An official website of the United States government
Phase separation plays crucial roles in both sustaining cellular function and perpetuating disease states. Despite extensive studies, our understanding of this process is hindered by low solubility of phase-separating proteins. One example of this is found in SR and SR-related proteins. These proteins are characterized by domains rich in arginine and serine (RS domains), which are essential to alternative splicing and in vivo phase separation. However, they are also responsible for a low solubility that has made these proteins difficult to study for decades. Here, we solubilize the founding member of the SR family, SRSF1, by introducing a peptide mimicking RS repeats as a co-solute. We find that this RS-mimic peptide forms interactions similar to those of the protein’s RS domain. Both interact with a combination of surface-exposed aromatic residues and acidic residues on SRSF1’s RNA Recognition Motifs (RRMs) through electrostatic and cation-pi interactions. Analysis of RRM domains from human SR proteins indicates that these sites are conserved across the protein family. In addition to opening an avenue to previously unavailable proteins, our work provides insight into how SR proteins phase separate and participate in nuclear speckles.
more »
« less
Controlled by disorder: Phosphorylation modulates SRSF1 domain availability for spliceosome assembly
Abstract Serine/arginine‐rich splicing factor 1 (SRSF1) is key in the mRNA lifecycle including transcription, splicing, nonsense‐mediated decay, and nuclear export. Consequently, its dysfunction is linked to cancers, viral evasion, and developmental disorders. The functionality of SRSF1 relies on its interactions with other proteins and RNA molecules. These processes are regulated by phosphorylation of its unstructured arginine/serine‐rich tail (RS). Here, we characterize how phosphorylation affects SRSF1's protein and RNA interaction and phase separation. Using NMR paramagnetic relaxation enhancement and chemical shift perturbation, we find that when unphosphorylated, SRSF1's RS interacts with its first RNA‐recognition motif (RRM1). Phosphorylation of RS decreases its interactions with the protein‐binding site of RRM1 and increases its interactions with the RNA‐binding site of RRM1. This change in SRSF1's intramolecular interactions increases the availability of protein‐interacting sites on RRM1 and weakens RNA binding of SRSF1. Phosphorylation alters the phase separation of SRSF1 by diminishing the role of arginine in intermolecular interactions. These findings provide an unprecedented view of how SRSF1 influences the early‐stage spliceosome assembly.
more »
« less
- Award ID(s):
- 2024964
- PAR ID:
- 10572578
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 34
- Issue:
- 3
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein.more » « less
-
Musier-Forsyth, Karin (Ed.)RNA-binding proteins play crucial roles in various cellular functions, and contain abundant disordered protein regions. The disordered regions in RNA-binding proteins are rich in repetitive sequences, such as poly-K/R, poly-N/Q, poly-A, and poly-G residues. Our bioinformatic analysis identified a largely neglected repetitive sequence family we define as electronegative clusters (ENCs) that contain acidic residues and/or phosphorylation sites. The abundance and length of ENCs exceed other known repetitive sequences. Despite their abundance, the functions of ENCs in RNA-binding proteins are still elusive. To investigate the impacts of ENCs on protein stability, RNA-binding affinity, and specificity, we selected one RNA-binding protein, the ribosomal biogenesis factor 15 (Nop15) as a model. We found that the Nop15 ENC increases protein stability and inhibits nonspecific RNA binding, but minimally interferes with specific RNA binding. To investigate the effect of ENCs on sequence specificity of RNA binding, we grafted an ENC to another RNA-binding protein, Ser/Arg-rich splicing factor 3 (SRSF3). Using RNA Bind-n-Seq, we found that the engineered ENC inhibits disparate RNA motifs differently, instead of weakening all RNA motifs to the same extent. The motif site directly involved in electrostatic interaction is more susceptible to the ENC inhibition. These results suggest that one of functions of ENCs is to regulate RNA binding via electrostatic interaction. This is consistent with our finding that ENCs are also overrepresented in DNA-binding proteins, while underrepresented in halophiles, in which nonspecific nucleic acid binding is inhibited by high concentrations of salts.more » « less
-
RNA molecules often play critical roles in assisting the formation of membraneless organelles in eukaryotic cells. Yet, little is known about the organization of RNAs within membraneless organelles. Here, using super-resolution imaging and nuclear speckles as a model system, we demonstrate that different sequence domains of RNA transcripts exhibit differential spatial distributions within speckles. Specifically, we image transcripts containing a region enriched in binding motifs of serine/arginine-rich (SR) proteins and another region enriched in binding motifs of heterogeneous nuclear ribonucleoproteins (hnRNPs). We show that these transcripts localize to the outer shell of speckles, with the SR motif-rich region localizing closer to the speckle center relative to the hnRNP motif-rich region. Further, we identify that this intra-speckle RNA organization is driven by the strength of RNA-protein interactions inside and outside speckles. Our results hint at novel functional roles of nuclear speckles and likely other membraneless organelles in organizing RNA substrates for biochemical reactions.more » « less
-
Abstract MOV10 is an RNA helicase that associates with the RNA‐induced silencing complex component Argonaute (AGO), likely resolving RNA secondary structures. MOV10 also binds the Fragile X mental retardation protein to block AGO2 binding at some sites and associates with UPF1, a principal component of the nonsense‐mediated RNA decay pathway. MOV10 is widely expressed and has a key role in the cellular response to viral infection and in suppressing retrotransposition. Posttranslational modifications of MOV10 include ubiquitination, which leads to stimulation‐dependent degradation, and phosphorylation, which has an unknown function. MOV10 localizes to the nucleus and/or cytoplasm in a cell type‐specific and developmental stage‐specific manner. Knockout ofMov10leads to embryonic lethality, underscoring an important role in development where it is required for the completion of gastrulation. MOV10 is expressed throughout the organism; however, most studies have focused on germline cells and neurons. In the testes, the knockdown ofMov10disrupts proliferation of spermatogonial progenitor cells. In brain, MOV10 is significantly elevated postnatally and binds mRNAs encoding cytoskeleton and neuron projection proteins, suggesting an important role in neuronal architecture. HeterozygousMov10mutant mice are hyperactive and anxious and their cultured hippocampal neurons have reduced dendritic arborization. Zygotic knockdown ofMov10inXenopus laeviscauses abnormal head and eye development and mislocalization of neuronal precursors in the brain. Thus, MOV10 plays a vital role during development, defense against viral infection and in neuronal development and function: its many roles and regulation are only beginning to be unraveled. This article is categorized under:RNA Interactions with Proteins and Other Molecules > RNA‐Protein ComplexesRNA Interactions with Proteins and Other Molecules > Protein‐RNA Interactions: Functional Implicationsmore » « less
