Interactome analyses revealed that the U1 snRNP machinery overlaps extensively with the RNAP II machinery and contains multiple ALS/SMA-causative proteins

Abstract

Mutations in multiple RNA/DNA binding proteins cause Amyotrophic Lateral Sclerosis (ALS). Included among these are the three members of the FET family (FUS, EWSR1 and TAF15) and the structurally similar MATR3. Here, we characterized the interactomes of these four proteins, revealing that they largely have unique interactors, but share in common an association with U1 snRNP. The latter observation led us to analyze the interactome of the U1 snRNP machinery. Surprisingly, this analysis revealed the interactome contains ~220 components, and of these, >200 are shared with the RNA polymerase II (RNAP II) machinery. Among the shared components are multiple ALS and Spinal muscular Atrophy (SMA)-causative proteins and numerous discrete complexes, including the SMN complex, transcription factor complexes, and RNA processing complexes. Together, our data indicate that the RNAP II/U1 snRNP machinery functions in a wide variety of molecular pathways, and these pathways are candidates for playing roles in ALS/SMA pathogenesis.

Figure 1

FET proteins and MATR3 associate with U1 snRNP. (a) Immunoprecipitations (IPs) were carried out with antibodies to FET proteins or MATR3 followed by analysis on a Coomassie-stained gel. Molecular weight markers and protein identified by mass spectrometry are indicated. (b) IPs were carried out from nuclear extract using a negative control antibody (EIF4A3) or an antibody to the SNRPC subunit of the U1 snRNP followed by Westerns with the indicated antibodies. (c) IPs were carried out with the indicated antibodies from nuclear extract treated with a U1 snRNA AMO or a negative control AMO followed by Western using the SNRPC antibody. (d) Same as (c) except that total RNAs from the IPs were examined on a denaturing gel stained with ethidium bromide.

Figure 2

Top hit interactors in the FET proteins and MATR3 interactomes. The top ranked (by total peptide number) proteins in each interactome are shown. The rank, HGNC official symbol, calculated molecular weight, best-known function, total and unique peptide counts are shown. Functions in splicing (pink), transcription (txn, orange), DNA damage response (green), neuronal (blue) and other (black) are indicated. The symbols of ALS-causative proteins are in red. The stars indicate U1 snRNP components.

Figure 3

Protein-protein interaction network of the FUS interactome. The network of the FUS interactome constructed using STRING database (confidence score >0.7) is shown. Disconnected nodes are omitted. Protein complexes and clusters of functionally related proteins are indicated by yellow circles and blue circles, respectively. The ALS and SMA-causative proteins are in red.

Figure 4

Protein-protein interaction network of the EWSR1 interactome. Same as Fig. 3, except that the network of the EWSR1 interactome is shown. The ALS and SMA causative proteins are in red.

Figure 5

Protein-protein interaction network of the TAF15 interactome. Same as Fig. 3, except that the network of the TAF15 interactome is shown. The ALS causative proteins are in red.

Figure 6

Protein-protein interaction network of the MATR3 interactome. Same as Fig. 3, except that the network of the MATR3 interactome is shown. The ALS causative proteins are in red.

Figure 7

Protein-protein interaction network of the U1 snRNP machinery. Same as Fig. 3, except that the network of the U1 snRNP interactome is shown.

Figure 8

The U1 snRNP machinery overlaps with the RNAP II machinery. (a) Venn diagram showing overlap of the U1 snRNP and RNAP II machineries. (b) HeLa cell nuclear extract was separated on a Sephacryl-S500 column. The indicated fractions were used for Western analyses with antibodies against RNAP II and U1 snRNP components (SNRPA and SNRPC). Fraction 25 is the void volume and 69 is the included volume. (c) IPs were carried out from nuclear extract using an antibody to the POLR2A subunit of the RNAP II (left panel) or an antibody to the SNRPC subunit of the U1 snRNP (right panel) as well as a negative control antibody (EIF4A3) followed by Westerns with antibodies to the DEAD box helicases (DHX9, DDX5 and DDX17). (d) IPs were carried out with the indicated antibodies from nuclear extract treated with a U1 snRNA AMO or a negative control AMO followed by Western using the SNRPC antibody.