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Article ; Online: Revised iCLIP-seq Protocol for Profiling RNA-protein Interaction Sites at Individual Nucleotide Resolution in Living Cells.

Nabeel-Shah, Syed / Greenblatt, Jack F

2023 Volume 13, Issue 11, Page(s) e4688

Abstract: Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins' (RBP) binding sites on target RNAs and to characterize the ...

Abstract	Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins' (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain
Language	English
Publishing date	2023-06-05
Publishing country	United States
Document type	Journal Article
ZDB-ID	2833269-6
ISSN	2331-8325 ; 2331-8325
ISSN (online)	2331-8325
ISSN	2331-8325
DOI	10.21769/BioProtoc.4688
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Article ; Online: Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.

Ni, Zuyao / Ahmed, Nujhat / Nabeel-Shah, Syed / Guo, Xinghua / Pu, Shuye / Song, Jingwen / Marcon, Edyta / Burke, Giovanni L / Tong, Amy Hin Yan / Chan, Katherine / Ha, Kevin C H / Blencowe, Benjamin J / Moffat, Jason / Greenblatt, Jack F

Nucleic acids research

2024

Abstract: Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA ... ...

Abstract	Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.
Language	English
Publishing date	2024-04-08
Publishing country	England
Document type	Journal Article
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkae240
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Article ; Online: RACS: rapid analysis of ChIP-Seq data for contig based genomes.

Saettone, Alejandro / Ponce, Marcelo / Nabeel-Shah, Syed / Fillingham, Jeffrey

BMC bioinformatics

2019 Volume 20, Issue 1, Page(s) 533

Abstract: Background: Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-Seq) is a widely-used molecular method to investigate the function of chromatin-related proteins by identifying their associated DNA sequences on a genomic scale. ChIP- ...

Abstract	Background: Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-Seq) is a widely-used molecular method to investigate the function of chromatin-related proteins by identifying their associated DNA sequences on a genomic scale. ChIP-Seq generates large quantities of data that is difficult to process and analyze, particularly for organisms with a contig-based sequenced genomes that typically have minimal annotation on their associated set of genes other than their associated coordinates primarily predicted by gene finding programs. Poorly annotated genome sequence makes comprehensive analysis of ChIP-Seq data difficult and as such standardized analysis pipelines are lacking. Results: We present a one-stop computational pipeline, "Rapid Analysis of ChIP-Seq data" (RACS), that utilizes traditional High-Performance Computing (HPC) techniques in association with open source tools for processing and analyzing raw ChIP-Seq data. RACS is an open source computational pipeline available from any of the following repositories https://bitbucket.org/mjponce/RACS or https://gitrepos.scinet.utoronto.ca/public/?a=summary&p=RACS . RACS is particularly useful for ChIP-Seq in organisms with contig-based genomes that have poor gene annotation to aid protein function discovery.To test the performance and efficiency of RACS, we analyzed ChIP-Seq data previously published in a model organism Tetrahymena thermophila which has a contig-based genome. We assessed the generality of RACS by analyzing a previously published data set generated using the model organism Oxytricha trifallax, whose genome sequence is also contig-based with poor annotation. Conclusions: The RACS computational pipeline presented in this report is an efficient and reliable tool to analyze genome-wide raw ChIP-Seq data generated in model organisms with poorly annotated contig-based genome sequence. Because RACS segregates the found read accumulations between genic and intergenic regions, it is particularly efficient for rapid downstream analyses of proteins involved in gene expression.
MeSH term(s)	Chromatin Immunoprecipitation Sequencing ; Chromosome Mapping ; Genome ; Genomics/methods ; Humans ; Molecular Sequence Annotation ; Sequence Analysis, DNA
Language	English
Publishing date	2019-10-29
Publishing country	England
Document type	Journal Article
ZDB-ID	2041484-5
ISSN	1471-2105 ; 1471-2105
ISSN (online)	1471-2105
ISSN	1471-2105
DOI	10.1186/s12859-019-3100-2
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Article: Exploring the Histone Acetylation Cycle in the Protozoan Model

Wahab, Suzanne / Saettone, Alejandro / Nabeel-Shah, Syed / Dannah, Nora / Fillingham, Jeffrey

Frontiers in cell and developmental biology

2020 Volume 8, Page(s) 509

Abstract: The eukaryotic histone acetylation cycle is composed of three classes of proteins, histone acetyltransferases (HATs) that add acetyl groups to lysine amino acids, bromodomain (BRD) containing proteins that are one of the most characterized of several ... ...

Abstract	The eukaryotic histone acetylation cycle is composed of three classes of proteins, histone acetyltransferases (HATs) that add acetyl groups to lysine amino acids, bromodomain (BRD) containing proteins that are one of the most characterized of several protein domains that recognize acetyl-lysine (Kac) and effect downstream function, and histone deacetylases (HDACs) that catalyze the reverse reaction. Dysfunction of selected proteins of these three classes is associated with human disease such as cancer. Additionally, the HATs, BRDs, and HDACs of fungi and parasitic protozoa present potential drug targets. Despite their importance, the function and mechanisms of HATs, BRDs, and HDACs and how they relate to chromatin remodeling (CR) remain incompletely understood.
Language	English
Publishing date	2020-06-30
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2737824-X
ISSN	2296-634X
ISSN	2296-634X
DOI	10.3389/fcell.2020.00509
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Article ; Online: Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation.

Nabeel-Shah, Syed / Garg, Jyoti / Ashraf, Kanwal / Jeyapala, Renu / Lee, Hyunmin / Petrova, Alexandra / Burns, James D / Pu, Shuye / Zhang, Zhaolei / Greenblatt, Jack F / Pearlman, Ronald E / Lambert, Jean-Philippe / Fillingham, Jeffrey

Epigenetics & chromatin

2023 Volume 16, Issue 1, Page(s) 10

Abstract: Background: Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. ... ...

Abstract	Background: Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts. Results: We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes. Conclusion: Our results provide an evolutionary perspective on H3.3's conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.
MeSH term(s)	Histones/genetics ; Histones/metabolism ; Gene Expression Regulation ; Chromatin/genetics ; Chromatin/metabolism ; Transcription, Genetic ; Cell Nucleus/metabolism
Chemical Substances	Histones ; Chromatin
Language	English
Publishing date	2023-04-07
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2462129-8
ISSN	1756-8935 ; 1756-8935
ISSN (online)	1756-8935
ISSN	1756-8935
DOI	10.1186/s13072-023-00484-9
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Article ; Online: Functional proteomics protocol for the identification of interaction partners in

Nabeel-Shah, Syed / Garg, Jyoti / Kougnassoukou Tchara, Pata-Eting / Pearlman, Ronald E / Lambert, Jean-Philippe / Fillingham, Jeffrey

STAR protocols

2021 Volume 2, Issue 1, Page(s) 100362

Abstract: We describe an optimized protocol for one-step affinity purification of FZZ-tagged proteins followed by mass spectrometry analysis for the identification of protein-protein interactions in the ciliate ... ...

Abstract	We describe an optimized protocol for one-step affinity purification of FZZ-tagged proteins followed by mass spectrometry analysis for the identification of protein-protein interactions in the ciliate protozoan
MeSH term(s)	Proteomics ; Protozoan Proteins/metabolism ; Tetrahymena thermophila/metabolism
Chemical Substances	Protozoan Proteins
Language	English
Publishing date	2021-03-04
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2021.100362
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Article: Functional Proteomics of Nuclear Proteins in

Saettone, Alejandro / Nabeel-Shah, Syed / Garg, Jyoti / Lambert, Jean-Philippe / Pearlman, Ronald E / Fillingham, Jeffrey

Genes

2019 Volume 10, Issue 5

Abstract: Identification and characterization of protein complexes and interactomes has been essential to the understanding of fundamental nuclear processes including transcription, replication, recombination, and maintenance of genome stability. Despite ... ...

Abstract	Identification and characterization of protein complexes and interactomes has been essential to the understanding of fundamental nuclear processes including transcription, replication, recombination, and maintenance of genome stability. Despite significant progress in elucidation of nuclear proteomes and interactomes of organisms such as yeast and mammalian systems, progress in other models has lagged. Protists, including the alveolate ciliate protozoa with
MeSH term(s)	Cell Nucleus/genetics ; Cell Nucleus/parasitology ; Chromatin/genetics ; Chromatin/parasitology ; Ciliophora Infections/genetics ; Ciliophora Infections/parasitology ; Cytoplasm/genetics ; Cytoplasm/parasitology ; Humans ; Nuclear Proteins/genetics ; Proteomics ; Tetrahymena thermophila/genetics ; Tetrahymena thermophila/pathogenicity
Chemical Substances	Chromatin ; Nuclear Proteins
Language	English
Publishing date	2019-05-01
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2527218-4
ISSN	2073-4425
ISSN	2073-4425
DOI	10.3390/genes10050333
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Article ; Online: A central chaperone-like role for 14-3-3 proteins in human cells.

Segal, Dmitri / Maier, Stefan / Mastromarco, Giovanni J / Qian, Wesley Wei / Nabeel-Shah, Syed / Lee, Hyunmin / Moore, Gaelen / Lacoste, Jessica / Larsen, Brett / Lin, Zhen-Yuan / Selvabaskaran, Abeeshan / Liu, Karen / Smibert, Craig / Zhang, Zhaolei / Greenblatt, Jack / Peng, Jian / Lee, Hyun O / Gingras, Anne-Claude / Taipale, Mikko

Molecular cell

2023 Volume 83, Issue 6, Page(s) 974–993.e15

Abstract: 14-3-3 proteins are highly conserved regulatory proteins that interact with hundreds of structurally diverse clients and act as central hubs of signaling networks. However, how 14-3-3 paralogs differ in specificity and how they regulate client protein ... ...

Abstract	14-3-3 proteins are highly conserved regulatory proteins that interact with hundreds of structurally diverse clients and act as central hubs of signaling networks. However, how 14-3-3 paralogs differ in specificity and how they regulate client protein function are not known for most clients. Here, we map the interactomes of all human 14-3-3 paralogs and systematically characterize the effect of disrupting these interactions on client localization. The loss of 14-3-3 binding leads to the coalescence of a large fraction of clients into discrete foci in a client-specific manner, suggesting a central chaperone-like function for 14-3-3 proteins. Congruently, the engraftment of 14-3-3 binding motifs to nonclients can suppress their aggregation or phase separation. Finally, we show that 14-3-3s negatively regulate the localization of the RNA-binding protein SAMD4A to cytoplasmic granules and inhibit its activity as a translational repressor. Our work suggests that 14-3-3s have a more prominent role as chaperone-like molecules than previously thought.
MeSH term(s)	Humans ; 14-3-3 Proteins/genetics ; 14-3-3 Proteins/metabolism ; HSP90 Heat-Shock Proteins/metabolism ; Molecular Chaperones/metabolism ; Protein Binding
Chemical Substances	14-3-3 Proteins ; HSP90 Heat-Shock Proteins ; Molecular Chaperones
Language	English
Publishing date	2023-03-14
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1415236-8
ISSN	1097-4164 ; 1097-2765
ISSN (online)	1097-4164
ISSN	1097-2765
DOI	10.1016/j.molcel.2023.02.018
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Article ; Online: RNA-binding proteins that lack canonical RNA-binding domains are rarely sequence-specific.

Ray, Debashish / Laverty, Kaitlin U / Jolma, Arttu / Nie, Kate / Samson, Reuben / Pour, Sara E / Tam, Cyrus L / von Krosigk, Niklas / Nabeel-Shah, Syed / Albu, Mihai / Zheng, Hong / Perron, Gabrielle / Lee, Hyunmin / Najafabadi, Hamed / Blencowe, Benjamin / Greenblatt, Jack / Morris, Quaid / Hughes, Timothy R

Scientific reports

2023 Volume 13, Issue 1, Page(s) 5238

Abstract: Thousands of RNA-binding proteins (RBPs) crosslink to cellular mRNA. Among these are numerous unconventional RBPs (ucRBPs)-proteins that associate with RNA but lack known RNA-binding domains (RBDs). The vast majority of ucRBPs have uncharacterized RNA- ... ...

Abstract	Thousands of RNA-binding proteins (RBPs) crosslink to cellular mRNA. Among these are numerous unconventional RBPs (ucRBPs)-proteins that associate with RNA but lack known RNA-binding domains (RBDs). The vast majority of ucRBPs have uncharacterized RNA-binding specificities. We analyzed 492 human ucRBPs for intrinsic RNA-binding in vitro and identified 23 that bind specific RNA sequences. Most (17/23), including 8 ribosomal proteins, were previously associated with RNA-related function. We identified the RBDs responsible for sequence-specific RNA-binding for several of these 23 ucRBPs and surveyed whether corresponding domains from homologous proteins also display RNA sequence specificity. CCHC-zf domains from seven human proteins recognized specific RNA motifs, indicating that this is a major class of RBD. For Nudix, HABP4, TPR, RanBP2-zf, and L7Ae domains, however, only isolated members or closely related homologs yielded motifs, consistent with RNA-binding as a derived function. The lack of sequence specificity for most ucRBPs is striking, and we suggest that many may function analogously to chromatin factors, which often crosslink efficiently to cellular DNA, presumably via indirect recruitment. Finally, we show that ucRBPs tend to be highly abundant proteins and suggest their identification in RNA interactome capture studies could also result from weak nonspecific interactions with RNA.
MeSH term(s)	Humans ; RNA-Binding Proteins/genetics ; RNA-Binding Proteins/metabolism ; RNA/metabolism ; Ribosomal Proteins/metabolism ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA-Binding Motifs/genetics ; Protein Binding ; Myogenic Regulatory Factors/metabolism
Chemical Substances	RNA-Binding Proteins ; RNA (63231-63-0) ; Ribosomal Proteins ; RNA, Messenger ; HABP4 protein, human ; Myogenic Regulatory Factors
Language	English
Publishing date	2023-03-31
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-023-32245-9
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Article ; Online: RACS

Alejandro Saettone / Marcelo Ponce / Syed Nabeel-Shah / Jeffrey Fillingham

BMC Bioinformatics, Vol 20, Iss 1, Pp 1-

rapid analysis of ChIP-Seq data for contig based genomes

2019 Volume 17

Abstract: Abstract Background Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-Seq) is a widely-used molecular method to investigate the function of chromatin-related proteins by identifying their associated DNA sequences on a genomic ... ...

Abstract	Abstract Background Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-Seq) is a widely-used molecular method to investigate the function of chromatin-related proteins by identifying their associated DNA sequences on a genomic scale. ChIP-Seq generates large quantities of data that is difficult to process and analyze, particularly for organisms with a contig-based sequenced genomes that typically have minimal annotation on their associated set of genes other than their associated coordinates primarily predicted by gene finding programs. Poorly annotated genome sequence makes comprehensive analysis of ChIP-Seq data difficult and as such standardized analysis pipelines are lacking. Results We present a one-stop computational pipeline, “Rapid Analysis of ChIP-Seq data” (RACS), that utilizes traditional High-Performance Computing (HPC) techniques in association with open source tools for processing and analyzing raw ChIP-Seq data. RACS is an open source computational pipeline available from any of the following repositories https://bitbucket.org/mjponce/RACS or https://gitrepos.scinet.utoronto.ca/public/?a=summary&p=RACS. RACS is particularly useful for ChIP-Seq in organisms with contig-based genomes that have poor gene annotation to aid protein function discovery.To test the performance and efficiency of RACS, we analyzed ChIP-Seq data previously published in a model organism Tetrahymena thermophila which has a contig-based genome. We assessed the generality of RACS by analyzing a previously published data set generated using the model organism Oxytricha trifallax, whose genome sequence is also contig-based with poor annotation. Conclusions The RACS computational pipeline presented in this report is an efficient and reliable tool to analyze genome-wide raw ChIP-Seq data generated in model organisms with poorly annotated contig-based genome sequence. Because RACS segregates the found read accumulations between genic and intergenic regions, it is particularly efficient for rapid downstream ...
Keywords	Chromatin immunoprecipitation ; Next generation sequencing ; Bioinformatics pipeline ; High-performance computing ; Tetrahymena thermophila ; Computer applications to medicine. Medical informatics ; R858-859.7 ; Biology (General) ; QH301-705.5
Subject code	000
Language	English
Publishing date	2019-10-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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