RNA targets of wild-type and mutant FET family proteins

Abstract

FUS, EWSR1 and TAF15, constituting the FET protein family, are abundant, highly conserved RNA-binding proteins with important roles in oncogenesis and neuronal disease, yet their RNA targets and recognition elements are unknown. Using PAR-CLIP, we defined global RNA targets for all human FET proteins and two ALS-causing human FUS mutants. FET members showed similar binding profiles, whereas FUS mutants showed a drastically altered binding pattern, consistent with changes in subcellular localization.

Figure 1

Protein-RNA interaction maps of FET (FUS, EWSR1, TAF15) proteins. (a) Phosphorimages of SDS-PAGE gels that resolved ³²P-labeled RNA–FLAGHA–FUS or EWSR1 or TAF15 PAR-CLIP immunoprecipitates. Excised regions are indicated by arrows. Protein identities of these bands were confirmed by mass spectrometry (not shown). Western blots (WB) were probed with an anti-HA antibody. (b) Hierarchical clustering diagram of binding patterns based on the number of reads per gene and Spearman correlation. Three unrelated reference datasets were included for comparison. Binding profiles were mean intensity normalized. Similar results were obtained when datasets were size-normalized (data not shown). Stable, constitutive expression of the indicated protein; inducible, inducible expression of the indicated protein. (c) Overlap frequencies based on the top 1000 crosslinked clusters (CCs) of each protein, based on the number of sequence reads. CCs were considered overlapping when center positions were within 10 nucleotides (nt). Scatter plots show the reproducibility in number of reads per overlapping site. Correlations (Pearson’s R) were calculated based on log-transformed values. (d) Venn diagrams that illustrate overlaps between genes targeted by the three FET proteins, as well as between FUS and mutant FUS. (e) Distribution of CCs across intronic and exonic regions of RefSeq mRNAs. (f) Positional distribution of CCs near intron-exon junctions show enriched binding upstream of the 3′ splice site (3′ SS, arrow). The Y-axis indicates the number of observed CCs per 4 nt segment. The P-value for observing a peak of similar magnitude or higher anywhere in a 10.000 nt region upstream of the SS was in all cases < 0.025 (based on randomization of CC positions within introns).

Figure 2

RNA-binding preferences of wild-type FET and mutant FUS proteins. (a) Genomic sequences of representative FET CCs. All clusters are present in all wild-type FET and mutant FUS datasets. Green shading indicates stems (“left” and “right”), orange shading the nucleotides closing the loop. The most frequent crosslinking positions are underscored. Gene name and number of reads are indicated. (b) Phosphorimage of native PAGE resolving complexes of recombinant FUS protein with different RNA oligoribonucleotides (all at 1 nM): GGU×12, AUU×12, SON (stem in natural left-right configuration as indicated in panel A), altered SON (non-complementary left-left stem), altered SON (non-complementary right-right stem), and altered SON (reconstituted right-left stem). Additionally, the effects of changing the “UA” opening the loop are shown (UA shifted, no UA in loop, 1st U deleted). Concentrations of FUS protein ranged from 1000 nM to 0 nM (lanes 1 to 10; fractions of bound vs. unbound protein can be found in Supplementary Data 3). Dissociation constants (Kd) are indicated; n.d., non-determinable. (c) Proposed model of the FET protein RRE. n is an integer ≥ 1 indicating variable stem and loop length.