Journal: Nucleic Acids Research

Article Title: G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA

doi: 10.1093/nar/gkaa475

Figure Lengend Snippet: GGGGCC repeats recruit NONO to model transcripts. ( A ) An eGFP-encoding model transcript system was used to test whether the presence of GGGGCC repeat sequence affects NONO-transcript association in a cellular context. Constructs contained an eGFP coding sequence transcribed under the control of a cytomegalovirus (CMV) promoter and bovine growth hormone polyadenylation signal (PAS), with experimental sequences inserted in the 5′-untranslated region. Two control transcripts were used: the Spacer construct included a 25-nucleotide spacer insert and the (TGGTCC) 8 construct included eight TGGTCC repeats, providing a mutated repeat sequence incapable of forming G-quadruplex structures. ( B ) RNA-IP for NONO was performed on HEK293T cells transfected with the model transcript constructs, followed by RT-qPCR to compare enrichment of the model transcripts. Transcript enrichment is expressed as percentage of input with SEM ( n = 3). ‘*’ indicates P < 0.05 (two-tailed t -test). ( C ) Western blot of NONO IP fractions, 5% input, and enriched fraction from IP with IgG control. Actin is blotted as a negative control for IP enrichment.

Article Snippet: Full-length NONO included a MYC/DDK-tag, was commercially produced and purified from HEK293T cells (Origene, TP306688), and was diluted for titrations in storage buffer (25 mM Tris-HCl pH 7.3, 100 mM glycine, 10% glycerol).

Techniques: Sequencing, Construct, Control, Transfection, Quantitative RT-PCR, Two Tailed Test, Western Blot, Negative Control

Journal: Nucleic Acids Research

Article Title: G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA

doi: 10.1093/nar/gkaa475

Figure Lengend Snippet: NONO preferentially binds GGGGCC repeat RNA in the G-quadruplex conformation. ( A ) Domain architecture of DBHS proteins and NONO(53-312) construct with shared DBHS core region colored by domain and low-complexity regions in grey. ( B ) EMSA with recombinant NONO(53-312) and (GGGGCC) 4 RNA in G-quadruplex and non-G-quadruplex conformations. 10 nM labelled (GGGGCC) 4 RNA was incubated with NONO(53-312) at the following concentrations (left to right): 0, 1.2 nM, 6.0 nM, 12 nM, 24 nM, 30 nM, 60 nM, 120 nM, 150 nM, 200 nM, 300 nM, 600 nM, 750 nM, 1.0 μM, 1.5 μM, 3.0 μM, 5.0 μM. For visualization purposes, images were adjusted independently to account for differences in fluorescence intensity between conformations. Quantification was performed prior to this adjustment. ( C ) Data fit of (B) with binding curves and SEM ( n = 3). In this and subsequent figures, error bars for some data points are within the size of the symbol and are therefore not visible.

Article Snippet: Full-length NONO included a MYC/DDK-tag, was commercially produced and purified from HEK293T cells (Origene, TP306688), and was diluted for titrations in storage buffer (25 mM Tris-HCl pH 7.3, 100 mM glycine, 10% glycerol).

Techniques: Construct, Recombinant, Incubation, Fluorescence, Binding Assay

Journal: Nucleic Acids Research

Article Title: G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA

doi: 10.1093/nar/gkaa475

Figure Lengend Snippet: NONO preferentially interacts with NEAT1 G-rich RNA in the G-quadruplex conformation. ( A ) Comparative enrichment of DBHS-family proteins by NEAT1 G-quadruplex RNA and non-G-quadruplex-forming G-rich RNA ‘Scrambled’. Mass spectrometry with TMT labelling was used for comparative analysis of proteins enriched from HEK293T lysate by pulldown with biotinylated RNA oligonucleotides. Enrichment is shown as mean and SEM of RNA-enriched abundance, relative to enrichment by beads, of each protein's peptides. ( B ) Western blot validation of RNA pulldown results. 2.5% input volume (‘In’) was loaded for reference. Pulldown with bead-only control (‘B’) was compared to NEAT1 G-quadruplex RNAs and non-G-quadruplex-forming G-rich (‘Scr’) RNA. Results shown are representative of three replicates. ( C ) EMSA with recombinant NONO(53-312) and NEAT1_22619 RNA in G-quadruplex and non-G-quadruplex conformations. 10 nM labelled NEAT1_22619 RNA was incubated with NONO(53-312) at the following concentrations (left to right): 0, 1.2 nM, 6.0 nM, 12 nM, 24 nM, 30 nM, 60 nM, 120 nM, 150 nM, 200 nM, 300 nM, 600 nM, 750 nM, 1.0 μM, 1.5 μM, 3.0 μM, 5.0 μM. ( D ) Data fit of (C) with binding curves and SEM ( n = 4). ( E ) Competition EMSA with recombinant NONO(53-312) and labelled NEAT1_22619 G-quadruplex. Labelled RNA only and labelled RNA with NONO(53-312) but no competitor are shown in lanes 1 and 2, respectively. Labelled NEAT1_22619 was pre-incubated with NONO(53-312) before addition of unlabelled competitor RNAs at 1-200× molar excess (lanes 3-7) relative to 10 nM labelled RNA. When present, NONO(53-312) was at 1.0 μM. Unlabelled competitors include NEAT1_22619 RNA in G-quadruplex conformation (upper), 5′-Splice Site RNA known to bind NONO via RRM1 (lower left), and (GGGGCC) 4 RNA in G-quadruplex conformation (lower right). All competitors were pre-annealed with 100 mM KCl. ( F ) Data fit of (E) with binding curves and SEM ( n = 3).

Article Snippet: Full-length NONO included a MYC/DDK-tag, was commercially produced and purified from HEK293T cells (Origene, TP306688), and was diluted for titrations in storage buffer (25 mM Tris-HCl pH 7.3, 100 mM glycine, 10% glycerol).

Techniques: Mass Spectrometry, Western Blot, Biomarker Discovery, Control, Recombinant, Incubation, Binding Assay

Journal: Nucleic Acids Research

Article Title: G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA

doi: 10.1093/nar/gkaa475

Figure Lengend Snippet: NONO binds to NEAT1 through G-quadruplex motifs. ( A ) Quadruplex-forming G-rich sequences (QGRS) mapped to NEAT1 are shown as tall bars (black). ( B ) NONO eCLIP data mapped to NEAT1. Reads are plotted by intensity and called peaks are shown as short bars. Tracks, from top to bottom: Merged peaks, determined by replication and adjustment for input (dark purple); Replicate 1 read intensity plot with called peaks directly beneath (purple); Replicate 2 read intensity plot with called peaks directly beneath (pink); size-matched paired input read intensity plot (grey). The statistical significance of overlap between QGRS and each set of peaks was determined using the two-tailed Fisher's exact test, with P -values shown in parentheses beneath each corresponding track label. ( C ) Circular dichroism of 5 μM NEAT1_425 and NEAT1_22619 G-quadruplex RNAs following addition of TMPyP4 at increasing concentrations. ( D ) NEAT1 enrichment by NONO RNA-IP with addition of TMPyP4. RNA-IP for NONO was performed on HEK293T cell lysate. Various concentrations of TMPyP4 were added to lysate before immunoprecipitation. RT-qPCR was performed to measure NEAT1 enrichment, expressed as percentage of input ( n = 4). '***' indicates P < 0.0005; 'n.s.' indicates P ≥ 0.05 (two-tailed t -test). ( E ) Western blot of RNA-IP samples described in (D) and 5% input. GAPDH was blotted as a negative control for IP enrichment.

Article Snippet: Full-length NONO included a MYC/DDK-tag, was commercially produced and purified from HEK293T cells (Origene, TP306688), and was diluted for titrations in storage buffer (25 mM Tris-HCl pH 7.3, 100 mM glycine, 10% glycerol).

Techniques: Two Tailed Test, Circular Dichroism, Immunoprecipitation, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Nucleic Acids Research

Article Title: G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA

doi: 10.1093/nar/gkaa475

Figure Lengend Snippet: Enrichment in G-quadruplex motifs is a conserved feature of NEAT1 homologues. ( A ) Human NEAT1 isoforms and scale bar. ( B ) Quadruplex-forming G-rich sequences (QGRS) on human NEAT1 (upper track) and on randomly generated sequences (lower tracks). QGRS are shown mapped to NEAT1 (upper track, light blue bars) and expanded below (upper track, short black bars). QGRS (tall black bars) were identified for five randomly generated sequences with length and GC content equal to NEAT1 (lower tracks). The number of QGRS identified on each sequence is shown to the left of the corresponding track. ( C ) Quadruplex-forming G-rich sequences predicted along the length of NEAT1 homologues. Identified QGRS are shown mapped to each NEAT1 homologue (light blue bars) and expanded below (short black bars). ( D ) QGRS enrichment among NEAT1 homologues. Sequences were randomly generated with the length and GC content of NEAT1 homologues and QGRS were identified. For each homologue, numbers of QGRS on five generated sequences are plotted with mean and SEM, with the number of QGRS on the corresponding homologue plotted for comparison. ( E ) Paraspeckle assembly begins with co-transcriptional recruitment of DBHS dimers (NONO and SFPQ homo- and heterodimers) to NEAT1, seeding formation of NEAT1 RNPs, the primary subunit of paraspeckles, and initiating paraspeckle formation. The elements of NEAT1 responsible for this recruitment have yet to be identified. G-quadruplex structures along the length of NEAT1 are recognized by NONO with structural specificity, play a critical role in NONO-NEAT1 association, and are a conserved feature among NEAT1 homologues. These findings position NEAT1 G-quadruplexes as a primary candidate for the NONO-recruiting elements of NEAT1.

Article Snippet: Full-length NONO included a MYC/DDK-tag, was commercially produced and purified from HEK293T cells (Origene, TP306688), and was diluted for titrations in storage buffer (25 mM Tris-HCl pH 7.3, 100 mM glycine, 10% glycerol).

Techniques: Generated, Sequencing, Comparison