Multi-condition affinity capture/MS analysis of LAP-tagged human NCBP1, NCBP2, and NCBP3

A 5′, 7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and thus a textbook aspect of co-transcriptional pre-mRNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). Recently, NCBP3 has been proposed to form an alternative, non-canonical CBC, together with NCBP1. NCBP3 has also been shown to interact with the canonical CBC along with the protein SRRT (aka ARS2), in a manner that is mutually exclusive with the RNA export factor, PHAX. Taken together, ambiguities and missing information in the bona fide physiological protein-protein associations of NCBP3 persist. In an effort to clarify the compositions of NCBP1-, 2-, and 3-related macromolecular assemblies, including their intersections and differences, we have applied our recently developed interactome screening platform (PMID: 25938370). Here the experimental design and data processing have been modified and updated to identify interactome differences between targets of affinity capture under a wide range of experimental conditions, followed by label-free quantitative mass spectrometry

LFQMS of RING2 (RING1B, RNF2) immunoprecipitation from MDA-MB-231 and T47D breast cancer cells

Polycomb repressive complex PRC1 is essential for gene regulation in numerous cell fate decisions. We show that RING1B (RING2, RNF2) and canonical PRC1 (cPRC1) genes are amplified and overexpressed in breast cancer (BC). Moreover, cPRC1 complexes functionally associate with genes regulated by cell type specific key transcription factors such as estrogen receptor (ER) in ER+ tumor cells and BRD4 in triple negative BC cells. cPRC1 is recruited to active enhancers in a manner independent of PRC2 and RING1B enzymatic activity. RING1B regulates enhancer activity and gene transcription not only by promoting the expression of BC oncogenes but also by regulating chromatin accessibility for oncogenic transcription factors. RING1B recruitment, and thus PRC1 association, to active enhancers occurs in multiple cancers

LFQMS of RING2 (RING1B, RNF2) immunoprecipitation from MDA-MB-231 and T47D breast cancer cells

Polycomb repressive complex PRC1 is essential for gene regulation in numerous cell fate decisions. We show that RING1B (RING2, RNF2) and canonical PRC1 (cPRC1) genes are amplified and overexpressed in breast cancer (BC). Moreover, cPRC1 complexes functionally associate with genes regulated by cell type specific key transcription factors such as estrogen receptor (ER) in ER+ tumor cells and BRD4 in triple negative BC cells. cPRC1 is recruited to active enhancers in a manner independent of PRC2 and RING1B enzymatic activity. RING1B regulates enhancer activity and gene transcription not only by promoting the expression of BC oncogenes but also by regulating chromatin accessibility for oncogenic transcription factors. RING1B recruitment, and thus PRC1 association, to active enhancers occurs in multiple cancers

Multi-condition affinity capture/MS analysis of LAP-tagged human NCBP1, NCBP2, and NCBP3

A 5′, 7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and thus a textbook aspect of co-transcriptional pre-mRNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). Recently, NCBP3 has been proposed to form an alternative, non-canonical CBC, together with NCBP1. NCBP3 has also been shown to interact with the canonical CBC along with the protein SRRT (aka ARS2), in a manner that is mutually exclusive with the RNA export factor, PHAX. Taken together, ambiguities and missing information in the bona fide physiological protein-protein associations of NCBP3 persist. In an effort to clarify the compositions of NCBP1-, 2-, and 3-related macromolecular assemblies, including their intersections and differences, we have applied our recently developed interactome screening platform (PMID: 25938370). Here the experimental design and data processing have been modified and updated to identify interactome differences between targets of affinity capture under a wide range of experimental conditions, followed by label-free quantitative mass spectrometry

Colorectal cancer anti-ORF1p LFQ IP-MS

Here, three colorectal tumors were subjected to anti-ORF1p LFQ, IP-MS: (A) Krukenberg Carcinoma, Ovary; (B) Metastatic Rectal Adenocarcinoma, Liver; (C) Adenocarcinoma, Colon. These were accompanied by matched normal IP controls and/or mouse IgG IP controls. The objective is to map LINE-1 RNP interactions in cancer

Colorectal cancer anti-ORF1p LFQ IP-MS

Here, three colorectal tumors were subjected to anti-ORF1p LFQ, IP-MS: (A) Krukenberg Carcinoma, Ovary; (B) Metastatic Rectal Adenocarcinoma, Liver; (C) Adenocarcinoma, Colon. These were accompanied by matched normal IP controls and/or mouse IgG IP controls. The objective is to map LINE-1 RNP interactions in cancer

Multi-condition affinity capture/MS analysis of LAP-tagged human NCBP1, NCBP2, and NCBP3

A 5′, 7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and thus a textbook aspect of co-transcriptional pre-mRNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). Recently, NCBP3 has been proposed to form an alternative, non-canonical CBC, together with NCBP1. NCBP3 has also been shown to interact with the canonical CBC along with the protein SRRT (aka ARS2), in a manner that is mutually exclusive with the RNA export factor, PHAX. Taken together, ambiguities and missing information in the bona fide physiological protein-protein associations of NCBP3 persist. In an effort to clarify the compositions of NCBP1-, 2-, and 3-related macromolecular assemblies, including their intersections and differences, we have applied our recently developed interactome screening platform (PMID: 25938370). Here the experimental design and data processing have been modified and updated to identify interactome differences between targets of affinity capture under a wide range of experimental conditions, followed by label-free quantitative mass spectrometry

Colorectal cancer anti-ORF1p LFQ IP-MS

Here, three colorectal tumors were subjected to anti-ORF1p LFQ, IP-MS: (A) Krukenberg Carcinoma, Ovary; (B) Metastatic Rectal Adenocarcinoma, Liver; (C) Adenocarcinoma, Colon. These were accompanied by matched normal IP controls and/or mouse IgG IP controls. The objective is to map LINE-1 RNP interactions in cancer

LFQMS of RING2 (RING1B, RNF2) immunoprecipitation from MDA-MB-231 and T47D breast cancer cells

Polycomb repressive complex PRC1 is essential for gene regulation in numerous cell fate decisions. We show that RING1B (RING2, RNF2) and canonical PRC1 (cPRC1) genes are amplified and overexpressed in breast cancer (BC). Moreover, cPRC1 complexes functionally associate with genes regulated by cell type specific key transcription factors such as estrogen receptor (ER) in ER+ tumor cells and BRD4 in triple negative BC cells. cPRC1 is recruited to active enhancers in a manner independent of PRC2 and RING1B enzymatic activity. RING1B regulates enhancer activity and gene transcription not only by promoting the expression of BC oncogenes but also by regulating chromatin accessibility for oncogenic transcription factors. RING1B recruitment, and thus PRC1 association, to active enhancers occurs in multiple cancers

LFQMS of RING2 (RING1B, RNF2) immunoprecipitation from MDA-MB-231 and T47D breast cancer cells

Polycomb repressive complex PRC1 is essential for gene regulation in numerous cell fate decisions. We show that RING1B (RING2, RNF2) and canonical PRC1 (cPRC1) genes are amplified and overexpressed in breast cancer (BC). Moreover, cPRC1 complexes functionally associate with genes regulated by cell type specific key transcription factors such as estrogen receptor (ER) in ER+ tumor cells and BRD4 in triple negative BC cells. cPRC1 is recruited to active enhancers in a manner independent of PRC2 and RING1B enzymatic activity. RING1B regulates enhancer activity and gene transcription not only by promoting the expression of BC oncogenes but also by regulating chromatin accessibility for oncogenic transcription factors. RING1B recruitment, and thus PRC1 association, to active enhancers occurs in multiple cancers