author(s):  
Lihong Zhou ◽  
Felicity Z. Watts
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Cell Cycle Progression ◽  
Dna Integrity ◽  
Main Function ◽  
Cycle Progression ◽  
D Cells ◽  
Modified Forms

Nedd8 is a ubiquitin-like modifier that is attached to the cullin components of E3 ubiquitin ligases. More recently, p53 has also been shown to be Nedd8-modified. Nedd8 attachment occurs in a manner similar to that observed for other ubiquitin-like modifiers. In the present study, we report on the characterization of Nep1, a deneddylating enzyme in fission yeast (Schizosaccharomyces pombe). Unlike loss of ned8, deletion of the nep1 gene is not lethal, although nep1.d cells are heterogeneous in length, suggesting a defect in cell-cycle progression. Viability of nep1.d cells is dependent on a functional spindle checkpoint but not on the DNA integrity checkpoint. Deletion of a related gene (nep2), either alone or in combination with nep1.d, also has little effect on cell viability. We show that Nep1 can deneddylate the Pcu1, Pcu3 and Pcu4 cullins in vitro and that its activity is sensitive to N-ethylmaleimide, consistent with the idea that it is a member of the cysteine protease family. nep1.d cells accumulate Nedd8-modified proteins, although these do not correspond to modified forms of the cullins, suggesting that, although Nep1 can deneddylate cullins in vitro, this is not its main function in vivo. Nep1 can be co-precipitated with the signalosome subunit Csn5. Nep1 itself is present in a high-molecular-mass complex, but the presence of this complex is not dependent on the production of intact signalosomes. Our results suggest that, in vivo, Nep1 may be responsible for deneddylating proteins other than cullins.

Genomic organization of the 5′ end of human β-ENaC and preliminary characterization of its promoter

2002 ◽  
Vol 282 (5) ◽  
pp. F898-F909 ◽  
Author(s):  
Christie P. Thomas ◽  
Randy W. Loftus ◽  
Kang Z. Liu ◽  
Omar A. Itani
Keyword(s):  
Genomic Organization ◽  
Human Kidney ◽  
Β Subunit ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Nuclear Extracts ◽  
Flanking Region ◽  
Rapid Amplification ◽  
Flanking Regions

The mRNA for the β-subunit of the epithelial Na+ channel (β-ENaC) is regulated developmentally and, in some tissues, in response to corticosteroids. To understand the mechanisms of transcriptional regulation of the human β-ENaC gene, we characterized the 5′ end of the gene and its 5′-flanking regions. Adaptor-ligated human kidney and lung cDNA were amplified by 5′ rapid amplification of cDNA ends, and transcription start sites of two 5′ variant transcripts were determined by nuclease protection or primer extension assays. Cosmid clones that contain the 5′ end of the gene were isolated, and analysis of these clones indicated that alternate first exons ∼1.5 kb apart and ∼ 45 kb upstream of a common second exon formed the basis of these transcripts. Genomic fragments that included the proximal 5′-flanking region of either transcript were able to direct expression of a reporter gene in lung epithelia and to bind Sp1 in nuclear extracts, confirming the presence of separate promoters that regulate β-ENaC expression.

scholarly journals The CWI Pathway: A Versatile Toolbox to Arrest Cell-Cycle Progression

2021 ◽  
Vol 7 (12) ◽  
pp. 1041
Author(s):  
Inma Quilis ◽  
Mercè Gomar-Alba ◽  
Juan Carlos Igual
Keyword(s):  
Cell Cycle ◽  
Cell Wall ◽  
Cellular Response ◽  
Cell Cycle Progression ◽  
Genetic Material ◽  
Regulatory System ◽  
Genotoxic Stress ◽  
Dna Integrity ◽  
Cycle Progression ◽  
Reciprocal Regulation

Cell-signaling pathways are essential for cells to respond and adapt to changes in their environmental conditions. The cell-wall integrity (CWI) pathway of Saccharomyces cerevisiae is activated by environmental stresses, compounds, and morphogenetic processes that compromise the cell wall, orchestrating the appropriate cellular response to cope with these adverse conditions. During cell-cycle progression, the CWI pathway is activated in periods of polarized growth, such as budding or cytokinesis, regulating cell-wall biosynthesis and the actin cytoskeleton. Importantly, accumulated evidence has indicated a reciprocal regulation of the cell-cycle regulatory system by the CWI pathway. In this paper, we describe how the CWI pathway regulates the main cell-cycle transitions in response to cell-surface perturbance to delay cell-cycle progression. In particular, it affects the Start transcriptional program and the initiation of DNA replication at the G1/S transition, and entry and progression through mitosis. We also describe the involvement of the CWI pathway in the response to genotoxic stress and its connection with the DNA integrity checkpoint, the mechanism that ensures the correct transmission of genetic material and cell survival. Thus, the CWI pathway emerges as a master brake that stops cell-cycle progression when cells are coping with distinct unfavorable conditions.

Characterization of the number and site of APC mutations in sporadic colorectal cancer.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 630-630 ◽  
Author(s):  
Ranjit Joseph ◽  
Paul Little ◽  
David N. Hayes ◽  
Michael Sangmin Lee
Keyword(s):  
Next Generation Sequencing ◽  
Apc Gene ◽  
Beta Catenin ◽  
Single Mutation ◽  
Next Generation ◽  
Primary Tumors ◽  
Apc Mutations ◽  
Apc Mutation ◽  
Generation Sequencing

630 Background: Truncating mutations in the adenomatous polyposis coli ( APC) gene are well-described events in the carcinogenesis of colorectal carcinomas (CRC) and may impact one or both APC alleles. These aberrations often fall within the mutation cluster region (MCR) of the APC gene to preserve a “just right” number of beta-catenin binding sites in the resulting mutant APC protein. Further clinical and genotypic characterization of CRCs based on number and site of mutations in APC determined using modern next generation sequencing methods is needed. Methods: Next generation sequencing of 70 CRC tumors was performed at a single institution via UNCseq to determine mutations in a panel of 247 oncogenes and tumor suppressors, including APC. RNASeq, DNA sequencing, and clinical characteristics from 224 colon and rectal cancer samples in The Cancer Genome Atlas (TCGA) project were also obtained. Results: In the UNCseq cohort, 58 patients (83%) had at least one inactivating APC mutation, and 33 (47%) had two mutations. Of those with at least one mutation, 81% had a mutation in the MCR (residues 1281-1556), but only 5/33 (15%) with two mutations had both in the MCR. In the TCGA cohort, 162 (72%) had at least one inactivating APC mutation, and 52 (23%) had two mutations. Of those with at least one mutation, 59% had a mutation in the MCR, but only 3/52 (6%) with two mutations had both in the MCR. Gene expression of APC was higher in those with no APC mutations vs. 1-2 mutations (p = 0.015), but was not significantly different between those with one vs. two mutations (p = 0.29). The absence of APC mutations was associated with microsatellite instability (p < 0.001) and with right-sided primary tumors (p = 0.001 by chi-square). Conclusions: CRCs have high frequency of biallelic APC mutations, and the majority of tumors with APC mutations had a single mutation within the MCR region of the APC gene. These genotypic factors may impact tumor biology and clinical features.

Abstract LB-159: Characterization of cell-cycle progression using a novel bi-cistronic lentiviral FUCCI reporter

2014 ◽  
Author(s):  
Gabriel Pineda ◽  
Florence Lambert-Fliszar ◽  
Gennarina L. Riso ◽  
Kathleen M. Kane ◽  
Catriona Jamieson
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cycle Progression

Digital Restriction Enzyme Analysis of Methylation (DREAM) by Next Generation Sequencing Yields High Resolution Maps of DNA Methylation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 567-567
Author(s):  
Jaroslav Jelinek ◽  
Shoudan Liang ◽  
Marcos R. H. Estecio ◽  
Rong He ◽  
Yue Lu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
High Resolution ◽  
Restriction Site ◽  
Cpg Islands ◽  
Normal Blood ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Generation Sequencing

Abstract Abstract 567 Methylation of CpG dinucleotides in DNA is a key epigenetic feature important for × chromosome inactivation, silencing of retrotransposons and genomic imprinting. DNA methylation undergoes complex changes in leukemia, most notably methylation of CpG islands at promoters and associated gene silencing. The direct comparison of epigenomes in normal and neoplastic blood cells will likely increase our understanding of the complex pathology of leukemia. We have developed a digital restriction enzyme analysis of methylation (DREAM) for quantitative mapping of DNA methylation with high resolution on the genome-wide scale. To perform the analysis, genomic DNA is sequentially digested with a pair of enzymes recognizing the same restriction site (CCCGGG) containing a CpG dinucleotide. The first enzyme, SmaI, cuts only at unmethylated CpG and leaves blunt ends. The second enzyme, XmaI, is not blocked by methylation and leaves a short 5' overhang. The enzymes thus create methylation-specific signatures at ends of digested DNA fragments. These are deciphered by next generation sequencing. Methylation levels for each sequenced restriction site are calculated based on the numbers of DNA molecules with the methylated or unmethylated signatures. Using the DREAM method and sequencing on the Illumina Gene Analyzer II platform, we analyzed DNA methylation in a normal adult blood sample. We acquired 32.5 million sequence tags; of these, 16.6 million were mapped to SmaI/XmaI sites unique in the human genome. With a threshold of minimum 5-fold coverage, we obtained quantitative information on the DNA methylation level of 85,171 CpG sites (23% of all genomic SmaI/XmaI sites) in 21,240 genes. The accuracy of DREAM methylation data was validated by a strong correlation with the bisulfite pyrosequencing analysis of 49 genes (R=0.83) and of spiked in plasmid DNA. In normal blood, methylation was strikingly bimodal with 39% sites showing methylation levels below 5% and 28% sites being hypermethylated at levels >95%. Methylation was largely absent within CpG islands (CGI) and more prevalent outside (non-CGI). Close to transcription start sites (within 500 bp), methylation >75% was found only in 0.65% of CGIs compared to 14% in non-CGIs (P<0.001). The methylated CGI promoters were significantly enriched for genes expressed in spermatogenesis and likely correspond to a class of potential cancer-testis antigens previously identified. Away from transcription start sites (>2 kb), methylation >75% was found in 24% of CGIs compared to 72% of non-CGIs (P<0.001). Transcription end regions were methylated in 20% in CGIs compared to 68% in non-CGIs (P<0.001). Also, we observed that 1.4% of CGIs had evidence of half methylation (35-65%), representing potentially imprinted genes. Indeed, this class includes known imprinted regions at chromosomes 8q24.3 and 11p15. Finally, we compared non-CGI promoters showing significant methylation to those free of methylation. Unmethylated promoters were more likely to be expressed in normal blood, and to encode for genes involved in metabolic processes and their regulation. In conclusion, high resolution quantitative methylation analysis is feasible using the DREAM method, and reveals important classes of genes based on methylation in normal blood. Disclosures: No relevant conflicts of interest to declare.

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