Crystal structure of the two N-terminal RRM domains of Pub1 and the poly(U)-binding properties of Pub1
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Abstract The insulin-like growth factor 2 mRNA binding protein (IGF2BP1) is a conserved RNA-binding protein that regulates RNA stability, localization, and translation. IGF2BP1 is part of various ribonucleoprotein (RNP) condensates regulating RNA outputs. However, the mechanism that regulates its assembly into condensates remains unknown. Here we found, using proteomics, that IGF2BP1 phosphorylation at S181 in a disordered linker is regulated in a stress-dependent manner. Phosphomimetic mutations in two disordered linkers, S181E and Y396E, modulated RNP condensate formation by IGF2BP1 without impacting its binding affinity for RNA. Intriguingly, the S181E mutant, which lies in linker 1, impaired IGF2BP1 condensate formation in vitro and in cells, whereas a Y396E mutant in the second linker increased condensate size and dynamics. Structural approaches showed that the first linker binds RNAs nonspecifically through its RGG/RG motif, an interaction weakened in the S181E mutant. Notably, linker 2 interacts with IGF2BP1’s folded domains and these interactions were partially impaired in the Y396E mutant. Our data reveal how phosphorylation modulates low affinity interaction networks in disordered linkers to regulate RNP condensate formation.
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We have previously identified the KH-type RNA-binding protein Rnc1 as an important regulator of the posttranscriptional expression of the MAPK phosphatase Pmp1 in fission yeast. Rnc1 localization in response to stress has not been elucidated thus far. Here, we report the dual roles of Rnc1 in assembly of stress granules (SGs), nonmembranous cytoplasmic foci composed of messenger ribonucleoproteins. Rnc1 can localize to poly(A)-binding protein (Pabp)-positive SGs upon various stress stimuli, including heat shock (HS) and arsenite treatment. Furthermore, Rnc1 deletion results in decreased SGs, indicating that Rnc1 is a new component and a regulator of SGs. Notably, Rnc1 translocates to the dot-like structures faster than Pabp, and this stress-induced Rnc1 translocation does not require its RNA-binding ability, as the Rnc1KH1,2,3GD mutant protein with impaired RNA-binding activity forms dots rather more efficiently than the wild-type Rnc1 upon HS. Interestingly, in the absence of stress, Rnc1 overproduction induced massive aggregation of Pabp-positive SGs and eIF2α phosphorylation. In clear contrast, overproduction of the Rnc1KH1,2,3GD mutant failed to induce Pabp aggregation and eIF2α phosphorylation, indicating that Rnc1 overproduction-induced SG assembly requires Rnc1 RNA-binding activity. Collectively, Rnc1 regulates SG assembly, dependently or independently of its RNA-binding activity.
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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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The processing and ultimately turnover of mRNA are complex processes that are highly regulated.Within a cell,RNA is usually not in a naked form.It forms ribonucleoprotein(RNP) complexes with various RNA-binding proteins(RBPs),thereby influencing processing and turnover events.Poly(C)-binding proteins(PCBPs),generally known as RBPs,interact in a sequence-specific fashion with single-stranded poly(C).They can be divided into two groups: hnRNP K and PCBP1-4.PCBPs and hnRNP K share a common structural motif,the hnRNP K homology(KH) domain,which provides a structural basis for mRNA binding.The KH-domains are components of a modular system,which enables the proteins to be engaged in both protein /nucleic acid and protein /protein interactions.The latter interactions are involved in cell signaling events.As components of different mRNA-protein complexes,PCBPs and hnRNP K have been identified as factors involved in regulating duplication,transcription,mRNA stability and translation.They have also been found related to some pathological processes,such as Hutchinson-Gilford Progeria syndrome,virus infections and malignancy.
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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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La-related proteins (LARPs) share a La motif (LaM) followed by an RNA recognition motif (RRM). Together these are termed the La-module that, in the prototypical nuclear La protein and LARP7, mediates binding to the UUU-3ʹOH termination motif of nascent RNA polymerase III transcripts. We briefly review La and LARP7 activities for RNA 3ʹ end binding and protection from exonucleases before moving to the more recently uncovered poly(A)-related activities of LARP1 and LARP4. Two features shared by LARP1 and LARP4 are direct binding to poly(A) and to the cytoplasmic poly(A)-binding protein (PABP, also known as PABPC1). LARP1, LARP4 and other proteins involved in mRNA translation, deadenylation, and decay, contain PAM2 motifs with variable affinities for the MLLE domain of PABP. We discuss a model in which these PABP-interacting activities contribute to poly(A) pruning of active mRNPs. Evidence that the SARS-CoV-2 RNA virus targets PABP, LARP1, LARP 4 and LARP 4B to control mRNP activity is also briefly reviewed. Recent data suggests that LARP4 opposes deadenylation by stabilizing PABP on mRNA poly(A) tails. Other data suggest that LARP1 can protect mRNA from deadenylation. This is dependent on a PAM2 motif with unique characteristics present in its La-module. Thus, while nuclear La and LARP7 stabilize small RNAs with 3ʹ oligo(U) from decay, LARP1 and LARP4 bind and protect mRNA 3ʹ poly(A) tails from deadenylases through close contact with PABP.Abbreviations: 5ʹTOP: 5ʹ terminal oligopyrimidine, LaM: La motif, LARP: La-related protein, LARP1: La-related protein 1, MLLE: mademoiselle, NTR: N-terminal region, PABP: cytoplasmic poly(A)-binding protein (PABPC1), Pol III: RNA polymerase III, PAM2: PABP-interacting motif 2, PB: processing body, RRM: RNA recognition motif, SG: stress granule.
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