Tau oligomers mediate aggregation of RNA‐binding proteins Musashi1 and Musashi2 inducing Lamin alteration

The exact mechanisms leading to neurodegeneration in Alzheimer's disease (AD) and other tauopathies are not yet entirely understood. However, it is known that several RNA‐binding proteins (RBPs) form toxic aggregates and also interact with tau in such granules in tauopathies, including AD. The Musashi (MSI) family of RBPs, consisting of two homologues: Musashi1 and Musashi2, have not been extensively investigated in neurodegenerative diseases. Here, using a tau inducible HEK (iHEK) model we investigate whether MSI proteins contribute to the aggregation of toxic tau oligomers (TauO). Wild‐type and mutant P301L tau iHEK cells are used to study the effect of different tau variants on the cellular localization of MSI proteins. Interestingly, we observe that tau co‐localizes with MSI in the cytoplasm and nuclei, altering the nuclear transport of MSI. Furthermore, incremental changes in the size and density of nuclear MSI/tau foci are observed. We also report here that TauO interact with MSI to cause the formation of distinct nuclear aggregates. Moreover, tau/MSI aggregates induce structural changes to LaminB1, leading to nuclear instability. These results illustrate a possible mechanism of neurodegeneration mediated by the aggregation of MSI proteins and TauO, suggesting that MSI plays a critical role in cellular dysfunction.     

动植物中RNA结合蛋白的研究进展

RNA结合蛋白(RNA-binding proteins)在转录后基因表达调节中起着重要的作用,它通过和RNA相互作用来调节细胞的功能。RNA结合蛋白参与RNA剪接、多聚腺苷化作用、序列编辑、RNA转运、维持RNA的稳定和降解、细胞内定位和翻译控制等RNA代谢的各个方面。主要介绍了RNA结合蛋白的结构、靶标RNA及RNA结合蛋白在动植物和疾病中的研究。     

RNA-binding proteins in neurological diseases

Emerging studies support that RNA-binding proteins(RBPs)play critical roles in human biology and pathogenesis.RBPs are essential players in RNA processing and metabolism,including pre-mRNA splicing,polyadenylation,transport,surveillance,mRNA localization,mRNA stability control,translational control and editing of various types of RNAs.Aberrant expression of and mutations in RBP genes affect various steps of RNA processing,altering target gene function.RBPs have been associated with various diseases,including neurological diseases.Here,we mainly focus on selected RNA-binding proteins including Nova-1/Nova-2,HuR/HuB/HuC/HuD,TDP-43,Fus,Rbfox1/Rbfox2,QKI and FMRP,discussing their function and roles in human diseases.     

Structure of the central RNA recognition motif of human TIA-1 at 1.95A resolution

T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in the cytoplasm, by recognizing uridine-rich sequences of RNAs. As a step towards understanding RNA recognition by this regulatory factor, the X-ray structure of the central RNA recognition motif (RRM2) of human TIA-1 is presented at 1.95 Å resolution. Comparison with structurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1-RRM2. The versatile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein-protein interactions mediated by the RNP motifs of TIA-1-RRM2. Importantly, the TIA-1-RRM2 structure reveals the locations of mutations responsible for inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior of the domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding.     

A Conserved Three-nucleotide Core Motif Defines Musashi RNA Binding Specificity

Musashi (MSI) family proteins control cell proliferation and differentiation in many biological systems. They are overexpressed in tumors of several origins, and their expression level correlates with poor prognosis. MSI proteins control gene expression by binding RNA and regulating its translation. They contain two RNA recognition motif (RRM) domains, which recognize a defined sequence element. The relative contribution of each nucleotide to the binding affinity and specificity is unknown. We analyzed the binding specificity of three MSI family RRM domains using a quantitative fluorescence anisotropy assay. We found that the core element driving recognition is the sequence UAG. Nucleotides outside of this motif have a limited contribution to binding free energy. For mouse MSI1, recognition is determined by the first of the two RRM domains. The second RRM adds affinity but does not contribute to binding specificity. In contrast, the recognition element for Drosophila MSI is more extensive than the mouse homolog, suggesting functional divergence. The short nature of the binding determinant suggests that protein-RNA affinity alone is insufficient to drive target selection by MSI family proteins.     

Structure-based prediction of RNA-binding domains and RNA-binding sites and application to structural genomics targets

Mechanistic understanding of many key cellular processes often involves identification of RNA binding proteins (RBPs) and RNA binding sites in two separate steps. Here, they are predicted simultaneously by structural alignment to known protein-RNA complex structures followed by binding assessment with a DFIRE-based statistical energy function. This method achieves 98% accuracy and 91% precision for predicting RBPs and 93% accuracy and 78% precision for predicting RNA-binding amino-acid residues for a large benchmark of 212 RNA binding and 6761 non-RNA binding domains (leave-one-out cross-validation). Additional tests revealed that the method makes no false positive prediction from 311 DNA binding domains but correctly detects six domains binding with both DNA and RNA. In addition, it correctly identified 31 of 75 unbound RNA-binding domains with 92% accuracy and 65% precision for predicted binding residues and achieved 86% success rate in its application to SCOP RNA binding domain superfamily (Structural Classification Of Proteins). It further predicts 25 targets as RBPs in 2076 structural genomics targets: 20 of 25 predicted ones (80%) are putatively RNA binding. The superior performance over existing methods indicates the importance of dividing structures into domains, using a Z-score to measure relative structural similarity, and a statistical energy function to measure protein-RNA binding affinity.     

Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.     

Global signatures of protein binding on structured RNAs in Saccharomyces cerevisiae

Protein binding is essential to the transport,decay and regulation of almost all RNA molecules.However,the structural preference of protein binding on RNAs and their cellular functions and dynamics upon changing environmental conditions are poorly understood.Here,we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins(RBPs)and structured RNAs in yeast at single-nucleotide resolution.We found that on average,in terms of percent total lengths,~15%of mRNA untranslated regions(UTRs),~37%of canonical non-coding RNAs(ncRNAs)and~11%of long ncRNAs(lncRNAs)are bound by proteins.The RBP binding sites,in general,tend to occur at single-stranded loops,with evolutionarily conserved signatures,and often facilitate a specific RNA structure conformation in vivo.We found that four nucleotide modifications of tRNA are significantly associated with RBP binding.We also identified various structural motifs bound by RBPs in the UTRs of mRNAs,associated with localization,degradation and stress responses.Moreover,we identified>200 novel lncRNAs bound by RBPs,and about half of them contain conserved secondary structures.We present the first ensemble pattern of RBP binding sites in the structured non-coding regions of a eukaryotic genome,emphasizing their structural context and cellular functions.     

X-ray crystallographic and NMR studies of protein-protein and protein-nucleic acid interactions involving the KH domains from human poly(C)-binding protein-2

Poly(C)-binding proteins (PCBPs) are KH (hnRNP K homology) domain-containing proteins that recognize poly(C) DNA and RNA sequences in mammalian cells. Binding poly(C) sequences via the KH domains is critical for PCBP functions. To reveal the mechanisms of KH domain-D/RNA recognition and its functional importance, we have determined the crystal structures of PCBP2 KH1 domain in complex with a 12-nucleotide DNA corresponding to two repeats of the human C-rich strand telomeric DNA and its RNA equivalent. The crystal structures reveal molecular details for not only KH1-DNA/RNA interaction but also protein-protein interaction between two KH1 domains. NMR studies on a protein construct containing two KH domains (KH1 + KH2) of PCBP2 indicate that KH1 interacts with KH2 in a way similar to the KH1-KH1 interaction. The crystal structures and NMR data suggest possible ways by which binding certain nucleic acid targets containing tandem poly(C) motifs may induce structural rearrangement of the KH domains in PCBPs; such structural rearrangement may be crucial for some PCBP functions.     

Impact of RNA–Protein Interaction Modes on Translation Control: The Versatile Multidomain Protein Gemin5

The fate of cellular RNAs is largely dependent on their structural conformation, which determines the assembly of ribonucleoprotein (RNP) complexes. Consequently, RNA‐binding proteins (RBPs) play a pivotal role in the lifespan of RNAs. The advent of highly sensitive in cellulo approaches for studying RNPs reveals the presence of unprecedented RNA‐binding domains (RBDs). Likewise, the diversity of the RNA targets associated with a given RBP increases the code of RNA–protein interactions. Increasing evidence highlights the biological relevance of RNA conformation for recognition by specific RBPs and how this mutual interaction affects translation control. In particular, noncanonical RBDs present in proteins such as Gemin5, Roquin‐1, Staufen, and eIF3 eventually determine translation of selective targets. Collectively, recent studies on RBPs interacting with RNA in a structure‐dependent manner unveil new pathways for gene expression regulation, reinforcing the pivotal role of RNP complexes in genome decoding.     

Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition

Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ∼3000 mRNAs at ∼9500 sites located in the 3′ UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors.     

NMR and small-angle scattering-based structural analysis of protein complexes in solution

Structural analysis of multi-domain protein complexes is a key challenge in current biology and a prerequisite for understanding the molecular basis of essential cellular processes. The use of solution techniques is important for characterizing the quaternary arrangements and dynamics of domains and subunits of these complexes. In this respect solution NMR is the only technique that allows atomic- or residue-resolution structure determination and investigation of dynamic properties of multi-domain proteins and their complexes. As experimental NMR data for large protein complexes are sparse, it is advantageous to combine these data with additional information from other solution techniques. Here, the utility and computational approaches of combining solution state NMR with small-angle X-ray and Neutron scattering (SAXS/SANS) experiments for structural analysis of large protein complexes is reviewed. Recent progress in experimental and computational approaches of combining NMR and SAS are discussed and illustrated with recent examples from the literature. The complementary aspects of combining NMR and SAS data for studying multi-domain proteins, i.e. where weakly interacting domains are connected by flexible linkers, are illustrated with the structural analysis of the tandem RNA recognition motif (RRM) domains (RRM1-RRM2) of the human splicing factor U2AF65 bound to a nine-uridine (U9) RNA oligonucleotide.     

紫外交联合并串联亲和纯化高效鉴定蛋白复合物组分的RNA结合活性

RNA结合蛋白在RNA的生成与代谢中发挥着重要作用.我们在近年报道的PAR-CLIP(photoactivatableribonucleoside-enhanced crosslinking and immunoprecipitation)技术的基础上建立了一套快速、有效鉴定RNA结合蛋白的实验方法:以串联亲和纯化替代一步免疫沉淀获得高纯度蛋白-RNA复合物;将Sypro Ruby蛋白染色与RNA放射自显影相结合判断复合物中哪种或哪些组分为RNA结合蛋白,该方法命名为紫外交联合并的串联亲和纯化(cross-linkingand tandem affinity purification,CLiTAP).运用该方法对布氏锥虫的三种锌指蛋白ZC3H7、ZC3H34和ZC3H5进行分析,发现ZC3H7作为帽结合蛋白复合物的核心组分具有很强的RNA结合能力;ZC3H34结合RNA能力较弱,但其互作蛋白具有强的RNA结合活性;相比之下,ZC3H5及其复合物组分皆无RNA结合活性.这些结果表明,CLiTAP与蛋白质鉴定方法相结合,能够有效鉴定靶蛋白复合物中的RNA结合蛋白种类,也为进一步定位RNA结合位点、研究RNA结合蛋白的结构及作用机制奠定了基础.