Killing the messenger: short RNAs that silence gene expression

Short interfering RNAs can be used to silence gene expression in a sequence-specific manner in a process that is known as RNA interference. The application of RNA interference in mammals has the potential to allow the systematic analysis of gene expression and holds the possibility of therapeutic gene silencing. Much of the promise of RNA interference will depend on the recent advances in short-RNA-based silencing technologies.     

Characterisation of the U83 and U84 small nucleolar RNAs: two novel 2'-O-ribose methylation guide RNAs that lack complementarities to ribosomal RNAs

In eukaryotic cells, the site-specific 2′-O-ribose methy-lation of ribosomal RNAs (rRNAs) and the U6 spliceosomal small nuclear RNA (snRNA) is directed by small nucleolar RNAs (snoRNAs). The C and D box-containing 2′-O-methylation guide snoRNAs select the correct substrate nucleotide through formation of a long 10–21 bp interaction with the target rRNA and U6 snRNA sequences. Here, we report on the characterisation of two novel mammalian C/D box snoRNAs, called U83 and U84, that contain all the elements that are essential for accumulation and function of 2′-O-methylation guide snoRNAs. However, in contrast to all of the known 2′-O-methylation guide RNAs, the human, mouse and pig U83 and U84 snoRNAs feature no antisense elements complementary to rRNA or U6 snRNA sequences. The human U83 and U84 snoRNAs are not associated with maturing nucleolar pre-ribosomal particles, suggesting that they do not function in rRNA biogenesis. Since artificial substrate RNAs complementary to the evolutionarily conserved putative substrate recognition motifs of the U83 and U84 snoRNAs were correctly 2′-O-methy-lated in the nucleolus of mouse cells, we suggest that the new snoRNAs act as 2′-O-methylation guides for cellular RNAs other then rRNAs and the U6 snRNA.     

The path leading to the discovery of cadherin: In retrospect

Problems of adhesion between cells are undoubtedly one of the key major issues in developmental biology and its related field. It is little doubt that cell adhesion is one of the most fundamental mechanisms underlying the morphogenesis in multicellular animals and that it is intrinsically related to the metastasis of cancer cells as well. The historical source of adhesion studies can be traced to Wilson's work using sponges published in 1907. The present article starts to outline briefly the conceptual history up to a rise of cell adhesion in the 1950s as a problem for understanding morphogenesis in general. A crucial landmark to a recent burst of the interest to adhesion mechanisms in terms of adhesion molecules is the discovery of a group of major molecules functioning cell-to-cell adhesion, cadherins, from a research group at Kyoto University, Japan, which was initiated by myself and very successfully continued by Takeichi. A main part of the present review is to record the path leading to this important discovery based on my own personal experience, including some retrospective anecdotes. The path was initiated with a series of very simple experiments of a naïve question; that is, to examine whether or not different divalent cations were required for cell adhesion in qualitatively different manners.     

Screening for transient biological interactions as applied to albumin ligands: a new concept for drug discovery

The notion that many biological interactions are based on transient binding (dissociation constants (K(d)) in the range of 10-0.01 mM) is familiar, yet the implications for biological sciences have been realized only recently. An important area of biological sciences is drug design, where the traditional "lock and key" view of binding has prevailed and drug candidates are usually selected on their merits as being tight binders. However, the rationale that transient interactions are of importance for drug discovery is slowly gaining acceptance. These interactions may relate not only to the desired target interaction but also to unwanted interactions creating, for example, toxicity problems. Here we demonstrate, in a high-throughput screening format, affinity selection of weak binders to a model target of albumin by zonal retardation chromatography. It is perceived that this approach can define the "transient drug" as a complement to current drug discovery procedures.     

Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a state-of-the-art tool for versatile genome editing that has advanced basic research dramatically, with great potential for clinic applications. The system consists of two key molecules: a CRISPR-associated (Cas) effector nuclease and a single guide RNA. The simplicity of the system has enabled the development of a wide spectrum of derivative methods. Almost any laboratory can utilize these methods, although new users may initially be confused when faced with the potentially overwhelming abundance of choices. Cas nucleases and their engineering have been systematically reviewed previously. In the present review, we discuss single guide RNA engineering and design strategies that facilitate more efficient, more specific and safer gene editing.     

The discovery of signal transduction by G proteins: a personal account and an overview of the initial findings and contributions that led to our present understanding

The realization that there existed a G-protein coupled signal transduction mechanism developed gradually and was initially the result of an ill fated quest for uncovering the mechanism of action of insulin, followed by a refocused research in many laboratories, including mine, on how GTP acted to increase hormonal stimulation of adenylyl cyclase. Independent research into how light-activated rhodopsin triggers a response in photoreceptor cells of the retina and the attendant biochemical studies joined midway and, without the left hand knowing well what the right hand was doing, preceded classical G protein research in identifying the molecular players responsible for signal transduction by G proteins.     

Argonaute: A scaffold for the function of short regulatory RNAs

Argonaute is the central protein component of RNA-silencing mechanisms. It provides the platform for target-mRNA recognition by short regulatory guide RNA strands and the Slicer catalytic activity for mRNA cleavage in RNA interference. Multiple Argonaute sub-families can be identified phylogenetically yet, despite this diversity, molecular and sequence analyses show that Argonaute proteins share common molecular properties and the capacity to function through a common mechanism. Recently, the members of the Piwi sub-family have been shown to interact with new classes of short regulatory RNAs, Piwi-interacting RNAs (piRNAs) and repeat-associated small interfering RNAs (rasiRNAs), which has implications for developmental processes and introduces a new dimension to the field of RNA silencing.     

The search for the salicylic acid receptor led to discovery of the SAR signal receptor

Systemic acquired resistance (SAR) is a state of heightened defense which is induced throughout a plant by an initial infection; it provides long-lasting, broad-spectrum resistance to subsequent pathogen challenge. Recently we identified a phloem-mobile signal for SAR which has been elusive for almost 30 years. It is methyl salicylate (MeSA), an inactive derivative of the defense hormone, salicylic acid (SA). This discovery resulted from extensive characterization of SA-binding protein 2 (SABP2), a protein whose high affinity for SA and extremely low abundance suggested that it might be the SA receptor. Instead we discovered that SABP2 is a MeSA esterase whose function is to convert biologically inactive MeSA in the systemic tissue to active SA. The accumulated SA then activates or primes defenses leading to SAR. SABP2''s esterase activity is inhibited in the initially/primary infected tissue by SA binding in its active site; this facilitates accumulation of MeSA, which is then translocated through the phloem to systemic tissue for perception and processing by SABP2 to SA. Thus, while SABP2 is not the SA receptor, it can be considered the receptor for the SAR signal. This study of SABPs not only illustrates the unexpected nature of scientific discoveries, but also underscores the need to use biochemical approaches in addition to genetics to address complex biological processes, such as disease resistance.Key words: salicylic acid, methyl salicylate, salicylic acid-binding proteins, systemic acquired resistance, methyl salicylate esteraseFor over a century, naturalists and scientists have observed that plants which survive an initial pathogen attack often develop enhanced resistance to subsequent infections. Systematic studies by Frank Ross in the early 1960s demonstrated that prior infection of tobacco plants by Tobacco Mosaic Virus (TMV) enhanced resistance in the systemic tissue to subsequent challenge by TMV or other pathogens, which he termed systemic acquired resistance.¹ In the later 1970s Kuc and others showed that development of SAR required movement of a signal made in the primary infected tissue through the phloem to the distal systemic tissue.²More recent studies starting in 1990 indicated that SA plays a critical role(s) in plant disease resistance.^3–5 For the past decade and a half we have used biochemical and genetic approaches to identify the components and molecular mechanisms involved in SA-mediated signal transduction. One approach was to biochemically identify proteins in tobacco which bound SA, with the hope that some would be SA effectors or targets and at least one would be a receptor for SA. This led to the identification of catalase, ascorbate peroxidase, SABP3, which is the chloroplastic carbonic anhydrase, and SABP2.^6–9 SA inhibits catalase''s and ascorbate peroxidase''s H₂O₂ scavenging activities; this inhibition may contribute to the oxidative burst that occurs after infection by avirulent microbes and the subsequent alteration in cellular redox state that facilitates relocation of the positive regulator protein NPR1 from the cytoplasm to the nucleus for activation of SA-responsive defense genes, such as PR-1.^5,10 While SA does not appear to alter carbonic anhydrase''s activity, altering carbonic anhydrase synthesis suppressed defense responses and/or disease resistance.^9,11,12SABP2 is a very low abundance (10 fmol/mg), soluble protein of ∼30 kDa that exhibits high affinity for SA (K_d = 90 nM).⁷ Because these properties suggested that SABP2 might be an SA receptor, we spent five years and overcame several setbacks to purify this protein and clone its gene.¹³ One major setback was a dramatic reduction and eventual discontinuation of funding by the National Science Foundation (NSF). This discontinuation reflected in part the historically low grant funding levels at U.S. government agencies due to the Iraq war. Another obstacle was the prevailing attitude that biochemical approaches were inefficient/ineffective. Indeed, some of the grant reviewers questioned why we were wasting our time using such a challenging approach when genetics would eventually reveal SABP2 function. Despite these obstacles, we succeeded in partially purifying SABP2, cloning its gene, and demonstrating that SABP2 has esterase/lipase activity and is involved in disease resistance, including SAR.¹³The second major breakthrough on the SABP2 project involved using a combination of biochemistry, enzymology and biophysics. X-ray crystallography revealed that SA was bound in SABP2''s active site; this suggested that SA binding would lead to inhibition of SABP2''s esterase activity as its active site is too small to accommodate both its substrate and SA. Biochemical analyses confirmed this hypothesis and also established that MeSA is SABP2''s likely substrate.¹⁴ Subsequent studies confirmed that MeSA is SABP2''s in planta substrate, while grafting experiments revealed that SABP2 is required in the systemic tissue for perception/processing of the SAR signal but not in the primary infected tissue for generation of the SAR signal.¹⁵ Structure-function analyses, based on SABP2''s enzymology and 3-D structure in complex with SA, revealed that SABP2''s MeSA esterase activity is required in the systemic tissue while SABP2''s SA-binding activity and the resulting feedback inhibition of its MeSA esterase activity are needed in the primary infected tissue for an effective SAR response. Together these results argued that MeSA is the long-sought mobile SAR signal. This was confirmed by quantification of MeSA and SA in the primary infected tissue, in phloem exudates from this tissue and in the systemic tissue of wild type and SAR-deficient mutant or transgenic plants. This conclusion was further supported by the demonstration (via RNAi-mediated gene silencing) that the enzyme responsible for MeSA production from SA, SA methyl transferase, is required in the SAR signal-generating, primary infected tissue, but not in the systemic tissue.¹⁵Subsequent analyses strongly suggests that MeSA also is an SAR signal in Arabidopsis¹⁶ and potato (Manosalva P, Park SW, Klessig DF, unpublished results). Since Arabidopsis contains five MeSA esterases and most of these must be silenced in order to inhibit SAR development,¹⁶ classical genetic analyses did not and would not have revealed the role of these genes in SAR. In sum, the results of this 15 plus year project illustrate that persistence, even in the face of adversity, may be necessary to succeed, and it can pay off in rather unexpected ways. Our results also demonstrate that it is important to use biochemical and biophysical approaches, in combination with genetics, to explore complex biological processes.