ms2-mbp fusion protein  (New England Biolabs)

Journal: bioRxiv

Article Title: RNA language models predict mutations that improve RNA function

doi: 10.1101/2024.04.05.588317

Figure Lengend Snippet: A. Matrix showing the overlap in the 200 positions with highest Jensen-Shannon divergence in finetuned (FT) model-generated versus pretrained (PT) model-generated sequences for the GNN, LM models, and in the hyperthermophilic vs. total GARNET sequences. B. Strategy for using log likelihoods of sequence generation from FT versus PT models, with WT E. coli serving as a normalization control. C. Cumulative plots of ΔΔlogP(FT-PT) of all single mutations to the E. coli 23S rRNA for each model. Each point represents the probability of generating a single nucleotide mutant E. coli 23S sequence from the FT model relative to the PT model, normalized to that of the WT sequence. Two mutations, U2477C and U2554C, are denoted in orange and green, respectively. D. Secondary structures of helices H89 and H92 of E. coli 23S rRNA. Positions that were mutated in this study are shown in red. E. Analysis of helix 89 for candidate thermostabilizing mutations. For each position, the most frequent nucleotide in FT generated sequences (top FT nucleotide) is grafted into the E. coli 23S rRNA sequence and used to calculate ΔΔlogP(FT-PT) for the 23S LM, 231-RNA LM, and GNN models. Positions where the top FT nucleotide differs from WT in at least one model are highlighted in gray. Base pairing positions are indicated on the left. F. Schematic for the heat-treatment in vitro translation assay. Purified 50S subunits are incubated at the indicated temperature, cooled to room temperature, and then added to a HiBit in vitro translation assay. The peptide complements an inactive protein fragment to form an active luciferase. G. Activity of pre-incubated ribosomes in the HiBit in vitro translation assay. WT and mutant 50S subunits all contain an MS2 tag ( Materials and Methods ). Relative activity is calculated as the slope of the initial increase in luminescence during translation and normalized to the WT value at the given temperature. Data and error bars represent the average and standard deviation of three reactions, respectively.

Article Snippet: 10 mg of MBP-MS2 protein was loaded onto a MBP Trap column (Cytiva) that was equilibrated with MS2-150 buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM EDTA, 2 mM 2-mercaptoethanol).

Techniques: Generated, Sequencing, Mutagenesis, In Vitro, Purification, Incubation, Luciferase, Activity Assay, Standard Deviation

Journal: bioRxiv

Article Title: The spliceosome assembles on excised linear introns to protect them from degradation

doi: 10.64898/2026.01.21.700889

Figure Lengend Snippet: a , Schematic for purification of the stable-intron complex from saturated cultures. MBP-MS2, Maltose-binding protein fused to MS2 coat protein; ProtA-PP7, 2X ProteinA fused to PP7 coat protein. b , Similar behaviors of tagged stable intron and endogenous stable intron. Shown is an RNA blot of a denaturing gel resolving total RNA from log-phase (LP) and saturated (S) cultures of a wildtype strain that expressed the PP7-GFP-SAC6-MS2 pre-mRNA described in (A). The blot was probed for the MS2 tag (top), and then reprobed for the endogenous SAC6 intron (upper middle), PP7 tag (lower middle), and 5.8S rRNA (bottom), which served as a loading control. c , Proteins in the stable-intron complex. Shown is an SDS-PAGE gel that resolves the cryo-EM sample, stained with Imperial Stain. Bands were labelled based on comparison to the distinct migration patterns of spliceosomal proteins observed in previous spliceosome preparations , . d , e , Enrichment of SAC6-MS2 stable intron ( d ) and snRNAs ( e ) in purified stable-intron complex. Shown is an RNA blot of a denaturing gel resolving the indicated proportion of the glycerol cushion fraction (Input) and the concentrated and dialyzed sample used for cryo-EM analysis (Cryo). The blot was probed using an oligonucleotide that hybridized to a sequence common to the endogenous SAC6 intron and the tagged SAC6-MS2 intron ( d ), and then stripped and reprobed for U1, U2, U4, U5 and U6 snRNAs simultaneously ( e ). Migration of markers with lengths indicated (nucleotides) is at the left. f , No detectable upstream exon in the purified stable-intron complex. Shown is a Urea-PAGE gel resolving the products of primer extension across the GFP-SAC6-MS2 exon–intron boundary at the 5′SS, performed in the absence of dGTP on RNA extracted from equal fractions of the glycerol cushion fraction (input) and IgG affinity flowthrough (exon depleted), and a five-fold fraction of the purified stable-intron complex (cryo-EM sample), as well as on in-vitro-transcribed RNAs representing the intron and the pre-mRNA (standards). Products terminating at the 5′-end of the excised intron (+6) represent the excised intron, and products terminating at the upstream C nucleotides of the 5′ exon (+11 and +20) represent the unspliced pre-mRNA. The +20-extension product was a result of reverse transcriptase readthrough of the first C at the +11 position, as verified by results from the in-vitro-transcribed standards. The in-vitro-transcribed intron standard consisted of the SAC6-MS2 intron with a precise 5′-end defined by hammerhead ribozyme cleavage, and the pre-mRNA standard consisted of an RNA spanning 166 nucleotides upstream to 66 nucleotides downstream of the intron.

Article Snippet: MBP-MS2 was purified essentially as described . pMBP-MS2 (a gift from Josep Vilardell, Addgene plasmid # 65104) was expressed in Rosetta2(DE3) E. coli (Novagen).

Techniques: Purification, Binding Assay, Northern blot, Control, SDS Page, Cryo-EM Sample Prep, Staining, Comparison, Migration, Sequencing, In Vitro, Reverse Transcription

Journal: Cell Regeneration

Article Title: Linc-RAM promotes muscle cell differentiation via regulating glycogen phosphorylase activity

doi: 10.1186/s13619-022-00109-8

Figure Lengend Snippet: Linc-RAM directly interacts with PYGM in the cytoplasm. A-F Linc-RAM in cytoplasmic (Cyto), nuclear-soluble (Nuc.Sol), and nuclear-insoluble (Nuc.Insol) fractions of C2C12 cells cultured in growth medium ( A-C ) and differentiation medium for 24 h ( D-F ), as determined by RT–qPCR. The GAPDH mRNA was used as a marker for the cytoplasmic fraction. Neat1 (nuclear paraspeckle assembly transcript 1) was used as a marker for the nuclear fraction. The data are representative of three independent experiments. G Schematic diagram showing the strategy applied to identify Linc-RAM-binding proteins using MS2-MBP-mediated RNA pull down. Three bacteriophage MS2 coat protein-binding sites (3 × MS2 hairpins) were fused to the 3′-end of Linc-RAM (Linc-RAM-3 × MS2). MS2-MBP represents a fusion protein comprising MS2 coat protein and maltose-binding protein. H A representative silver-stained SDS-PAGE gel showing the bands that differed (red arrow) between Linc-RAM-3 × MS2 (Linc-RAM) and the 3 × MS2 control (Ctrl). The differential bands were individually extracted and subjected to mass spectrometry (MS) analysis. I , J RNA immunoprecipitation (RIP) analysis to validate the physical interaction between Linc-RAM and PYGM in C2C12 cells cultured in differentiation medium for 24 h. Native ( I ) or UV-crosslinked ( J ) C2C12 cells differentiated for 24 h were immunoprecipitated using anti-PYGM, anti-MyoD, and IgG antibodies. Linc-RAM in immunoprecipitates was examined by RT–qPCR. GAPDH served as a negative control. MyoD antibodies served as a positive control, as we previously reported that Linc-RAM binds MyoD (Yu et al., ). K Representative RNA electrophoretic mobility shift assay (EMSA) results obtained using biotin-labeled Linc-RAM and different doses of recombinant GST-PYGM fusion protein (50 ng, 100 ng, 200 ng). The biotin-labeled Linc-RAM and recombinant PYGM protein complex were resolved on a 5% native polyacrylamide gel and subsequently the Linc-RAM/PYGM complex was detected by HRP-Streptavidin. Recombinant GST protein (GST-only) served as a negative control. L Representative RNA EMSA results obtained using biotin-labeled Linc-RAM and recombinant GST-PYGM fusion protein (200 ng). As competitors, non-labeled Linc-RAM probes were added to confirm the binding specificity. The presented values reflect the means ± SE obtained from three independent experiments

Article Snippet: Subsequently, cytoplasmic fractions from 1 × 10 7 cells were incubated with 4 μg of purified recombinant MS2-MBP protein in 0.1% NP-40 lysis buffer containing a protease inhibitor cocktail at 4 °C for 3 h. Then 100 μl of pretreated amylase magnetic beads (NEB, E8035S) were added and incubated for additional 1 h at 4 °C.

Techniques: Cell Culture, Quantitative RT-PCR, Marker, Binding Assay, Protein Binding, Staining, SDS Page, Mass Spectrometry, Immunoprecipitation, Negative Control, Positive Control, Electrophoretic Mobility Shift Assay, Labeling, Recombinant

Journal: Science Advances

Article Title: Establishing RNA-RNA interactions remodels lncRNA structure and promotes PRC2 activity

doi: 10.1126/sciadv.abc9191

Figure Lengend Snippet: ( A ) Model of B1-mediated HOTAIR RNA-RNA interactions with nascent target RNA leading to PRC2 activity and gene silencing. ( B ) In vitro RNA pulldown with MS2-tagged HOTAIR or Anti-luc with recombinant B1 or A2. “A2+” is 3× the concentration of A2. Minus MS2-MBP fusion protein pulldown was included to account for background bead binding. Western blot analysis was performed using A2/B1 antibody. RNA recovery was quantified by quantitative polymerase chain reaction (qPCR; n = 3). ( C ) Crystal structure of RRM domains of A2/B1 in complex with 10-mer RNA (yellow) . Two molecules of the tandem RRMs are shown in purple/green. Blue circles highlight the N terminus. Adapted from ( http://creativecommons.org/licenses/by/4.0/ ). ( D ) Schematic of RNA-RNA interaction assay: RAT-tagged JAM2 and HOTAIR in vitro transcriptions (IVTs) incubated ± recombinant hnRNP B1. JAM2 tethered by PP7 coat protein on magnetic beads. Recovery of HOTAIR by JAM2 pulldown was quantified by RT-qPCR and protein by Western blot. ( E ) Assays from (D) with HOTAIR or Anti-luc RNA ± hnRNP B1 or A2 ( n = 6). ( F ) As in (E) with full-length HOTAIR, HOTAIR with the JAM2 interaction site deleted or mutated ± hnRNP B1 ( n = 3). Error bars in (E) and (F) represent SDs. P values were determined using two-way analysis of variance (ANOVA) and two-tailed Student’s t tests. n.s., not significant; WT, wild type; Pol II, RNA Polymerase II.

Article Snippet: At the same time, 300 nM MS2-MBP was prebound to 20 μl of amylose resin (New England Biolabs) in EMB 300 Buffer, RNase inhibitor (New England Biolabs), and 1% BSA and rotated at room temperature for 15 min.

Techniques: Activity Assay, In Vitro, Recombinant, Concentration Assay, Binding Assay, Western Blot, Real-time Polymerase Chain Reaction, Incubation, Magnetic Beads, Quantitative RT-PCR, Two Tailed Test