pmad plasmids  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: RBM41 is a paralog of the U11/U12-65K protein. ( A ) Domain structures of human U11/U12-65K and RBM41 proteins. ( B ) Pairwise sequence alignment of RBM41 and U11/U12-65K. Local sequence alignment was carried out using Matcher and visualized using ESPript 3.0 . Identical residues are shown in white text with blue background and similar residues in blue text with white background. Protein secondary structure elements extracted from NMR structures (U11/U12-65K: 5OBN, RBM41: 2CPX) are shown below the alignment. ( C ) Structure of the U11/U12-65K C-terminal RRM (5OBN) showing identical and similar residues between RBM41 and U11/U12-65K. ( D ) AlphaFold-predicted structure of human RBM41 colored for sequence conservation. Conservation is based on a multiple sequence alignment of RBM41 orthologues from 15 animal species . Conservation was mapped to the structure with ESPript 3.0 and structure rendered using PyMOL. ( E ) Phylogenetic profile of RBM41 compared to the known minor spliceosome-specific proteins and minor and major spliceosomal snRNAs.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 C-terminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( ).

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: RBM41 interacts with the U12 and U6atac snRNAs in vitro . ( A ) Consensus RNA motifs bound by RBM41 in vitro and matching sequences in the U12 and U6atac snRNAs. The consensus motif (obtained from ENCODE database , entry ENCSR637HFY) determined by Ray et al. using the RNAcompete method is shown. ( B ) RNA hairpins used in EMSA experiments and their location in the U12 and U6atac snRNAs. ( C ) EMSA analysis of RBM41 and U11/U12-65K RRM binding to U12 (top panel) and U6atac snRNA (bottom panel) hairpins. EMSA was carried out using recombinant RBM41 RRM (residues 267–413) or 65K C-terminal RRM (residues 380–517) and 32 P-labeled U12, U6atac or negative control RNA hairpins shown in panel B. ( D ) Binding curves and dissociation constants for the interaction of RBM41 and 65K RRMs with U12 and U6atac hairpins. The inset shows a low protein concentration range (0–10 μM) of the same binding curves.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 C-terminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( ).

Techniques: In Vitro, Binding Assay, Recombinant, Labeling, Negative Control, Protein Concentration

Journal: Nucleic Acids Research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: RBM41 specifically associates with minor spliceosomal snRNPs. ( A ) RNA immunoprecipitation with V5-tagged RBM41 and 65K. V5-RBM41 or V5-65K expression vector or empty vector were transfected into HEK293 cells. 24 h later, RNA immunoprecipitation with anti-V5 antibody or control antibody was carried out in native conditions and co-immunoprecipitated RNA analyzed by northern blot using the indicated probes. ( B ) RNA immunoprecipitation with endogenous RBM41. RIP was carried out in native conditions in either HeLa nuclear extract (left) or HEK293 total lysate (right) using an antibody against endogenous RBM41 or control antibody. ( C ) V5-RBM41 constructs used for RNA immunoprecipitation in panel D. ( D ) Effect of truncations and RRM mutations on the snRNP association of RBM41.V5-tagged RBM41 constructs shown in C were transfected into HEK293 cells and RNA immunoprecipitation carried out using anti-V5 or control antibody.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 C-terminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( ).

Techniques: RNA Immunoprecipitation, Expressing, Plasmid Preparation, Transfection, Control, Immunoprecipitation, Northern Blot, Construct

Journal: Nucleic Acids Research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: RBM41 and U11/U12-65K partition into distinct snRNP complexes. ( A ) Glycerol gradient analysis of RBM41 and U11/U12-65K in HeLa nuclear extract. Nuclear extract was loaded on top of a 10–30% glycerol gradient. After ultracentrifugation, the gradient was fractionated, protein and RNA isolated and analyzed by western and northern blot using the antibodies and probes indicated on the left. Location of the U11, U12 and U6atac mono-snRNPs, U11/U12 di-snRNP and U4atac/U6atac di-snRNP are inferred based on the snRNA profiles. ( B ) Domain structures of MAC-tagged RBM41 and 65K constructs used for BioID. N-terminal MAC tag is not drawn to scale. ( C ) Spectral count fold changes for U11/U12 di-snRNP proteins in BioID datasets. ( D ) Immunoprecipitation of U11 and U12 snRNAs by anti-31K, anti-48K, anti-59K, anti-65K and anti-Sm antibodies in HEK293 total lysate followed by Northern blot detection of the U11 and U12 snRNAs.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 C-terminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( ).

Techniques: Isolation, Western Blot, Northern Blot, Construct, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: RBM41 interacts with DHX8 and localizes to Cajal bodies. ( A ) Spectral counts for DHX8 in RBM41 and U11/U12-65K BioID datasets. ( B ) Immunoprecipitation with anti-V5 or control antibody followed by western blot in Flp-In™ T-REx™ 293 cell lines expressing V5-RBM41 or V5-65K. The asterisk indicates a non-specific band detected in both control and anti-31K IPs and likely represents cross-reaction of the anti-rabbit secondary antibody with light chain from the IP antibody. ( C ) RNA immunoprecipitation with exogenously expressed V5-tagged proteins followed by RT-PCR. The indicated pCI-neo constructs for expressing V5-tagged proteins or empty pCI-neo vectors were transfected into HEK293 cells. 24 h later, RIP was carried out using anti-V5 antibody and RNA extracted from the beads analyzed by RT-PCR. Amplification across the branch junction was used to detect U2- and U12-type intron lariats and lariat intermediates from the following introns: SPCS2 introns 3–4 (U12) and 2–3 (U2), SUDS3 introns 7–8 (U12) and 9–10 (U2), WDR11 introns 28–29 (U12) and 27–28 (U2). ( D ) RNA immunoprecipitation with endogenous RBM41 in HEK293 cells followed by RT-PCR. ( E ) Spectral counts for coilin in RBM41 and U11/U12-65K BioID datasets. ( F ) Anti-RBM41 immunofluorescence in HEK293 cells transfected with a vector for expressing coilin-GFP.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 C-terminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( ).

Techniques: Immunoprecipitation, Control, Western Blot, Expressing, RNA Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Construct, Transfection, Amplification, Immunofluorescence, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: RBM41 knockout influences the splicing of U12-type introns. ( A ) Western blot analysis of RBM41 knockout and matching control cell lines used in the RNAseq analysis. ( B ) Comparison of the statistically significant (Whippet Probability > 0.9) alternative splicing events in the genes containing only U2-type introns and events either within or near proximity (immediate up- or downstream exons and introns) of the U12-type introns. AA - alternative acceptor, AD – alternative donor, CE – core exon. ( C ) Representative sashimi plots showing Intron retention ( NOL11 ), Alternative U12-type 3′ss choice ( THOC2 ) and loss of both exon skipping and alternative U12-type 3′ss usage in RBM41 knockout cells ( TCTN1 ). The percentages refer to the intron retention levels ( NOL11 ), the alternative 3′ splice usage levels ( THOC2 ) or exon skipping levels ( TCTN1 ) as indicated by the arches in the Sashimi plot. ( D ) Validation of the THOC2 and TCTN1 alternative splicing changes using a set of three independent RBM41 knockout cell lines and their matching controls.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 C-terminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( ).

Techniques: Knock-Out, Western Blot, Control, Comparison, Alternative Splicing, Biomarker Discovery

Journal: Nucleic Acids Research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: A model of the dynamic exchanges between RBM41 and 65K binding to U12 snRNA during the splicing cycle. In the U11/U12 di-snRNP, the 3′-terminal stem-loop is bound by U11/U12-65K, which mediates the connection between the U11 and U12 snRNPs. U11/U12-65K likely remains bound to the stem-loop throughout minor spliceosome assembly and activation but is exchanged for RBM41 during or after the catalytic steps of splicing. After spliceosome disassembly, RBM41 remains bound to the post-spliceosomal U12 mono-snRNP and accompanies it to Cajal bodies. During U11/U12 di-snRNP recycling, which likely takes place in Cajal bodies, RBM41 is again replaced by U11/U12-65K at the 3′-terminal stem-loop.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 C-terminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( ).

Techniques: Binding Assay, Activation Assay

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 2. RBM41 interacts with the U12 and U6atac snRNAs in vitro . ( A ) Consensus RNA motifs bound by RBM41 in vitro and matching sequences in the U12 and U6atac snRNAs. The consensus motif (obtained from ENCODE database ( 81 ), entry ENCSR637HFY) determined by Ray et al. ( 58 ) using the RNAcompete method is shown. ( B ) RNA hairpins used in EMSA experiments and their location in the U12 and U6atac snRNAs. ( C ) EMSA analysis of RBM41 and U11 / U12-65K RRM binding to U12 (top panel) and U6atac snRNA (bottom panel) hairpins. EMSA was carried out using recombinant RBM41 RRM (residues 267–413) or 65K C-terminal RRM (residues 380–517) and 32 P-labeled U12, U6atac or negative control RNA hairpins shown in panel B. ( D ) Binding curves and dissociation constants for the interaction of RBM41 and 65K RRMs with U12 and U6atac hairpins. The inset shows a low protein concentration range (0–10 μM) of the same binding curves.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: In Vitro, Binding Assay, Recombinant, Labeling, Negative Control, Protein Concentration

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 3. RBM41 specifically associates with minor spliceosomal snRNPs. ( A ) RNA immunoprecipitation with V5-tagged RBM41 and 65K. V5-RBM41 or V5-65K expression vector or empty vector were transfected into HEK293 cells. 24 h later, RNA immunoprecipitation with anti-V5 antibody or control antibody was carried out in native conditions and co-immunoprecipitated RNA analyzed by northern blot using the indicated probes. ( B ) RNA immunoprecipitation with endogenous RBM41. RIP was carried out in native conditions in either HeLa nuclear extract (left) or HEK293 total lysate (right) using an antibody against endogenous RBM41 or control antibody. ( C ) V5-RBM41 constructs used for RNA immunoprecipitation in panel D. ( D ) Effect of truncations and RRM mutations on the snRNP association of RBM41.V5-tagged RBM41 constructs shown in C were transfected into HEK293 cells and RNA immunoprecipitation carried out using anti-V5 or control antibody.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: RNA Immunoprecipitation, Expressing, Plasmid Preparation, Transfection, Control, Immunoprecipitation, Northern Blot, Construct

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 4. RBM41 and U11 / U12-65K partition into distinct snRNP comple x es. ( A ) Glycerol gradient analysis of RBM41 and U11 / U12-65K in HeLa nuclear extract. Nuclear extract was loaded on top of a 10–30% glycerol gradient. After ultracentrifugation, the gradient was fractionated, protein and RNA isolated and analyzed by western and northern blot using the antibodies and probes indicated on the left. Location of the U11, U12 and U6atac mono-snRNPs, U11 / U12 di-snRNP and U4at ac / U6at ac di-snRNP are inferred based on the snRNA profiles. ( B ) Domain str uct ures of MAC-tagged RBM41 and 65K constructs used for BioID. N-terminal MAC tag is not drawn to scale. ( C ) Spectral count fold changes for U11 / U12 di-snRNP proteins in BioID datasets. ( D ) Immunoprecipitation of U11 and U12 snRNAs by anti-31K, anti-48K, anti-59K, anti-65K and anti-Sm antibodies in HEK293 total lysate f ollo w ed b y Northern blot detection of the U11 and U12 snRNAs.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: Isolation, Western Blot, Northern Blot, Construct, Immunoprecipitation

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 5. RBM41 interacts with DHX8 and localizes to Cajal bodies. ( A ) Spectral counts for DHX8 in RBM41 and U11 / U12-65K BioID datasets. ( B ) Immunoprecipitation with anti-V5 or control antibody f ollo w ed b y w estern blot in Flp-In™T-REx™293 cell lines e xpressing V5-RBM41 or V5-65K. T he asterisk indicates a non-specific band detected in both control and anti-31K IPs and likely represents cross-reaction of the anti-rabbit secondary antibody with light chain from the IP antibody. ( C ) RNA immunoprecipitation with e x ogenously e xpressed V5-tagged proteins f ollo w ed b y R T-PCR. T he indicated pCI-neo constructs for expressing V5-tagged proteins or empty pCI-neo vectors were transfected into HEK293 cells. 24 h later, RIP was carried out using anti-V5 antibody and RNA extracted from the beads analyzed by RT-PCR. Amplification across the branch junction was used to detect U2- and U12-type intron lariats and lariat intermediates from the f ollo wing introns: SPCS2 introns 3–4 (U12) and 2–3 (U2), SUDS3 introns 7–8 (U12) and 9–10 (U2), WDR11 introns 28–29 (U12) and 27–28 (U2). ( D ) RNA immunoprecipitation with endogenous RBM41 in HEK293 cells f ollo w ed b y R T-PCR. ( E ) Spectral counts for coilin in RBM41 and U11 / U12-65K BioID datasets. ( F ) Anti-RBM41 immunofluorescence in HEK293 cells transfected with a vector f or e xpressing coilin-GFP.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: Immunoprecipitation, Control, RNA Immunoprecipitation, Construct, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Immunofluorescence, Plasmid Preparation

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 6. RBM41 knockout influences the splicing of U12-type introns. ( A ) Western blot analysis of RBM41 knockout and matching control cell lines used in the RNAseq analysis. ( B ) Comparison of the statistically significant (Whippet Probability > 0.9) alternative splicing events in the genes containing only U2-type introns and e v ents either within or near proximity (immediate up- or downstream exons and introns) of the U12-type introns. AA - alternative acceptor, AD – alternative donor, CE – core e x on. ( C ) R epresentativ e sashimi plots showing Intron retention ( NOL11 ), Alternative U12-type 3 ′ ss choice ( THOC2 ) and loss of both exon skipping and alternative U12-type 3 ′ ss usage in RBM41 knockout cells ( TCTN1 ). The percentages refer to the intron retention le v els ( NOL11 ), the alternative 3 ′ splice usage le v els ( TH OC2 ) or e x on skipping le v els ( TCTN1 ) as indicated by the arches in the Sashimi plot. ( D ) Validation of the THOC2 and TCTN1 alternative splicing changes using a set of three independent RBM41 knockout cell lines and their matching controls.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: Knock-Out, Western Blot, Control, Comparison, Alternative Splicing, Biomarker Discovery