Arginine-enriched mixed-charge domains provide cohesion for nuclear speckle condensation
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Abstract Low-complexity protein domains promote the formation of various biomolecular condensates. However, in many cases, the precise sequence features governing condensate formation and identity remain unclear. Here, we investigate the role of intrinsically disordered mixed-charge domains (MCDs) in nuclear speckle condensation. Proteins composed exclusively of arginine/aspartic-acid dipeptide repeats undergo length-dependent condensation and speckle incorporation. Substituting arginine with lysine in synthetic and natural speckle-associated MCDs abolishes these activities, identifying a key role for multivalent contacts through arginine’s guanidinium ion. MCDs can synergise with a speckle-associated RNA recognition motif to promote speckle specificity and residence. MCD behaviour is tuneable through net-charge: increasing negative charge abolishes condensation and speckle incorporation. By contrast, increasing positive charge through arginine leads to enhanced condensation, speckle enlargement, decreased splicing factor mobility, and defective mRNA export. Together, these results identify key sequence determinants of MCD-promoted speckle condensation, and link the speckle’s dynamic material properties with function in mRNA processing.Keywords:
RNA recognition motif
Linker
Splicing factor
Splicing factor RBM10 and its close homologues RBM5 and RBM6 govern the splicing of oncogenes such as Fas, NUMB, and Bcl-X. The molecular architecture of these proteins includes zinc fingers (ZnFs) and RNA recognition motifs (RRMs). Three of these domains in RBM10 that constitute the RNA binding part of this splicing factor were found to individually bind RNAs with micromolar affinities. It was thus of interest to further investigate the structural basis of the well-documented high-affinity RNA recognition by RMB10. Here, we investigated RNA binding by combinations of two or three of these domains and discovered that a polypeptide containing RRM1, ZnF1, and RRM2 connected by their natural linkers recognizes a specific sequence of the Fas exon 6 mRNA with an affinity of 20 nM. Nuclear magnetic resonance structures of the RBM10 domains RRM1 and ZnF1 and the natural V354del isoform of RRM2 further confirmed that the interactions with RNA are driven by canonical RNA recognition elements. The well-known high-fidelity RNA splice site recognition by RBM10, and probably by RBM5 and RBM6, can thus be largely rationalized by a cooperative binding action of RRM and ZnF domains.
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TheArabidopsisSR45 splicing factor bridges the splicing machinery and the exon-exon junction complex
Abstract The Arabidopsis splicing factor serine/arginine-rich 45 (SR45) contributes to several biological processes. The sr45-1 loss-of-function mutant exhibits delayed root development, late flowering, unusual numbers of floral organs, shorter siliques with decreased seed sets, narrower leaves and petals, and altered metal distribution. SR45 bears a unique RNA recognition motif (RRM) flanked by one serine/arginine-rich (RS) domain on both sides. Here, we studied the function of each of SR45 domains by examining their involvement in: (i) the spatial distribution of SR45, (ii) the establishment of a protein-protein interaction network including spliceosomal and exon-exon junction complex (EJC) components, and (iii) the RNA binding specificity. We report that the endogenous SR45 promoter is active during vegetative and reproductive growth, and that the SR45 protein localizes in the nucleus. We demonstrate that the C-terminal arginine/serine-rich domain is a determinant of nuclear localization. We show that the SR45 RNA recognition motif (RRM) domain specifically binds purine-rich RNA motifs via three residues (H101, H141, Y143), and is also involved in protein-protein interactions. We further show that SR45 bridges both mRNA splicing and surveillance machineries as a partner of EJC core components and peripheral factors, which requires phosphoresidues likely phosphorylated by kinases from both CLK and SRPK families. Our findings provide insights into the contribution of each SR45 domain to both spliceosome and EJC assemblies. Highlight The contribution of the Arabidopsis SR45 splicing factor individual domains to its nuclear localization, ability to contact in planta novel protein partners and specifically bind RNA motifs was examined.
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The sequence-specific recognition of RNA by proteins is mediated through various RNA binding domains, with the RNA recognition motif (RRM) being the most frequent and present in >50% of RNA-binding proteins (RBPs). Many RBPs contain multiple RRMs, and it is unclear how each RRM contributes to the binding specificity of the entire protein. We found that RRMs within the same RBP (i.e., sibling RRMs) tend to have significantly higher similarity than expected by chance. Sibling RRM pairs from RBPs shared by multiple species tend to have lower similarity than those found only in a single species, suggesting that multiple RRMs within the same protein might arise from domain duplication followed by divergence through random mutations. This finding is exemplified by a recent RRM domain duplication in DAZ proteins and an ancient duplication in PABP proteins. Additionally, we found that different similarities between sibling RRMs are associated with distinct functions of an RBP and that the RBPs tend to contain repetitive sequences with low complexity. Taken together, this study suggests that the number of RBPs with multiple RRMs has expanded in mammals and that the multiple sibling RRMs may recognize similar target motifs in a cooperative manner.
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Many splicing factors in vertebrate nuclei belong to a class of evolutionarily conserved proteins containing arginine/serine (RS) or serine/arginine (SR) domains. Previously, we demonstrated the existence of SR splicing factors in plants. In this article, we report on a novel member of this splicing factor family from Arabidopsis designated atRSp31. It has one N-terminal RNA recognition motif and a C-terminal RS domain highly enriched in arginines. The RNA recognition motif shows significant homology to all animal SR proteins identified to date, but the intermediate region does not show any homology to any other known protein. Subsequently, we characterized two cDNAs from Arabidopsis that are highly homologous to atRSp31 (designated atRSp35 and atRSp41). Their deduced amino acid sequences indicate that these proteins constitute a new family of RS domain splicing factors. Purified recombinant atRSp31 is able to restore splicing in SR protein-deficient human S100 extracts. This indicates that atRSp31 is a true plant splicing factor and plays a crucial role in splicing, similar to that of other RS splicing factors. All of the three genes are differentially expressed in a tissue-specific manner. The isolation of this new plant splicing factor family enlarges the essential group of RS domain splicing factors. Furthermore, because no animal equivalent to this protein family has been identified to date, our results suggest that these proteins play key roles in constitutive and alternative splicing in plants.
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Serine/arginine (SR) proteins, one of the major families of alternative-splicing regulators in Eukarya, have two types of RNA-recognition motifs (RRMs): a canonical RRM and a pseudo-RRM. Although pseudo-RRMs are crucial for activity of SR proteins, their mode of action was unknown. By solving the structure of the human SRSF1 pseudo-RRM bound to RNA, we discovered a very unusual and sequence-specific RNA-binding mode that is centered on one α-helix and does not involve the β-sheet surface, which typically mediates RNA binding by RRMs. Remarkably, this mode of binding is conserved in all pseudo-RRMs tested. Furthermore, the isolated pseudo-RRM is sufficient to regulate splicing of about half of the SRSF1 target genes tested, and the bound α-helix is a pivotal element for this function. Our results strongly suggest that SR proteins with a pseudo-RRM frequently regulate splicing by competing with, rather than recruiting, spliceosome components, using solely this unusual RRM.
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In recent years, much progress has been made in elucidating the functional roles of plant glycine-rich RNA-binding proteins (GR-RBPs) during development and stress responses. Canonical GR-RBPs contain an RNA recognition motif (RRM) or a cold-shock domain (CSD) at the N-terminus and a glycine-rich domain at the C-terminus, which have been associated with several different RNA processes, such as alternative splicing, mRNA export and RNA editing. However, many aspects of GR-RBP function, the targeting of their RNAs, interacting proteins and the consequences of the RNA target process are not well understood. Here, we discuss recent findings in the field, newly defined roles for GR-RBPs and the actions of GR-RBPs on target RNA metabolism.
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RNA-binding proteins are a large group of structurally diverse molecules that associate with nascent transcripts and other proteins through their structural and functional domains. The presence of multiple copies and various arrangements of the different structural domains are important in generating functional versatility as well as defining specificity for these RNA-binding proteins. As a result, RNA-binding proteins often perform both specific and redundant or overlapping functions in multiple aspects of ribonucleic acid metabolism. The heterogeneous nuclear ribonucleoproteins (hnRNPs) and the serine/arginine-rich (SR) proteins are two of the most abundant groups of RNA-binding proteins that share similar functional properties (binding to nascent transcripts, antagonistic effect on alternative splicing, and multiple roles in various aspects of RNA metabolism). These two groups of proteins, especially the hnRNP A/B subgroup and the core SR proteins, also share common features within their protein structure, which are comprised of one or more RNA recognition motifs (RRMs) linked to an auxiliary domain that is glycine-rich or arginine/serine-rich, respectively. Despite the similarities, members from the hnRNPs and the SR proteins also perform specific RNA regulatory functions. Functional studies have repeatedly emphasized that RNA-binding specificity is achieved by the combinatorial effect of the different domains within the RNA-binding protein structure. The RRM is a well-studied RNA-binding motif that is common to many RBPs. The less-studied auxiliary domain, especially the glycine-rich domain, is known to be involved in protein-protein and protein-RNA interactions. Although specific functions are assigned for the different domains of hnRNPs A/B and SR proteins, these domains are generally studied and characterized apart. Hence, in order to understand the significance of modularity in contributing to both specific and non-specific functions of an RNA-binding protein, it is important to understand the relationships between their protein domains and how these domains cooperate as a complete unit. To examine similarities and differences between the RNA-binding proteins that contribute to both functional redundancy and specificity, sequence and phylogenetic analyses at the protein, domain and functional motif levels were performed in Chapter 3 to gain a broader understanding of their evolutionary relationships. We found that there are significant sequence similarities between the different groups of RNA-binding proteins studied, including the hnRNPs and SR proteins, which share common RNA-binding RRMs. Phylogenetic analyses of the RRM domains between these proteins however, suggest a diversification of the domain sequences in the early evolution of metazoans, and subsequently strong selective pressure to maintain overall domain structure and their consensus motif sequences. Hence, although RRMs are structurally and functionally conserved, there are limited sequence similarities within the RRMs between different RNA-binding proteins that can contribute to target selectivity. This diversification, therefore, is thought to allow RRM-containing RNA-binding proteins to have redundant as well as specific roles in co- and post-transcriptional processing. In addition, the presence of the different auxiliary domains that link with these RRMs further facilitates the functional specialization of the different RNA-binding proteins in RNA regulation. The importance of the auxiliary domains, especially the glycinenrich and the arginine/serine-rich domain from hnRNPs and SR proteins, in specifying biochemical activities of a RNA-binding protein were further addressed in Chapter 4 and 5. We created a series of chimeric proteins consisting of domain swaps between different hnRNP A/B paralogues, and between hnRNPs A/B and SR proteins, to examine the contributions of the different functional domains in sub-nuclear localization of these proteins. We found that the glycine-rich domain of hnRNPs A/B, rather than the RRMs, has a dominant role in determining the nuclear localization and the enrichment of the wild-type and heterologous proteins in different subnuclear compartments. As well, both the RRMs and the arginine/serine-rich domain are irreplaceable in specifying the functional characteristics of the SR proteins. Furthermore, we confirmed that the glycine-rich domain is important in mediating the co-localization of hnRNPs A/B with the paraspeckle protein p54nrb within the subnuclear paraspeckle compartment. Interestingly, immunoprecipitation and RT-PCR analyses suggested that the hnRNPs A/B directly interact with the paraspeckle structural component NEAT1 long non-coding transcript, but not p54nrb. Hence, our protein subnuclear localization studies have collectively suggested that the auxiliary domain is an essential determinant in conferring specificity to the RNA-binding proteins in RNA regulation. In conclusion, this study highlights the significance of the cooperation between the different functional domains, especially the RRMs and the auxiliary domain, in both redundant and specific functions of the different RNA-binding proteins. In particular, our results have reinforced the view that specific RNA-binding protein functions are determined by the combination of different structural domains function in concert as a complete unit. Altogether, this study provides a better understanding of how RNA-binding proteins benefit from the complex modular networks between the different domains that contribute to both sequence-specific and non-specific activities of these proteins in essential gene regulation.
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Abstract The Arabidopsis splicing factor serine/arginine-rich 45 (SR45) contributes to several biological processes. The sr45-1 loss-of-function mutant exhibits delayed root development, late flowering, unusual numbers of floral organs, shorter siliques with decreased seed sets, narrower leaves and petals, and altered metal distribution. SR45 bears a unique RNA recognition motif (RRM) flanked by one serine/arginine-rich (RS) domain on both sides. Here, we studied the function of each SR45 domains by examining their involvement in: (i) the spatial distribution of SR45; (ii) the establishment of a protein–protein interaction network including spliceosomal and exon–exon junction complex (EJC) components; and (iii) the RNA binding specificity. We report that the endogenous SR45 promoter is active during vegetative and reproductive growth, and that the SR45 protein localizes in the nucleus. We demonstrate that the C-terminal arginine/serine-rich domain is a determinant of nuclear localization. We show that the SR45 RRM domain specifically binds purine-rich RNA motifs via three residues (H101, H141, and Y143), and is also involved in protein–protein interactions. We further show that SR45 bridges both mRNA splicing and surveillance machineries as a partner of EJC core components and peripheral factors, which requires phosphoresidues probably phosphorylated by kinases from both the CLK and SRPK families. Our findings provide insights into the contribution of each SR45 domain to both spliceosome and EJC assemblies.
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