Journal: Scientific Reports

Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

doi: 10.1038/s41598-018-25204-2

Figure Lengend Snippet: NHD minigene shows exon 3 skipping. ( A ) Schematic diagram of the TREM2 exon 2–4 minigene. The minigene is inserted downstream of the EGFP cDNA. The positions of the primer set used to detect splicing are indicated by arrows. A red arrowhead indicates the position of the 5′ splice site mutation (c.482 + 2 T > C) found in NHD. ( B ) Splicing assay of WT and NHD minigenes transfected into HEK cells. Splicing patterns were detected by RT-PCR using the primer set indicated in ( A ). ( C ) Unmodified U1 and U7 snRNAs did not alter the splicing pattern of the NHD minigene. The NHD minigene was transfected with either an empty vector, unmodified U1 snRNA, or unmodified U7 snRNA. Splicing patters were detected as indicated in ( B ). Exon 3 inclusion was not induced by co-expression of U1 or U7 snRNA. Original gel images are shown in Supplementary Fig. .

Article Snippet: We used the following antibodies: anti-human TREM2 (goat polyclonal, R&D Systems, Minneapolis, MN, USA), anti-HSP60 (goat polyclonal, Santa Cruz Biotechnology, Dallas, TX, USA), and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology).

Techniques: Mutagenesis, Splicing Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Expressing

Journal: Scientific Reports

Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

doi: 10.1038/s41598-018-25204-2

Figure Lengend Snippet: Correction of exon 3 skipping by modified U1 and U7 snRNAs. ( A ) The left panel shows the sequence of the 5′ region of the unmodified (WT) and modified U1 snRNAs used in this study. The mutated nucleotide in the NHD minigene is shown in blue. Nucleotides of the U1 snRNA constructs complementary to the 5′ splice site region of TREM2 intron 3 are indicated in red. The right panel shows the results of a splicing assay using modified U1 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected by RT-PCR and agarose gel electrophoresis. U1mut3 increased exon 3 inclusion. ( B ) The left panel shows a schematic diagram of the modified U7 snRNA used in this study. U7A–U7E were designed to hybridize to different regions in exon 3 of TREM2 . Refer to the text for the details regarding SmOPT and ESE (exonic splicing enhancer). The right panel shows the results of a splicing assay using the modified U7 snRNA. The NHD minigene was transfected with either an empty vector or modified constructs. Splicing patterns were detected as in ( A ). U7A and U7B did not increase exon 3 inclusion when used separately or simultaneously. ( C ) Co-expression of U1 and U7 constructs to modulate exon 3 splicing. Cells were transfected with the NHD minigene together with the U1 and U7 constructs as indicated. Splicing patterns were detected as in ( A ). Original gel images are shown in Supplementary Fig. .

Article Snippet: We used the following antibodies: anti-human TREM2 (goat polyclonal, R&D Systems, Minneapolis, MN, USA), anti-HSP60 (goat polyclonal, Santa Cruz Biotechnology, Dallas, TX, USA), and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology).

Techniques: Modification, Sequencing, Construct, Splicing Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing

Journal: Scientific Reports

Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

doi: 10.1038/s41598-018-25204-2

Figure Lengend Snippet: Modified U1 snRNA-mediated correction of TREM2 mis-splicing associated with NHD. ( A ) Structure of the fl-WT and fl-NHD minigenes integrated into the genome of HEK293 cells using the Flp-In system. Congenic cell lines stably harboring either minigene were established. The splicing pattern of TREM 2 in these cell lines are shown at the bottom. ( B ) TREM2 splicing patterns of the fl-NHD cells. For fl-NHD cells, transfection of U1mut3 induced the spliced product containing exon 3. ( C ) Western blot analysis of fl-WT and fl-NHD cells and parental HEK cells. Either U1mut3 or U1mut4 were co-transfected with U7A and/or U7B into fl-NHD cells. HSP60 was used as a loading control. ( D ) U1mut3 dose-dependently induced TREM2 protein expression in fl-NHD cells. Increasing amounts of U1mut3 were transfected into fl-NHD cells and TREM2 expression was detected by western blotting. ( E ) Immunofluorescence of fl-WT and fl-NHD cells using an anti-TREM2 antibody (red). U1mut3 induced TREM2 protein expression in fl-NHD cells. Nuclei were stained with DAPI. Scale bar: 100 μm. ( F ) Transient co-expression of U1mut3 and fl-NHD restored TREM2 protein expression. HEK cells transfected with the indicated constructs were analyzed by western blotting. Original gel images and western blot data are shown in Supplementary Fig. .

Article Snippet: We used the following antibodies: anti-human TREM2 (goat polyclonal, R&D Systems, Minneapolis, MN, USA), anti-HSP60 (goat polyclonal, Santa Cruz Biotechnology, Dallas, TX, USA), and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology).

Techniques: Modification, Stable Transfection, Transfection, Western Blot, Control, Expressing, Immunofluorescence, Staining, Construct

Journal: Scientific Reports

Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

doi: 10.1038/s41598-018-25204-2

Figure Lengend Snippet: Exon 3 of TREM2 is flanked by a weak 5′ splice site that predisposes it to exon skipping. ( A ) Splice site scores of TREM2 5′ splice sites calculated using an online tool (see Methods). The 5′ splice site of exon 3 showed the lowest score. ( B ) Sequence of the 5′ splice site flanking to TREM2 exon 3 with and without mutations. NHD contains a GT-to-GC mutation. GC-opt contains a GT-to-GC mutation but is otherwise an optimized sequence of a 5′ splice site complementary to unmodified U1 snRNA. The red letters indicate nucleotides complementary to U1 snRNA. ( C ) Splicing patterns of TREM2 minigenes in HEK cells. The GC-opt minigene showed predominant inclusion of exon 3. Original gel images are shown in Supplementary Fig. .

Article Snippet: We used the following antibodies: anti-human TREM2 (goat polyclonal, R&D Systems, Minneapolis, MN, USA), anti-HSP60 (goat polyclonal, Santa Cruz Biotechnology, Dallas, TX, USA), and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology).

Techniques: Sequencing, Mutagenesis

Journal: Scientific Reports

Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

doi: 10.1038/s41598-018-25204-2

Figure Lengend Snippet: Exon 3 splicing of TREM2 is a determinant of its protein expression. ( A ) Schematic model of TREM2 exon 3 splicing. Exon 3 inclusion leads to expression of full-length TREM2 protein. Exon 3 skipping would result in either expression of a TREM2 isoform lacking exon 3 or degradation of mRNA via nonsense-mediated mRNA decay (NMD) due to production of a premature termination codon in exon 4. ( B ) Exon 3 is alternatively spliced in THP-1 cells. The splicing patterns of TREM2 in fl-WT and THP-1 cells with or without cycloheximide treatment, an NMD inhibitor, are shown. ( C ) Schematic illustration of U7-ex3-skip containing antisense sequences to both the branch point of intron 2 and exon/intron junction of exon 3. ( D ) fl-TREM2 (WT) cells were treated with U7-ex3-skip and/or CHX and the splicing patterns were analyzed by RT-PCR. The exon 3-skipped pattern was increased by U7-ex3-skip and further enhanced by CHX, suggesting that some of the spliced products of exon 3 skipping were degraded by NMD. ( E ) Western blot analysis of TREM2 expression in fl-WT cells with or without transfection of U7-ex3-skip (left panel). The bar chart shows the quantification of the western blot results (mean ± SE). *P = 0.003 in a two-tailed t -test (n = 6). Original gel images and western blot data are shown in Supplementary Fig. .

Article Snippet: We used the following antibodies: anti-human TREM2 (goat polyclonal, R&D Systems, Minneapolis, MN, USA), anti-HSP60 (goat polyclonal, Santa Cruz Biotechnology, Dallas, TX, USA), and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Two Tailed Test

Journal: Scientific Reports

Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

doi: 10.1038/s41598-018-25204-2

Figure Lengend Snippet: Induction of exon 3 inclusion of fl-WT minigene increased TREM2 protein expression. ( A ) Structure of the 5′ end of unmodified U1 snRNA (cntl) and modified U1 construct based on U1mut3 but matches wild-type (WT) exon 3. ( B ) Splicing assay of WT(ex2-4) minigene transfected with unmodified U1 snRNA or the U1-ex3 construct in HEK cells. ( C ) U1-ex3 increased TREM2 protein expression from the fl-WT minigene in HEK cells. Western blot analysis of TREM2 expression (left panel). Bar chart shows the quantification of the western blot results (mean ± SE, n = 4). *P = 0.00013 (- vs . ex3) and P = 0.0007 (cntl vs . ex3) in Tukey’s multiple comparison test. Original gel images and western blot data are shown in Supplementary Fig. .

Article Snippet: We used the following antibodies: anti-human TREM2 (goat polyclonal, R&D Systems, Minneapolis, MN, USA), anti-HSP60 (goat polyclonal, Santa Cruz Biotechnology, Dallas, TX, USA), and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology).

Techniques: Expressing, Modification, Construct, Splicing Assay, Transfection, Western Blot, Comparison

Journal: Scientific Reports

Article Title: Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation

doi: 10.1038/s41598-018-25204-2

Figure Lengend Snippet: Suboptimal 5′ splice site of exon 3 underlies its alternative splicing and a predisposition to the NHD-associated mutation. Weak splicing signals are associated with alternative splicing. The 5′ splice site of TREM2 exon 3 is a weak splicing signal, which accounts for its alternative splicing. An NHD-associated GT-to-GC mutation (c.482 + 2 T > C) caused complete skipping of this exon. However, even in the presence of the GT-to-GC mutation, the GC-opt minigene showed nearly complete inclusion of exon 3, suggesting that the GT-to-GC mutation could have less deleterious effects if exon 3 had a stronger 5′ splice site.

Article Snippet: We used the following antibodies: anti-human TREM2 (goat polyclonal, R&D Systems, Minneapolis, MN, USA), anti-HSP60 (goat polyclonal, Santa Cruz Biotechnology, Dallas, TX, USA), and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology).

Techniques: Alternative Splicing, Mutagenesis