Journal: bioRxiv

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

doi: 10.1101/2023.10.12.562036

Figure Lengend Snippet: (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 ( Robert et al ., 2014 ). 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, a gift from Markku Varjosalo) using Gateway cloning as described ( Liu et al ., 2020 ).

Techniques: Sequencing

Journal: bioRxiv

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

doi: 10.1101/2023.10.12.562036

Figure Lengend Snippet: Multiple sequence alignment of human RBM41, U11/U12-65K, U1A, U2B″ and Drosophila melanogaster SNF (sans fille) RNA recognition motifs. Alignment was carried out using MAFFT and visualized using ESPript 3.0. Protein secondary structure information was extracted from the following PDB structures: 6F4I, 1NU4, 1A9N, 2CPX, 5OBN. The conserved YQF triad residues are indicated with blue stars.

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, a gift from Markku Varjosalo) using Gateway cloning as described ( Liu et al ., 2020 ).

Techniques: Sequencing

Journal: bioRxiv

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

doi: 10.1101/2023.10.12.562036

Figure Lengend Snippet: Multiple sequence alignment of RBM41 orthologs from 15 animal species. Alignment was carried out using MAFFT and visualized using ESPript 3.0. Secondary structures extracted from AlphaFold-predicted human RBM41 structure are shown. Sequences used for the alignment are listed in Supplementary Table 2.

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, a gift from Markku Varjosalo) using Gateway cloning as described ( Liu et al ., 2020 ).

Techniques: Sequencing

Journal: bioRxiv

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

doi: 10.1101/2023.10.12.562036

Figure Lengend Snippet: (A) Consensus RNA motifs bound by RBM41 in vitro and matching sequences in the U12 and U6atac snRNAs. The consensus motif determined by Ray et al. ( Ray et al ., 2013 ) 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, a gift from Markku Varjosalo) using Gateway cloning as described ( Liu et al ., 2020 ).

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

Journal: bioRxiv

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

doi: 10.1101/2023.10.12.562036

Figure Lengend Snippet: (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, a gift from Markku Varjosalo) using Gateway cloning as described ( Liu et al ., 2020 ).

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

Journal: bioRxiv

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

doi: 10.1101/2023.10.12.562036

Figure Lengend Snippet: (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 Norther 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, a gift from Markku Varjosalo) using Gateway cloning as described ( Liu et al ., 2020 ).

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

Journal: bioRxiv

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

doi: 10.1101/2023.10.12.562036

Figure Lengend Snippet: Localization MAC-tagged constructs in Flp-in 293 cell lines. Expression was induced with tetracycline for 24 h and immunofluorescence carried out with anti-HA antibody. (B) Immunofluorescence staining of HEK293 cells for with anti-U11/U12-65K and anti-RBM41 antibodies.

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, a gift from Markku Varjosalo) using Gateway cloning as described ( Liu et al ., 2020 ).

Techniques: Construct, Expressing, Immunofluorescence, Staining

Journal: bioRxiv

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

doi: 10.1101/2023.10.12.562036

Figure Lengend Snippet: (A) Spectral counts for DHX8 in RBM41 and U11/U12-65K BioID datasets. (B) 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). (C) RNA immunoprecipitation with endogenous RBM41 in HEK293 cells followed by RT-PCR. (D) Spectral counts for coilin (COIL) in RBM41 and U11/U12-65K BioID datasets. (E) 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, a gift from Markku Varjosalo) using Gateway cloning as described ( Liu et al ., 2020 ).

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

Journal: bioRxiv

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

doi: 10.1101/2023.10.12.562036

Figure Lengend Snippet: (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 - Cassette 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 ). (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, a gift from Markku Varjosalo) using Gateway cloning as described ( Liu et al ., 2020 ).

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

Journal: bioRxiv

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

doi: 10.1101/2023.10.12.562036

Figure Lengend Snippet: The sequeces of individual HEK293 the RBM41 Knock-Out clones. The targets of the Guide RNAs in RBM41 exons 2 and 3 are indicated in the WT sequece. Clones C11, D1, and D14 were used in the RNAseq analysis. C9, C10, and D4 were used in RT-PCR validation experiments.

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, a gift from Markku Varjosalo) using Gateway cloning as described ( Liu et al ., 2020 ).

Techniques: Knock-Out, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery