Electrostatic modulation of hnRNPA1 low‐complexity domain liquid–liquid phase separation and aggregation

Abstract

Membrane‐less organelles and RNP granules are enriched in RNA and RNA‐binding proteins containing disordered regions. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key regulating protein in RNA metabolism, localizes to cytoplasmic RNP granules including stress granules. Dysfunctional nuclear‐cytoplasmic transport and dynamic phase separation of hnRNPA1 leads to abnormal amyloid aggregation and neurodegeneration. The intrinsically disordered C‐terminal domain (CTD) of hnRNPA1 mediates both dynamic liquid–liquid phase separation (LLPS) and aggregation. While cellular phase separation drives the formation of membrane‐less organelles, aggregation within phase‐separated compartments has been linked to neurodegenerative diseases. To understand some of the underlying mechanisms behind protein phase separation and LLPS‐mediated aggregation, we studied LLPS of hnRNPA1 CTD in conditions that probe protein electrostatics, modulated specifically by varying pH conditions, and protein, salt and RNA concentrations. In the conditions investigated, we observed LLPS to be favored in acidic conditions, and by high protein, salt and RNA concentrations. We also observed that conditions that favor LLPS also enhance protein aggregation and fibrillation, which suggests an aggregation pathway that is LLPS‐mediated. The results reported here also suggest that LLPS can play a direct role in facilitating protein aggregation, and that changes in cellular environment that affect protein electrostatics can contribute to the pathological aggregation exhibited in neurodegeneration.

m6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress

“…The strongest interaction was observed with m 6 A-modified Fragment 6, and weak binding to unmodified Fragment 6 (Figure 1B, WT). WT TLS/FUS did not bind to Fragment 3, regardless of m 6 A modification. On the other hand, ALSrelated TLS/FUS mutants demonstrated lower binding specificity compared to WT.…”

“…We first examined if RNA m 6 A modification could alter the interaction between RNA and TLS/FUS. For this purpose, we generated two RNA fragments derived from pncRNA-D, an lncRNA expressed from cyclin D1 promoter (Figure 1A, black box, [29]).…”

“…For this purpose, we generated two RNA fragments derived from pncRNA-D, an lncRNA expressed from cyclin D1 promoter (Figure 1A, black box, [29]). In a previous study, we defined GGACU motif at the 467 nt of pncRNA-D is m 6 A modified, and 20 nt RNA fragment (named Fragment 6, Figure 1A) around this motif robustly binds to TLS/FUS [30]. This time, we selected an additional GGACC motif at the position of 249 nt of pncRNA-D, which is speculated to be m 6 A modified (Fragment 3, Figure 1A).…”

“…LLPS has received much attention in the field of RNA binding proteins (RBPs). Many RBPs have an intrinsically disordered region (IDR), the region which does not fold into highorder structures [5][6][7][8]. IDRs interact by themselves, forming membraneless particles, which could isolate contents from external environments.…”

“…m 6 A modification is one of the most abundant RNA modifications found in mRNAs as well as in noncoding RNAs. Its function covers RNA degradation, translational inhibition, and alteration of interaction with proteins [26].…”