We globally analyzed FUS-mediated regulations of gene expressions and alternative splicing events using exon arrays. In addition, we globally mapped RNA-FUS interactions in vivo by HITS-CLIP and analyzed position dependence of FUS-binding to gene expressions and alternative splicing events.
Collation of the exon-level analysis of exon arrays and CLIP-tags enabled us to draw a normalized complexity map (). The map disclosed scattered binding of FUS to the upstream and downstream introns with conspicuous binding peaks close to the 3′ end of the downstream intron. A similar peak close to the 3′ end of the downstream intron is observed with PTB and MBNL1 but not with CUGBP123,24
. The underlying mechanisms shared by these RNA-binding molecules, however, remain elusive.
Although we could not detect FUS-binding consensus motifs in our CLIP-tags, we found that FUS-binding regions readily form secondary structures. Our observation is consistent with previous reports that FUS binds to stem-and-loop structures19
and that FUS has a weakly enriched motif endowed with G/C nucleotides, which is present in less than 10% of the FUS-bound sites25
. In vitro
SLELX analysis determined that FUS-binding motif is GGUG26,27
, however, has no GGUG motif but is able to specifically bind to FUS28
. We assume that FUS binds to specific RNA targets with specific secondary structures, and simple analysis of primary sequences is unlikely to be sufficient.
Divergent transcriptions including bidirectional transcriptions at the promoter regions of protein-coding genes are widely recognized29,30
, although its biological significance is poorly understood. Collation of the gene-level analysis of exon arrays and CLIP-tags on the antisense strands at the promoter regions revealed that binding of FUS to the promoter antisense strand downregulates transcriptions of the coding sense strand (). FUS interacts with CBP and p300 in the presence of ncRNA and inhibits HAT followed by repression of transcription of Ccnd1
, but the origin of ncRNA was not scrutinized10
. Our global analysis suggests that binding of FUS to the promoter antisense strand and the subsequent downregulation of transcription of the sense-coding strand is likely to be instrumental in some but not all genes. Recently, Tan and colleagues reported that FUS binds to single-strand DNA at the promoter region, and up- or down-regulates transcriptions31
. In our CLIP experiments, we needed to degrade DNA using DNase to make a precipitate with anti-FUS antibody. Lack of DNA in our RNA-protein complex was also indicated by that a high concentration of RNase decreased the molecular weight of the RNA-protein complex to that of FUS alone (). The underlying mechanisms shared by FUS-binding to single-strand DNA and to the promoter antisense RNA strand need to be further studied.
Involvement of TDP43 and FUS in the pathogenesis of ALS and FTLD suggests that the two diseases are likely to be caused by aberrations of RNA metabolisms32
. Four additional RNA-binding proteins are causally associated with ALS: senataxin (SETX
), elongation protein 3 (ELP3
), and survival motor neuron (SMN
. In the present study, we identified that FUS facilitates skipping of Mapt
exon 10 in primary cortical neurons (Supplementary Fig. S2
encodes Tau protein and inclusion of exon 10 yields 4-repeat Tau (RD4), whereas skipping of exon 10 generates 3-repeat Tau (RD3). It has been reported that the RD4-to-RD3 ratio is increased in neurodegenerative disorders including PSP and FTLD34,35
. Recently, a large hexanucleotide repeat expansion in intron 1 of C9ORF72
has been reported in both familial ALS and familial FTLD36,37
. Abnormally expanded repeats sequestrate MBNL1 in myotonic dystrophy38
, spinocerebellar ataxia type 839
, and Huntington′s disease-like 240
, and cause RNA gain-of-function pathologies, in which MBNL1 is sequestrated to abnormally expanded repeats, which compromises physiological functions of MBNL1. Thus, aberrations of RNA metabolisms are likely to be a common underlying mechanism shared by familial and sporadic ALS/FLTD. We hope that the global expression profiling and the global CLIP-mapping of FUS in our studies further facilitate discovery of the underlying pathophysiology leading to ALS/FTLD.