Arginine-enriched mixed-charge domains provide cohesion for nuclear speckle condensation

Abstract

15Low-complexity protein domains promote the formation of various biomolecular 16condensates. However, in many cases, the precise sequence features governing 17 condensate formation and identity remain unclear. Here, we investigate the role of 18 intrinsically disordered mixed-charge domains (MCDs) in nuclear speckle 19 condensation. Proteins composed exclusively of arginine/aspartic-acid dipeptide 20 repeats undergo length-dependent condensation and speckle incorporation. 21Substituting arginine with lysine in synthetic and natural speckle-associated MCDs 22 abolishes these activities, identifying a key role for multivalent contacts through 23 arginine's guanidinium ion. MCDs can synergise with a speckle-associated RNA 24 recognition motif to promote speckle specificity and residence. MCD behaviour is 25 tuneable through net-charge: increasing negative charge abolishes condensation and 26 speckle incorporation. By contrast, increasing positive charge through arginine leads 27 to enhanced condensation, speckle enlargement, decreased splicing factor mobility, 28 and defective mRNA export. Together, these results identify key sequence 29 determinants of MCD-promoted speckle condensation, and link the speckle's dynamic 30 material properties with function in mRNA processing. 31 32 33 34 2015; Nott et al., 2015; Patel et al., 2015). Multivalent interactions among repetitive 58 protein-protein or protein-RNA interaction domains / motifs are the key determinants 59 of LLPS (Dennis, 2015; Fung et al., 2018). Multivalent contacts can be achieved with 60 tandem repeats of folded domains (Li et al., 2012), through low-complexity (LC) 61intrinsically disordered regions (IDRs) (Halfmann, 2016;Lin et al., 2015; Mittag and 62 Parker, 2018), or a combination of the two (Mitrea et al., 2016; Zhang et al., 2015). In 63 these systems, individual contacts are relatively weak. However, with increasing 64 valence, the combined effect of many weak interactions overcome the entropic cost of 65 (Hyman et al., 2014). In many cases LC-IDRs have been shown to be necessary 66 and sufficient for LLPS. This raises key questions regarding the types of residues that 67 promote intermolecular contacts, and the extent to which these determine condensate 68 LLPSResults 103 104 Mixed-charge domains (MCDs) undergo condensation and localize to nuclear 105 speckles 106We previously showed that an arginine and aspartic acid (RD)-enriched MCD 107 from the fungal SPA-5 (SPA-5 MCD ) protein forms a viscoelastic hydrogel. In the fungus, 108 this activity is associated with the formation of cytoplasmic plugs that gate cell-to-cell 109 channels (Lai et al., 2012). To explore the role of such sequences in animal cells, we 110 expressed SPA-5 MCD as an mGFP-fusion in HeLa cells (SPA-5 MCD -mGFP). In this 111 context, SPA-5 MCD -mGFP accumulates in nuclear speckles as revealed by co-112 localization with the speckle marker serine/arginine-rich splicing factor 1 (SRSF1) 113( Figures 1A and S1A). SPA-5 MCD is enriched in RD dipeptide repeats. However, it als...

Nuclear RNA concentration coordinates RNA production with cell size in human cells

“…To test the hypothesis of a negative feedback of nuclear RNA concentration on transcription in an orthogonal manner, we made use of artificial arginine-enriched mixed-charge domain (R-MCD) proteins, which were recently found to drive nuclear mRNA retention (Greig et al, 2020). When expressed in cells, positively charged R-MCD +0.1 -mGFP and R-MCD +0.2 -mGFP localised to nuclear speckles, leading to dose-and charge-dependent nuclear accumulation and cytoplasmic depletion of mRNA (Figure 6G-H, S11E-F), in line with previous data.…”

Middle-Down Proteomics Reveals Dense Sites of Methylation and Phosphorylation in Arginine-Rich RNA-Binding Proteins

“…The “BAD” acronym of LC1/BAD stands for “basic–acidic dipeptide”, containing dipeptide repeats of a basic (K/R) residue adjacent to an acidic residue (D/E). The U1-70K LC1/BAD domain has both BAD and RS motifs and can form condensates via liquid–liquid phase separation (LLPS) and eventually aggregates in vitro and in vivo. , The LC1/BAD domain of U1-70K is of particular biological interest due to its central role in U1-70K nuclear localization, granule formation, and coaggregation with tau in Alzheimer’s disease. ,, On PeptideAtlas.org, there are currently over 50 000 peptide spectral matches (PSMs) to U1-70K using standard bottom-up approaches with complete trypsin digestion, mapping approximately 70% of the protein . Despite this number of PSMs, the LC1/BAD domain has only 7.5% coverage (6 out of 80 residues). , In comparison, the PTM repository Phosphosite.org shows several phosphorylated peptides within the LC1/BAD domain, albeit with few repeated observations .…”

“…The U1-70K LC1/BAD domain has both BAD and RS motifs and can form condensates via liquid−liquid phase separation (LLPS) and eventually aggregates in vitro and in vivo. 4,5 The LC1/BAD domain of U1-70K is of particular biological interest due to its central role in U1-70K nuclear localization, granule formation, and coaggregation with tau in Alzheimer's disease. 3,52,59 On PeptideAtlas.org, there are currently over 50 000 peptide spectral matches (PSMs) to U1-70K using standard bottom-up approaches with complete trypsin digestion, mapping approximately 70% of the protein.…”

“…In addition to regulating localization and molecular interactions, PTMs within Arg-rich domains may also trigger toxic gain-of-function pathological properties in neurodegenerative disease. For example, the net positive charge within the disordered LC1/BAD domain of U1-70K mediates the condensation, insolubility, and, eventually, aggregation of U1-70K. , Importantly, phosphorylation PTMs within LC1/BAD may be a mechanism to regulate the condensation behavior of U1-70K by altering the overall charge within the LC1/BAD domain. Similarly, phosphorylation within the LC domain of FUS acts to prevent LLPS, aggregation, and toxicity in vitro .…”

“…One class of these RBPs contains highly repetitive complementary repeats of basic (K/R) and acidic (D/E) residues that we have previously referred to as basic–acidic dipeptide (BAD) domains . Importantly, BAD domains facilitate protein condensation and eventual aggregation , and, in the context of Alzheimer’s disease, regulate interactions with the pathological Tau protein . A second subset, related to the BAD proteins, are the arginine/serine-rich (RS) domains that are ubiquitous in the serine/arginine (SR) family of proteins .…”