Small RNAs in Escherichia coli

Bacterial cells contain several small RNAs (sRNAs) that are not translated. These stable, abundant RNAs act by multiple mechanisms, such as RNA-RNA basepairing, RNA-protein interactions and intrinsic RNA activity, and regulate diverse cellular functions, including RNA processing, mRNA stability, translation, protein stability and secretion.     

More than a bystander: RNAs specify multifaceted behaviors of liquid-liquid phase-separated biomolecular condensates

Cells contain a myriad of membraneless ribonucleoprotein (RNP) condensates with distinct compositions of proteins and RNAs. RNP condensates participate in different cellular activities, including RNA storage, mRNA translation or decay, stress response, etc. RNP condensates are assembled via liquid-liquid phase separation (LLPS) driven by multivalent interactions. Transition of RNP condensates into bodies with abnormal material properties, such as solid-like amyloid structures, is associated with the pathogenesis of various diseases. In this review, we focus on how RNAs regulate multiple aspects of RNP condensates, such as dynamic assembly and/or disassembly and biophysical properties. RNA properties – including concentration, sequence, length and structure – also determine the phase behaviors of RNP condensates. RNA is also involved in specifying autophagic degradation of RNP condensates. Unraveling the role of RNA in RNPs provides novel insights into pathological accumulation of RNPs in various diseases. This new understanding can potentially be harnessed to develop therapeutic strategies.     

Regulation of innate immunity through RNA structure and the protein kinase PKR

Molecular recognition of RNA structure is key to innate immunity. The protein kinase PKR differentiates self from non-self by recognition of molecular patterns in RNA. Certain biological RNAs induce autophosphorylation of PKR, activating it to phosphorylate eukaryotic initiation factor 2α (eIF2α), which leads to inhibition of translation. Additional biological RNAs inhibit PKR, while still others have no effect. The aim of this article is to develop a cohesive framework for understanding and predicting PKR function in the context of diverse RNA structure. We present effects of recently characterized viral and cellular RNAs on regulation of PKR, as well as siRNAs. A central conclusion is that assembly of accessible long double-stranded RNA (dsRNA) elements within biological RNAs plays a key role in regulation of PKR kinase. Strategies for forming such elements include RNA dimerization, formation of symmetrical helical defects, A-form dsRNA mimicry, and coaxial stacking of helices.     

The Pendulum Model for Genome Compositional Dynamics:from the Four Nucleotides to the Twenty Amino Acids

The genetic code serves as one of the natural links for life’s two conceptual frameworks-the informational and operational tracks- bridging the nucleotide sequence of DNA and RNA to the amino acid sequence of protein and thus its structure and function.On the informational track,DNA and its four building blocks have four basic variables:order,length,GC and purine contents;the latter two exhibit unique characteristics in prokaryotic genomes where protein-coding sequences dominate.Bridging the two tracks,tRNAs and their aminoacyl tRNA synthases that interpret each codon-nucleotide triplet,together with ribosomes,form a complex machinery that translates genetic information encoded on the messenger RNAs into proteins.On the operational track,proteins are selected in a context of cellular and organismal functions constantly.The principle of such a functional selection is to minimize the damage caused by sequence alteration in a seemingly random fashion at the nucleotide level and its function-altering consequence at the protein level;the principle also suggests that there must be complex yet sophisticated mechanisms to protect molecular interactions and cellular processes for cells and organisms from the damage in addition to both immediate or short-term eliminations and long-term selections.The twocentury study of selection at species and population levels has been leading a way to understand rules of inheritance and evolution at molecular levels along the informational track,while ribogenomics,epigenomics and other operationally-defined omics(such as the metabolite-centric metabolomics) have been ushering biologists into the new millennium along the operational track.     

Conservation of RNA-protein interactions among picornaviruses.

Picornavirus genomes encode unique 5' noncoding regions (5' NCRs) which are approximately 600 to 1,300 nucleotides in length, contain multiple upstream AUG codons, and display the ability to form extensive secondary structures. A number of recent reports have shown that picornavirus 5' NCRs are able to facilitate cap-independent internal initiation of translation. This mechanism of translation occurs in the absence of viral gene products, suggesting that the host cell contains the necessary components for the cap-independent internal initiation of translation of picornavirus RNAs as well as cellular mRNAs. In an attempt to identify some of the perhaps novel cellular proteins involved in this newly discovered mechanism of translation, we utilized RNA mobility shifts assays to identify and characterize interactions that occur between the 5'NCR of poliovirus type 1 (PV1) and cellular proteins. In this report, we describe two separate interactions between RNA structures from the 5' NCR of PV1 and proteins present in extracts from HeLa cells as well as other cell types. We describe the interaction between nucleotides 186 to 220 (stem-loop D) and a cellular protein(s) present in HeLa cell extracts. Mutational analysis of this stem-loop structure suggests that maintenance of a base-paired structure in the lower stem is necessary to present the sequences which directly interact with the protein(s). We also describe the interaction between nucleotides 220 to 460 (stem-loop E) and a cellular protein present in HeLa cell extracts. This RNA binding activity fractionates to a specific ammonium sulfate fraction (A cut) of a ribosomal salt wash. Mutational analysis of the stem-loop E structure suggests that the preservation of an extensive RNA structure is necessary for a strong interaction with the cellular protein(s), although smaller RNAs derived from this region of the 5' NCR can interact to lesser extents. Finally, we show that both of these RNA-protein interactions are conserved among the closely related enteroviruses PV1 and coxsackievirus type B3, human rhinovirus type 14, and the more distantly related cardiovirus Theiler's murine encephalomyelitis virus, suggesting that such RNA-protein interactions serve basic functions which are conserved and utilized by each of these picornaviruses.     

Regulation of RpoS by a novel small RNA: the characterization of RprA

Translational regulation of the stationary phase sigma factor RpoS is mediated by the formation of a double-stranded RNA stem-loop structure in the upstream region of the rpoS messenger RNA, occluding the translation initiation site. The interaction of the rpoS mRNA with a small RNA, DsrA, disrupts the double-strand pairing and allows high levels of translation initiation. We screened a multicopy library of Escherichia coli DNA fragments for novel activators of RpoS translation when DsrA is absent. Clones carrying rprA (RpoS regulator RNA) increased the translation of RpoS. The rprA gene encodes a 106 nucleotide regulatory RNA. As with DsrA, RprA is predicted to form three stem-loops and is highly conserved in Salmonella and Klebsiella species. Thus, at least two small RNAs, DsrA and RprA, participate in the positive regulation of RpoS translation. Unlike DsrA, RprA does not have an extensive region of complementarity to the RpoS leader, leaving its mechanism of action unclear. RprA is non-essential. Mutations in the gene interfere with the induction of RpoS after osmotic shock when DsrA is absent, demonstrating a physiological role for RprA. The existence of two very different small RNA regulators of RpoS translation suggests that such additional regulatory RNAs are likely to exist, both for regulation of RpoS and for regulation of other important cellular components.     

Prp43/DHX15 exemplify RNA helicase multifunctionality in the gene expression network

Dynamic regulation of RNA folding and structure is critical for the biogenesis and function of RNAs and ribonucleoprotein (RNP) complexes. Through their nucleotide triphosphate-dependent remodelling functions, RNA helicases are key modulators of RNA/RNP structure. While some RNA helicases are dedicated to a specific target RNA, others are multifunctional and engage numerous substrate RNAs in different aspects of RNA metabolism. The discovery of such multitasking RNA helicases raises the intriguing question of how these enzymes can act on diverse RNAs but also maintain specificity for their particular targets within the RNA-dense cellular environment. Furthermore, the identification of RNA helicases that sit at the nexus between different aspects of RNA metabolism raises the possibility that they mediate cross-regulation of different cellular processes. Prominent and extensively characterized multifunctional DEAH/RHA-box RNA helicases are DHX15 and its Saccharomyces cerevisiae (yeast) homologue Prp43. Due to their central roles in key cellular processes, these enzymes have also served as prototypes for mechanistic studies elucidating the mode of action of this type of enzyme. Here, we summarize the current knowledge on the structure, regulation and cellular functions of Prp43/DHX15, and discuss the general concept and implications of RNA helicase multifunctionality.     

The N-terminal K homology domain of the poly(rC)-binding protein is a major determinant for binding to the poliovirus 5'-untranslated region and acts as an inhibitor of viral translation

The poly(rC)-binding proteins (PCBP1 and PCBP2) are RNA-binding proteins whose RNA recognition motifs are composed of three K homology (KH) domains. These proteins are involved in both the stabilization and translational regulation of several cellular and viral RNAs. PCBP1 and PCBP2 specifically interact with both the 5'-element known as the cloverleaf structure and the large stem-loop IV RNA of the poliovirus 5'-untranslated region. We have found that the first KH domain of PCBP2 (KH1) specifically interacts with the viral RNAs, and together with viral protein 3CD, KH1 forms a high affinity ternary ribonucleoprotein complex with the cloverleaf RNA, resembling the full-length PCBP protein. Furthermore, KH1 acts as a dominant-negative mutant to inhibit translation from a poliovirus reporter gene in both Xenopus laevis oocytes and HeLa cell in vitro translation extracts.     

RNA结合蛋白Sam68及其功能

细胞通过基因表达调控来应对外界刺激,其中影响mRNA稳定性及翻译效率的转录后调控发挥重要作用。RNA结合蛋白(RNA binding proteins, RBPs)是介导转录后调控的重要分子,Sam68（SRC associated in mitosis of 68 kD）是集信号转导特性与RNA激活功能于一身的RNA结合蛋白,参与转录、可变剪接及核输出等mRNA 的代谢过程,且Sam68可通过信号通路参与细胞应答、细胞周期调控和疾病发生等。最新研究表明,Sam68可通过非编码RNAs(noncoding RNA, ncRNAs)参与表观遗传、转录与转录后调控。本文在介绍Sam68结构和转录后修饰的基础上,着重讨论Sam68在信号转导、可变剪接、ncRNAs代谢、疾病发生等方面的最新研究进展。     

The metaphorical swiss army knife: The multitude and diverse roles of HEAT domains in eukaryotic translation initiation

Biomolecular associations forged by specific interaction among structural scaffolds are fundamental to the control and regulation of cell processes. One such structural architecture, characterized by HEAT repeats, is involved in a multitude of cellular processes, including intracellular transport, signaling, and protein synthesis. Here, we review the multitude and versatility of HEAT domains in the regulation of mRNA translation initiation. Structural and cellular biology approaches, as well as several biophysical studies, have revealed that a number of HEAT domain-mediated interactions with a host of protein factors and RNAs coordinate translation initiation. We describe the basic structural architecture of HEAT domains and briefly introduce examples of the cellular processes they dictate, including nuclear transport by importin and RNA degradation. We then focus on proteins in the translation initiation system featuring HEAT domains, specifically the HEAT domains of eIF4G, DAP5, eIF5, and eIF2Bϵ. Comparative analysis of their remarkably versatile interactions, including protein–protein and protein–RNA recognition, reveal the functional importance of flexible regions within these HEAT domains. Here we outline how HEAT domains orchestrate fundamental aspects of translation initiation and highlight open mechanistic questions in the area.     

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 a Packaged Cellular mRNA in Virions of Rous Sarcoma Virus

A novel messenger activity has been identified by in vitro translation of the 70S virion RNAs of a variety of avian leukosis and avian sarcoma viruses. When the 70S virion RNA complex was heat dissociated and the polyadenylated RNA was fractionated on neutral sucrose gradients, a polypeptide of 34,000 daltons (34K) was observed in the translation products of 18S polyadenylic acid-containing virion RNA. Aside from the p60(src)-related subgenomic messenger activities, this was the only prominent messenger activity that sedimented at <20S. It was determined that the 34K protein was not virally coded because (i) messenger activity for the 34K protein was not generated by mild alkaline hydrolysis of 35S genomic RNA, (ii) the 34K proteins synthesized in response to different virion RNAs had identical tryptic peptide maps, and (iii) the tryptic peptide map of the 34K protein coded for by virion RNA was identical to that of a major in vitro translation product of 34,000 daltons made from 18S uninfected chick cell polyadenylated RNA. The 18S RNA was shown to be contained within virion particles, rather than part of a cellular structure copurifying with virus preparations, by demonstrating the presence of 34K messenger activity in virion cores made from detergent-disrupted virus. This cellular mRNA, however, was not observed in the virion RNAs of Rous-associated virus types 0 and 2 avian leukosis viruses and therefore is not packaged by all avian retroviruses. Since no other cellular message has been detected by this assay, it seems likely that the 34K mRNA found in 70S virion RNA is the result of selective packaging of an abundant host cell mRNA by certain avian retroviruses.     

Small RNAs for defence and regulation in archaea

Non-coding RNAs are key players in many cellular processes within organisms from all three domains of life. The range and diversity of small RNA functions beyond their involvement in translation and RNA processing was first recognized for eukaryotes and bacteria. Since then, small RNAs were also found to be abundant in archaea. Their functions include the regulation of gene expression and the establishment of immunity against invading mobile genetic elements. This review summarizes our current knowledge about small RNAs used for regulation and defence in archaea.