Journal: Viruses

Article Title: Murine Leukemia Virus Uses TREX Components for Efficient Nuclear Export of Unspliced Viral Transcripts

doi: 10.3390/v6031135

Figure Lengend Snippet: A model of gammaretroviral RNA export. Both unspliced and spliced transcripts require UAP56 and NXF1. THOC5 and THOC7 are required for the nuclear export unspliced viral transcripts. SD: splice donor; SA: splice acceptor.

Article Snippet: Rabbit anti-NXF1 antibody was purchased from Sigma.

Techniques:

Journal: Nature Communications

Article Title: Nuclear export and translation of circular repeat-containing intronic RNA in C9ORF72-ALS/FTD

doi: 10.1038/s41467-021-25082-9

Figure Lengend Snippet: a Two-color smFISH in +(GGGGCC) 70 splicing reporter cells transfected with nontargeting control (top) or NXF1-targeting (bottom) siRNA. Magenta: intron (MBS); cyan: exon (PBS); blue: DAPI. Scale bar: 5 μm. The boxes were zoomed and shown on the right, scale bar: 1 μm. Arrow: intron; arrowhead: exon. Dash line: nuclear boundary. Quantification shown in panel b . b Scatter plot of cytosolic intron vs exon numbers in each cell with either nontargeting control siRNA (top) or NXF1-targeting siRNA (bottom) transfection. Each dot represented one cell (total 418 cells for control and 490 for NXF1 siRNA, from three biological replicates). c HeLa Flp-In bicistronic splicing reporter cells (C9R-NLuc is located in the C9ORF72 intron 1, flanked by C9 exon 1 and 2, pA: poly-A tail) were induced to express translation reporters by doxycycline after two days of siRNA transfection, and luciferase activities were measured after another 24 h. NLuc signals were normalized to FLuc in each sample and the relative expression was compared to nontargeting siRNA control. * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed t- test. Data are mean ± SEM. from three biological replicates. The exact P values for frame-GA from left to right are 0.0001, 0.0012, 0.0036, 0.0001, 0.00002, 0.0003. The exact P values for frame-GP from left to right are 0.009, 0.0093, 0.033, 0.0046, 0.0085, 0.0417. d C9ORF72-ALS iPSNs were transfected with NXT1, NXF1 or nontargeting siRNA at day 16 post-differentiation. Poly-GP was measured by ELISA after another 16 days of differentiation and maturation. Different shapes of datapoints represent independent cell lines. Data are presented as mean ± SEM. from three control and three C9ORF72-ALS cell lines. * P < 0.05 by One-way ANOVA followed by Dunn’s post hoc. e Working model: G-rich repeats stabilize introns in circular form and mediate the nuclear export. In C9ORF72-ALS/FTD, the circular intron with GGGGCC exp is the template for RAN translation in the cytoplasm, which can be upregulated by stress stimuli. The NXF1-NXT1 pathway plays an important role in the nuclear export of the circular intron, mediated by the GGGGCC exp . Source data are provided as a Source Data File.

Article Snippet: The primary antibodies included NXF1 (1:1000, Bethyl Laboratories, #A300-914A), β-actin (1:1000, Cell Signaling Technology, #3700).

Techniques: Transfection, Control, Luciferase, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Cell Biology

Article Title: Intranuclear binding in space and time of exon junction complex and NXF1 to premRNPs/mRNPs in vivo

doi: 10.1083/jcb.201412017

Figure Lengend Snippet: Immuno-EM analyses of NXF1, UPF2, and UPF3 in BR mRNPs in the interchromatin. (A) Section through a polytene cell. Part of the cytoplasm (Cyt) and the nuclear membrane (NE) are visible. The interchromatin (Interchrom) accounts for large volumes devoid of chromatin. Part of chromosome IV (Chrom IV) with its BR genes (BR) is seen. In the BR loci, the volumes occupied by transcriptionally active gene loops are delimited by broken lines. No., nucleolus. (B) BR mRNPs in the interchromatin. A BR mRNP containing NXF1 (gold particle) is marked (arrowhead). A BR mRNP, not labeled by the anti-NXF1 antibodies, is also observed (arrow). Parts of the cytoplasm (Cyt) and nuclear membrane (NE) are visible. Two docked BR mRNPs contain NXF1 (gold particles, arrowheads). (C) Three BR mRNPs in the interchromatin are detected. (top) Anti-UPF3 antibodies label one out of two BR mRNPs (gold particle, arrowhead). Unlabeled BR mRNP are indicated (arrow). (bottom) Anti-UPF2 antibodies label a BR mRNP (gold particle, arrowhead). (D) Anti-NXF1 antibodies do no stain BR premRNPs. (E) Anti-UPF2 antibodies do not stain BR mRNPs. (F) Anti-UPF3 antibodies do not stain BR mRNPs. Bars: (A) 2.5 µm; (B–F) 50 nm.

Article Snippet: Polyclonal antibodies against Y14, Mago, NXF1, UPF2, and UPF3 were raised in rabbits (Agrisera).

Techniques: Membrane, Labeling, Staining

Journal: The Journal of Cell Biology

Article Title: Intranuclear binding in space and time of exon junction complex and NXF1 to premRNPs/mRNPs in vivo

doi: 10.1083/jcb.201412017

Figure Lengend Snippet: Immunogold labeling of EJC-associated proteins in the BR mRNPs in the interchromatin and docked at the NPC

Article Snippet: Polyclonal antibodies against Y14, Mago, NXF1, UPF2, and UPF3 were raised in rabbits (Agrisera).

Techniques: Labeling

Journal: The Journal of Cell Biology

Article Title: Intranuclear binding in space and time of exon junction complex and NXF1 to premRNPs/mRNPs in vivo

doi: 10.1083/jcb.201412017

Figure Lengend Snippet: Distribution of immunogold labeling of EJC-associated proteins at the nuclear membrane: nuclear side, middle, and cytoplasmic side

Article Snippet: Polyclonal antibodies against Y14, Mago, NXF1, UPF2, and UPF3 were raised in rabbits (Agrisera).

Techniques: Labeling, Membrane

Journal: The Journal of Cell Biology

Article Title: Intranuclear binding in space and time of exon junction complex and NXF1 to premRNPs/mRNPs in vivo

doi: 10.1083/jcb.201412017

Figure Lengend Snippet: Model of BR mRNP acquisition of EJC core, NXF1, UPF2, and UPF3 in relation to transcription, processing, and export. (a) At the BR gene (gray line), premRNPs (light blue) are assembled, and the premRNAs are largely spliced. In connection with splicing, the EJC core (purple circles) becomes associated with the premRNP. The EJC core remains associated with the mRNP in the interchromatin and during export. (b) New BR mRNPs (light blue) are released into the interchromatin and move in all directions. (c) They become part of an interchromatin pool of BR mRNPs. In the interchromatin, the BR mRNPs bind NXF1, UPF2, and UPF3 (yellow and orange crosses). At a given moment 10–25% of the BR mRNPs contain these proteins. The pool contains old BR mRNPs (dark blue, boxed by dark blue line) and new BR mRNPs (light blue, boxed by light blue line). NXF1, UPF2, and UPF3 bind to both old and new BR mRNPs. Some BR mRNPs have recruited the proteins at an earlier time point (orange crosses), and some BR mRNPs have just recruited the proteins (yellow crosses). In steady state, the pool receives newly synthesized BR mRNPs and loses an approximately equal number through export to the cytoplasm. Both new and old BR mRNPs are exported if they contain NXF1, UPF, and UPF3 (boxed by black line). (d) BR mRNPs that are exported are a mixture of those that have contained the proteins for some time and those that have just obtained them. (e) BR mRNPs that do not yet contain NXF1, UPF2, and UPF3 can diffuse and interact with the basket of the NPC, but they are rejected and return into the interchromatin.

Article Snippet: Polyclonal antibodies against Y14, Mago, NXF1, UPF2, and UPF3 were raised in rabbits (Agrisera).

Techniques: Synthesized