Neuronal RNA granule contains ApCPEB1, a novel cytoplasmic polyadenylation element binding protein, in Aplysia sensory neuron

The cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) binds to CPE containing mRNAs on their 3'' untranslated regions (3''UTRs). This RNA binding protein comes out many important tasks, especially in learning and memory, by modifying the translational efficiency of target mRNAs via poly (A) tailing. Overexpressed CPEB has been reported to induce the formation of stress granules (SGs), a sort of RNA granule in mammalian cell lines. RNA granule is considered to be a potentially important factor in learning and memory. However, there is no study about RNA granule in Aplysia. To examine whether an Aplysia CPEB, ApCPEB1, forms RNA granules, we overexpressed ApCPEB1-EGFP in Aplysia sensory neurons. Consistent with the localization of mammalian CPEB, overexpressed ApCPEB1 formed granular structures, and was colocalized with RNAs and another RNA binding protein, ApCPEB, showing that ApCPEB1 positive granules are RNA-protein complexes. In addition, ApCPEB1 has a high turnover rate in RNA granules which were mobile structures. Thus, our results indicate that overexpressed ApCPEB1 is incorporated into RNA granule which is a dynamic structure in Aplysia sensory neuron. We propose that ApCPEB1 granule might modulate translation, as other RNA granules do, and furthermore, influence memory.     

RNA-binding residues in sequence space: Conservation and interaction patterns

RNA-binding proteins (RBPs) perform fundamental and diverse functions within the cell. Approximately 15% of proteins sequences are annotated as RNA-binding, but with a significant number of proteins without functional annotation, many RBPs are yet to be identified. A percentage of uncharacterised proteins can be annotated by transferring functional information from proteins sharing significant sequence homology. However, genomes contain a significant number of orphan open reading frames (ORFs) that do not share significant sequence similarity to other ORFs, but correspond to functional proteins. Hence methods for protein function annotation that go beyond sequence homology are essential. One method of annotation is the identification of ligands that bind to proteins, through the characterisation of binding site residues. In the current work RNA-binding residues (RBRs) are characterised in terms of their evolutionary conservation and the patterns they form in sequence space. The potential for such characteristics to be used to identify RBPs from sequence is then evaluated.In the current work the conservation of residues in 261 RBPs is compared for (a) RBRs vs. non-RBRs surface residues, and for (b) specific and non-specific RBRs. The analysis shows that RBRs are more conserved than other surface residues, and RBRs hydrogen-bonded to the RNA backbone are more conserved than those making hydrogen bonds to RNA bases. This observed conservation of RBRs was then used to inform the construction of RBR sequence patterns from known protein–RNA structures. A series of RBR patterns were generated for a case study protein aspartyl-tRNA synthetase bound to tRNA; and used to differentiate between RNA-binding and non-RNA-binding protein sequences. Six sequence patterns performed with high precision values of >80% and recall values 7 times that of an homology search. When the method was expanded to the complete dataset of 261 proteins, many patterns were of poor predictive value, as they had not been manipulated on a family-specific basis. However, two patterns with precision values ≥85% were used to make function predictions for a set of hypothetical proteins. This revealed a number of potential RBPs that require experimental verification.     

Synthesis and protein binding studies of a peptide fragment of clathrin assembly protein AP180 bearing an O-linked β-N-acetylglucosaminyl-6-phosphate modification

A novel post-translational modification of threonine, β-N-acetylglucosaminyl-phosphate, was recently discovered on assembly protein AP180, a protein which plays a crucial role in clathrin coated vesicle formation in synaptic vesicle endocytosis (SVE). Herein, we report studies aimed at probing the effect of this modification on binding to proteins in rat brain lysate using pull down experiments with peptide fragments of AP180.     

Segmental,Domain‐Selective Perdeuteration and Small‐Angle Neutron Scattering for Structural Analysis of Multi‐Domain Proteins

Multi‐domain proteins play critical roles in fine‐tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi‐domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA‐1 using Sortase A mediated protein ligation. We show that domain‐selective perdeuteration combined with contrast‐matched small‐angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi‐domain proteins and changes induced by ligand binding.     

Segmental,Domain‐Selective Perdeuteration and Small‐Angle Neutron Scattering for Structural Analysis of Multi‐Domain Proteins

Multi‐domain proteins play critical roles in fine‐tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi‐domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA‐1 using Sortase A mediated protein ligation. We show that domain‐selective perdeuteration combined with contrast‐matched small‐angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi‐domain proteins and changes induced by ligand binding.     

Exploring assembly energetics of the 30S ribosomal subunit using an implicit solvent approach

To explore the relationship between the assembly of the 30S ribosomal subunit and interactions among the constituent components, 16S RNA and proteins, relative binding free energies of the T. thermophilus 30S proteins to the 16S RNA were studied based on an implicit solvent model of electrostatic, nonpolar, and entropic contributions. The late binding proteins in our assembly map were found not to bind to the naked 16S RNA. The 5' domain early kinetic class proteins, on average, carry the highest positive charge, get buried the most upon binding to 16S RNA, and show the most favorable binding. Some proteins (S10/S14, S6/S18, S13/S19) have more stabilizing interactions while binding as dimers. Our computed assembly map resembles that of E. coli; however, the central domain path is more similar to that of A. aeolicus, a hyperthermophilic bacteria.     

In Situ Self-Assembly of Fluorogenic RNA Nanozipper Enables Real-Time Imaging of Single Viral mRNA Translation

Real-time visualization of individual viral mRNA translation activities in live cells is essential to obtain critical details of viral mRNA dynamics and to detect its transient responses to environmental stress. Fluorogenic RNA aptamers are powerful tools for real-time imaging of mRNA in live cells, but monitoring the translation activity of individual mRNAs remains a challenge due to their intrinsic photophysical properties. Here, we develop a genetically encoded turn-on 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI)-binding RNA nanozipper with superior brightness and high photostability by in situ self-assembly of multiple nanozippers along single mRNAs. The nanozipper enables real-time imaging of the mobility and dynamic translation of individual viral mRNAs in live cells, providing information on the spatial dynamics and translational elongation rate of viral mRNAs.     

核糖核酸-蛋白质复合物规模化富集与鉴定技术的研究进展

核糖核酸(RNA)在细胞中并非单独存在,从它们产生到被降解的过程中与大量蛋白质发生相互作用,RNA结合蛋白(RNA-binding proteins, RBPs)能与RNA结合形成RNA-蛋白质复合物(RP复合物),并以这种复合物的形式发挥生理功能。RNAs或RBPs任一组分的异常与缺失都会影响RP复合物的正常生理功能,从而导致疾病的发生,如代谢异常、肌肉萎缩症、自身免疫性疾病和癌症。因此,定性定量分析RBPs及其在正常细胞和肿瘤细胞中与RNAs靶标之间的复杂相互作用网络有助于挖掘RP复合物在肿瘤发生发展中的作用,开发肿瘤生物标志物和新的治疗方式。要深入研究和理解RNAs与RBPs的相互作用网络,须依赖组学技术对RP复合物进行大规模鉴定。而作为在组学层面系统性解析RP复合物组成、含量和功能的第一步,大规模富集RP复合物极具挑战性。为了解决这一难题,研究者们发展了各种富集鉴定策略。该文针对RP复合物富集策略的最新进展进行了综述,包括紫外光交联和免疫沉淀(crosslinking and immunoprecipitation, CLIP)及其衍生技术、基于“点击化学”的富集策略和基于相分离的富集策略,比较分析了它们的技术原理、优缺点,以方便研究者们选择合适的策略来解决感兴趣的生物学问题。该文最后总结了当前的RP复合物富集方法仍然存在富集效率低和操作繁琐等亟需解决的技术挑战,为富集策略的发展提供了研究方向。     

Profiling of Ribose Methylations in RNA by High‐Throughput Sequencing

Ribose methylations are the most abundant chemical modifications of ribosomal RNA and are critical for ribosome assembly and fidelity of translation. Many aspects of ribose methylations have been difficult to study due to lack of efficient mapping methods. Here, we present a sequencing‐based method (RiboMeth‐seq) and its application to yeast ribosomes, presently the best‐studied eukaryotic model system. We demonstrate detection of the known as well as new modifications, reveal partial modifications and unexpected communication between modification events, and determine the order of modification at several sites during ribosome biogenesis. Surprisingly, the method also provides information on a subset of other modifications. Hence, RiboMeth‐seq enables a detailed evaluation of the importance of RNA modifications in the cells most sophisticated molecular machine. RiboMeth‐seq can be adapted to other RNA classes, for example, mRNA, to reveal new biology involving RNA modifications.     

RNA-mediated sequestration of the RNA helicase eIF4A by Pateamine A inhibits translation initiation

Eukaryotic initiation factor 4A (eIF4A) is a member of the DEAD-box family of putative RNA helicases whose members are involved in many aspects of RNA metabolism. eIF4A is thought to facilitate binding of 43S preinitiation complexes to mRNAs by unwinding secondary structures present in the 5' untranslated region. Pateamine A, a small-molecule inhibitor of translation initiation, acts in an unusual manner by stimulating eIF4A activity. Herein, we report the elucidation of pateamine's mode of action. We demonstrate that Pateamine A is a chemical inducer of dimerization that forces an engagement between eIF4A and RNA and prevents eIF4A from participating in the ribosome-recruitment step of translation initiation.     

Homogenous time resolved fluorescence assay to identify modulators of cap-dependent translation initiation

Eukaryotic initiation factor (eIF) 4F plays a key role in recruiting 40S ribosomes and associated factors to mRNA templates during translation initiation. The function of this heterotrimeric complex is to deliver an RNA helicase to the 5' cap proximal region of mRNAs in preparation for ribosome binding. To study the interaction between subunits of this complex, as well as identify small molecules that could interfere with their association, we developed a time resolved fluorescence assay that allows monitoring of interactions between two subunits of eIF4F. We have performed a small molecule chemical screen of >73,000 compounds using this assay.