Cell-free Formation of RNA Granules: Bound RNAs Identify Features and Components of Cellular Assemblies

Abstract

Cellular granules lacking boundary membranes harbor RNAs and their associated proteins and play diverse roles controlling the timing and location of protein synthesis. Formation of such granules was emulated by treatment of mouse brain extracts and human cell lysates with a biotinylated isoxazole (b-isox) chemical. Deep sequencing of the associated RNAs revealed an enrichment for mRNAs known to be recruited to neuronal granules used for dendritic transport and localized translation at synapses. Precipitated mRNAs contain extended 3' UTR sequences and an enrichment in binding sites for known granule-associated proteins. Hydrogels composed of the low complexity (LC) sequence domain of FUS recruited and retained the same mRNAs as were selectively precipitated by the b-isox chemical. Phosphorylation of the LC domain of FUS prevented hydrogel retention, offering a conceptual means of dynamic, signal-dependent control of RNA granule assembly.

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

“…In the case of FUS, multisite phosphorylation of the LC dramatically increases the effective number of negatively charged residues in the domain. Complementing previous findings that DNA‐PK treatment disrupts FUS LC recruitment to hydrogel models of granules (Han et al , 2012), here we demonstrate that phosphorylation and phosphomimetic substitution of FUS LC disrupts domain self‐interaction and LLPS. The LC is required for nuclear self‐association of FUS into chromatin‐binding dynamic puncta (Yang et al , 2014) and cytoplasmic granules (Shelkovnikova et al , 2014), as well as robust recruitment at sites of DNA damage (Mastrocola et al , 2013; Altmeyer et al , 2015).…”

“…However, DNA‐PK treatment in vitro followed by either tandem mass spectroscopy or Edman sequencing resulted in identification of only five phosphorylation sites in FUS: S26, S42, S61, S84, and S131 (Gardiner et al , 2008; Han et al , 2012). None of the four conserved TQ sites were detected.…”

“…We and others have suspected that the complete lack of positively charged residues in FUS LC obscures phosphorylation detection by standard proteomic workflows due to lack of positive residues both for efficient positive ion formation for mass spectrometry and for trypsin cleavage sites (Knight et al , 2003). Therefore, to determine the full possible extent of the FUS LC phosphorylation by DNA‐PK, we used in vitro DNA‐PK phosphorylation as described previously (Gardiner et al , 2008; Han et al , 2012; Deng et al , 2014) and detected phosphosites by two‐dimensional solution NMR, which offers direct residue resolution. The appearance of many robust peaks in the phosphoserine and importantly in the phosphothreonine regions of the 1 H 15 N heteronuclear single quantum coherence (HSQC) spectrum immediately suggests FUS can be phosphorylated at more positions than the five previously reported serines (Fig 1B).…”

“…PIKKs phosphorylate their protein targets at serine or threonine residues followed by glutamine (S/TQ; Kim et al , 1999; O'Neill et al , 2000). Indeed, the FUS LC domain has twelve S/TQ motifs (8 SQ, 4 TQ), but previous in vitro DNA‐PK phosphorylation of FUS identified only pS26, pS42, pS61, pS84, and pS131 (Gardiner et al , 2008; Han et al , 2012). …”

“…Interestingly, in vitro phosphorylation of recombinant, isolated FUS LC reduces its ability to bind hydrogels formed from amyloid‐like fibrils of purified recombinant, unphosphorylated FUS LC (Han et al , 2012). However, the effect of FUS phosphorylation on LLPS has not yet been determined.…”