Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions - PubMed (original) (raw)

Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions

Shinsuke Ishigaki et al. Sci Rep. 2012.

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Abstract

FUS is an RNA-binding protein that regulates transcription, alternative splicing, and mRNA transport. Aberrations of FUS are causally associated with familial and sporadic ALS/FTLD. We analyzed FUS-mediated transcriptions and alternative splicing events in mouse primary cortical neurons using exon arrays. We also characterized FUS-binding RNA sites in the mouse cerebrum with HITS-CLIP. We found that FUS-binding sites tend to form stable secondary structures. Analysis of position-dependence of FUS-binding sites disclosed scattered binding of FUS to and around the alternatively spliced exons including those associated with neurodegeneration such as Mapt, Camk2a, and Fmr1. We also found that FUS is often bound to the antisense RNA strand at the promoter regions. Global analysis of these FUS-tags and the expression profiles disclosed that binding of FUS to the promoter antisense strand downregulates transcriptions of the coding strand. Our analysis revealed that FUS regulates alternative splicing events and transcriptions in a position-dependent manner.

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Figures

Figure 1

Figure 1. Experimental schemes.

(a) Mouse primary cortical neurons are prepared and infected with lentivirus expressing two different shRNA against FUS (shFus1 and shFus2) and control shRNA (shCont). Total RNA is isolated and analyzed by the Affymetrix Mouse Exon Array. (b) Fus is efficiently knocked down in primary cortical neurons, which is evaluated by real-time qRT-PCR. Bars indicate the mean and SD of three experiments. (c) Immunohistchimical analysis using anti-FUS antibody on primary cortical neurons silenced by shFus1, shFus2, and shCont. Cells are fixed and immunostained with anti-FUS antibody, anti-βTubulin antibody, and DAPI. (d) Mouse cerebrum derived from a 12-week-old C57Bl/6 mouse is UV-irradiated at 400 mJ and FUS-bound RNA segments are immuno-precipitated. High-throughput 50 bp single-end sequencing is performed using the SOLiD 3 sequencer.

Figure 2

Figure 2. Four representative FUS-mediated alternative splicing events.

The top panels show the positions of CLIP-tags and exon-intron structures. The second panels represent schematic splicing changes mediated by FUS. shCont and shFus lead to the upper and lower splicing events, respectively. The third panels show representative RT-PCR of the indicated exons. The experiments are repeated in quadruplicate using four independent sets of samples. The last panels show densitometric quantification of RT-PCR (n = 4; mean and SD).

Figure 3

Figure 3. Annotation mapping of FUS CLIP-tags.

(a) Distributions of FUS CLIP-tags. Binding regions are mapped to CDS (coding sequence), 5′ and 3′ UTRs, introns, intergenic regions including tRNA and rRNA genes according to the ENSEMBL version e!61 annotation based on the mouse genome assembly NCBI build 37.1/mm9. Pie-charts show ratios of binding regions mapped to the indicated regions. (b) Distribution of FUS CLIP-tags normalized for the length of each annotation. (c) Distribution of FUS CLIP-tags mapped to the relative positions of each gene. The broken line indicates 12,508 genes with constitutive transcriptional start/end sites, and the solid line indicates 7,477 genes with alternative transcriptional start/end sites. (d) Probability density function of the minimum free energies, δG, of 30-mer stretches of CLIP-tagged regions. CLIP-tagged regions and controls are shown in red and blue lines, respectively.

Figure 4

Figure 4. Mapping of CLIP-tags on exon-intron structures.

(a) Distribution of CLIP-tags on constitutively or alternatively spliced exons and the flanking intronic regions. The abscissa indicates an intron-exon-intron structure. The sizes of all the exons are normalized to 150 nucleotides. The number of exonic CLIP-tags is also normalized accordingly. Intronic CLIP-tags within 1,000 nucleotides upstream or downstream of exons are indicated. The number of CLIP-tags is normalized for the number of transcripts belonging to each category of constitutive and alternative exons. (b) Normalized complexity map of FUS-dependent splice sites. shFus-mediated alternative splicing events are compiled. Arrows point to conspicuous peaks at ∼500 nt upstream of the 3′ end of the downstream intron. Shaded areas indicate an average of 100 sets of normalized complexity of 20 randomly selected constitutive exons.

Figure 5

Figure 5. CLIP-tags on the promoter antisense strand and gene expression profiles.

(a) The numbers of CLIP-tags on the antisense strand at 1 to 700 nucleotides upstream of the transcription start sites of each gene are divided into three categories according to the partitioning functionality of the JMP 8.0 software. Fold-changes of gene expression levels with shFus1 and shFus2 are calculated for each category. Means and SEs are plotted. The numbers of transcription start sites are 2390 for no tag, 53 for 1-250 tags, and 18 for more than 250 tags. The number of CLIP-tags represents the total number of nucleotides covered by the tags. No statistical difference is observed for each dataset with the one-way ANOVA analysis. (b) Four representative genes, Ptprn2, Xrn1, Gak, and Glt1d1, for which more than 250 CLIP-tags are bound to the promoter antisense strand, are validated by real-time qPCR. Changes in gene expression levels in cortical neurons after knocking down Fus with shFus1 and shFus2 are indicated by means and SDs (n = 3).

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