The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex

The repeat-associated mysterious proteins (RAMPs) comprise the most abundant family of proteins involved in prokaryotic immunity against invading genetic elements conferred by the clustered regularly interspaced short palindromic repeat (CRISPR) system. Cas6 is one of the first characterized RAMP proteins and is a key enzyme required for CRISPR RNA maturation. Despite a strong structural homology with other RAMP proteins that bind hairpin RNA, Cas6 distinctly recognizes single-stranded RNA. Previous structural and biochemical studies show that Cas6 captures the 5' end while cleaving the 3' end of the CRISPR RNA. Here, we describe three structures and complementary biochemical analysis of a noncatalytic Cas6 homolog from Pyrococcus horikoshii bound to CRISPR repeat RNA of different sequences. Our study confirms the specificity of the Cas6 protein for single-stranded RNA and further reveals the importance of the bases at Positions 5-7 in Cas6-RNA interactions. Substitutions of these bases result in structural changes in the protein-RNA complex including its oligomerization state.     

RAMPs and G protein coupled receptors

RAMPs (receptor activity-modifying proteins) were discovered in 1998 as accessory proteins needed to the functionnal activity of CGRP (calcitonin gene-related peptide) receptors. Three RAMPs generated by three different genes are known in human, rat and mice. The coding sequences of such genes are described, but as yet, regulation sequences are unknown. RAMPs interact with GPCR (G protein-coupled receptors) of class II. In the case of the calcitonin/CGRP peptide family, RAMPs determine the functionnal specificity of the receptor, glycosylate and translocate the receptor to the cell surface. CGRP receptors are observed in presence of the RAMP1/calcitonin receptor-like receptor (CRLR), but the association of RAMP2 or RAMP3 with CRLR generates an adrenomedullin receptor. The calcitonin receptor (CTR) is translocated alone to the cell surface, but interactions of RAMPs with CTR forms amylin receptors. If RAMPs can interact with glucagon, parathyroid hormone and VIP/PACAP (vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide (VPACR1)) receptors, the functionnal specificity of these receptors remains unaltered. However, the complex VPACR1/RAMP2 enhances specifically the phosphoinoside signaling pathway.     

RNA:DNA hybrids are a novel molecular pattern sensed by TLR9

The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen‐associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral‐derived sequences efficiently induce pro‐inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88‐dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease.     

Prevalent RNA recognition motif duplication in the human genome

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.     

Conservation and Variability in the Structure and Function of the Cas5d Endoribonuclease in the CRISPR-Mediated Microbial Immune System

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form an RNA-mediated microbial immune system against invading foreign genetic elements. Cas5 proteins constitute one of the most prevalent Cas protein families in CRISPR–Cas systems and are predicted to have RNA recognition motif (RRM) domains. Cas5d is a subtype I-C-specific Cas5 protein that can be divided into two distinct subgroups, one of which has extra C-terminal residues while the other contains a longer insertion in the middle of its N-terminal RRM domain. Here, we report crystal structures of Cas5d from Streptococcus pyogenes and Xanthomonas oryzae, which respectively represent the two Cas5d subgroups. Despite a common domain architecture consisting of an N-terminal RRM domain and a C-terminal β-sheet domain, the structural differences between the two Cas5d proteins are highlighted by the presence of a unique extended helical region protruding from the N-terminal RRM domain of X. oryzae Cas5d. We also demonstrate that Cas5d proteins possess not only specific endoribonuclease activity for CRISPR RNAs but also nonspecific double-stranded DNA binding affinity. These findings suggest that Cas5d may play multiple roles in CRISPR-mediated immunity. Furthermore, the specific RNA processing was also observed between S. pyogenes Cas5d protein and X. oryzae CRISPR RNA and vice versa. This cross-species activity of Cas5d provides a special opportunity for elucidating conserved features of the CRISPR RNA processing event.     

cDNA cloning and characterization of Drb1, a new member of RRM-type neural RNA-binding protein

Neural RNA recognition motif (RRM)-type RNA-binding proteins play essential roles in neural development. To search for a new member of neural RRM-type RNA-binding protein, we screened rat cerebral expression library with polyclonal antibody against consensus RRM sequences. We have cloned and characterized a rat cDNA that belongs to RRM-type RNA-binding protein family, which we designate as drb1. Orthologs of drb1 exist in human and mouse. The predicted amino acid sequence reveals an open reading frame of 476 residues with a corresponding molecular mass of 53kDa and consists of four RNA-binding domains. drb1 gene is specifically expressed in fetal (E12, E16) rat brain and gradually reduced during development. In situ hybridization demonstrated neuron-specific signals in fetal rat brain. RNA-binding assay indicated that human Drb1 protein possesses binding preference on poly(C)RNA. These results indicate that Drb1 is a new member of neural RNA-binding proteins, which expresses under spatiotemporal control.     

Proteome-Level Analysis Indicates Global Mechanisms for Post-Translational Regulation of RRM Domains

RRM, or RNA-recognition motif, domains are the largest class of single-stranded RNA binding domains in the human proteome and play important roles in RNA processing, splicing, export, stability, packaging, and degradation. Using a current database of post-translational modifications (PTMs), ProteomeScout, we found that RRM domains are also one of the most heavily modified domains in the human proteome. Here, we present two interesting findings about RRM domain modifications, found by mapping known PTMs onto RRM domain alignments and structures. First, we find significant overlap of ubiquitination and acetylation within RRM domains, suggesting the possibility for ubiquitination-acetylation crosstalk. Additionally, an analysis of quantitative study of ubiquitination changes in response to proteasome inhibition highlights the uniqueness of RRM domain ubiquitination – RRM domain ubiquitination decreases in response to proteasome inhibition, whereas the majority of sites increase. Second, we found conservation of tyrosine phosphorylation within the RNP1 and RNP2 consensus sequences, which coordinate RNA binding – suggesting a possible role for regulation of RNA binding by tyrosine kinase signaling. These observations suggest there are unique regulatory mechanisms of RRM function that have yet to be uncovered and that the RRM domain represents a model system for further studies on understanding PTM crosstalk.     

Flow cytometric analysis of the calcitonin receptor-like receptor domains responsible for cell-surface translocation of receptor activity-modifying proteins

The three receptor activity-modifying proteins (RAMPs1, -2, and -3) associate with a wide variety of G protein-coupled receptors (GPCRs), including calcitonin receptor-like receptor (CRLR). In this study, we used flow cytometry to measure RAMP translocation to the cell surface as a marker of RAMP-receptor interaction. Because VPAC2 does not interact with RAMPs, although, like CRLR, it is a Family B peptide hormone receptor, we constructed a set of chimeric CRLR/VPAC2 receptors to evaluate the trafficking interactions between CRLR domains and each RAMP. We found that CRLR regions extending from transmembrane domain 1 (TM1) through TM5 are necessary and sufficient for the transport of RAMPs to the plasma membrane. In addition, the extracellular N-terminal domain of CRLR, its 3rd intracellular loop and/or TM6 were also important for the cell-surface translocation of RAMP2, but not RAMP1 or RAMP3. Other regions within CRLR were not involved in trafficking interactions with RAMPs. These findings provide new insight into the trafficking interactions between accessory proteins such as RAMPs and their receptor partners.     

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms.     

Receptor activity modifying proteins

Our understanding of G protein-coupled receptor (GPCR) function has recently expanded to encompass novel protein interactions that underlie both cell-surface receptor expression and the exhibited phenotype. The most notable examples are those involving receptor activity modifying proteins (RAMPs). RAMP association with the calcitonin (CT) receptor-like receptor (CRLR) traffics this receptor to the cell surface where individual RAMPs dictate the expression of unique phenotypes. A similar function has been ascribed to RAMP interaction with the CT receptor (CTR) gene product. This review examines our current state of knowledge of the mechanisms underlying RAMP function.     

DEAD-box RNA解旋酶家族在天然免疫应答信号通路中的作用

病毒入侵宿主细胞时,宿主细胞启动抑制病毒复制的免疫机制.同样,病毒也会利用多种手段去逃避先天免疫感应机制的监测以及宿主细胞对外来者的降解,同时还会操纵宿主细胞为自身的增殖提供便利.DEAD-box解旋酶家族是一类存在于宿主细胞中的功能蛋白,它们在转录、剪接、mRNA的合成和翻译等多种细胞过程中起着关键作用.该家族成员拥...     

Novel receptor partners and function of receptor activity-modifying proteins

The receptor activity-modifying proteins (RAMPs) comprise a family of three accessory proteins that heterodimerize with the calcitonin receptor-like receptor (CL receptor) or with the calcitonin receptor (CTR) to generate different receptor phenotypes. However, RAMPs are more widely distributed across cell and tissue types than the CTR and CL receptor, suggesting additional roles for RAMPs in cellular processes. We have investigated the potential for RAMP interaction with a number of Class II G protein-coupled receptors (GPCRs) in addition to the CL receptor and the CTR. Using immunofluorescence confocal microscopy, we demonstrate, for the first time, that RAMPs interact with at least four additional receptors, the VPAC1 vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor with all three RAMPs; the glucagon and PTH1 parathyroid hormone receptors with RAMP2; and the PTH2 receptor with RAMP3. Unlike the interaction of RAMPs with the CL receptor or the CTR, VPAC1R-RAMP complexes do not show altered phenotypic behavior compared with the VPAC1R alone, as determined using radioligand binding in COS-7 cells. However, the VPAC1R-RAMP2 heterodimer displays a significant enhancement of agonist-mediated phosphoinositide hydrolysis with no change in cAMP stimulation compared with the VPAC1R alone. Our findings identify a new functional consequence of RAMP-receptor interaction, suggesting that RAMPs play a more general role in modulating cell signaling through other GPCRs than is currently appreciated.     

受体活性修饰蛋白研究进展

受体活性修饰蛋白(receptor activity-modifying proteins,RAMPs)属于单跨膜蛋白家族,分三个结构域,RAMP的N端和跨膜区决定本身的功能和受体表型,胞内C端对于配体的信号传导和受体循环有重要作用。目前发现有三个成员:RAMP1、RAMP2和RAMP3。RAMPs通过改变G蛋白偶联受体的糖基化,作用于配体结合区域来调节受体表型。RAMP1与降钙素受体样受体(calcitonin receptor like receptor,CRLR)结合表现出降钙素基因相关肽(calcitonin gene-related peptide,CGRP)受体表型:RAMP2和RAMP3与CRLR结合则对肾上腺髓质素(adrenomedullin,AM)表现高亲和力,与降钙素受体(calcitonin receptor,CTR)结合则作为胰淀粉样酶(amylin,AMY)受体。由此可见,RAMPs不仅调节受体与配体结合,还影响细胞内的蛋白相互作用调节细胞内信号传导来影响细胞的增殖、迁移、分化等生物学特性。RAMPs还对心血管系统的病理生理有重要调节作用。     

Visualization of the calcitonin receptor-like receptor and its receptor activity-modifying proteins during internalization and recycling

Expression of the calcitonin receptor-like receptor (CRLR) and its receptor activity modifying proteins (RAMPs) can produce calcitonin gene-related peptide (CGRP) receptors (CRLR/RAMP1) and adrenomedullin (AM) receptors (CRLR/RAMP2 or -3). A chimera of the CRLR and green fluorescent protein (CRLR-GFP) was used to study receptor localization and trafficking in stably transduced HEK 293 cells, with or without co-transfection of RAMPs. CRLR-GFP failed to generate responses to CGRP or AM without RAMPs. Furthermore, CRLR-GFP was not found in the plasma membrane and its localization was unchanged after agonist exposure. When stably coexpressed with RAMPs, CRLR-GFP appeared on the cell surface and was fully active in intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs. This internalization was strongly inhibited by hypertonic medium (0.45 m sucrose) and paralleled localization of rhodamine-labeled transferrin, suggesting that CRLR endocytosis occurred predominantly through a clathrin-dependent pathway. A significant proportion of CRLR was targeted to lysosomes upon binding of the ligands, and recycling of the internalized CRLR was not efficient. In HEK 293 cells stably expressing CRLR-GFP and Myc-RAMPs, these rhodamine-labeled RAMPs were co-localized with CRLR-GFP in the presence and absence of the ligands. Thus, the CRLR is endocytosed together with RAMPs via clathrin-coated vesicles, and both the internalized molecules are targeted to the degradative pathway.     

Covariance analysis of RNA recognition motifs identifies functionally linked amino acids.

The RNA recognition motif (RRM) is one of the most common eukaryotic protein motifs. RRM sequences form a conserved globular structure known as the RNA-binding domain (RBD) or the ribonucleoprotein domain. Many proteins that contain RRM sequences bind RNA in a sequence-specific manner. To investigate the basis for the RNA-binding specificity of RRMs, we subjected 330 aligned RRM sequences to covariance analysis. The analysis revealed a single network of covariant amino acid pairs comprising the buried core of the RBD and a surface patch. Structural studies have implicated a subset of these residues in RNA binding. The covariance linkages identify a larger set of amino acid residues, including some not directly in contact with bound RNA, that may influence RNA-binding specificity.     

Adrenomedullin and related peptides: receptors and accessory proteins.

Adrenomedullin (AM), alpha- and beta-calcitonin gene-related peptide (CGRP), amylin and calcitonin (CT) are structurally and functionally related peptides. The structure of a receptor for CT (CTR) was elucidated in 1991 through molecular cloning, but the structures of the receptors for the other three peptides had yet to be elucidated. The discovery of receptor-activity-modifying proteins (RAMP) 1 and -2 and their co-expression with an orphan receptor, calcitonin receptor-like receptor (CRLR) has led to the elucidation of functional CGRP and AM receptors, respectively. RAMP1 and -3 which are co-expressed with CTR revealed two amylin receptor isotypes. Molecular interactions between CRLR and RAMPs are involved in their transport to the cell surface. Heterodimeric complexes between CRLR or CTR and RAMPs are required for ligand recognition.