Pieces Of RNA Research Articles

Disrupted tRNA Gene Diversity and Possible Evolutionary Scenarios

The following unusual tRNAs have recently been discovered in the genomes of Archaea and primitive Eukaryota: multiple-intron-containing tRNAs, which have more than one intron; split tRNAs, which are produced from two pieces of RNA transcribed from separate genes; tri-split tRNAs, which are produced from three separate genes; and permuted tRNA, in which the 5' and 3' halves are encoded with permuted orientations within a single gene. All these disrupted tRNA genes can form mature contiguous tRNA, which is aminoacylated after processing by cis or trans splicing. The discovery of such tRNA disruptions has raised the question of when and why these complex tRNA processing pathways emerged during the evolution of life. Many previous reports have noted that tRNA genes contain a single intron in the anticodon loop region, a feature common throughout all three domains of life, suggesting an ancient trait of the last universal common ancestor. In this context, these unique tRNA disruptions recently found only in Archaea and primitive Eukaryota provide new insight into the origin and evolution of tRNA genes, encouraging further research in this field. In this paper, we summarize the phylogeny, structure, and processing machinery of all known types of disrupted tRNAs and discuss possible evolutionary scenarios for these tRNA genes.

Formation of the First Peptide Bond: The Structure of EF-P Bound to the 70 S Ribosome

Elongation factor P (EF-P) is an essential protein that stimulates the formation of the first peptide bond in protein synthesis. Here we report the crystal structure of EF-P bound to the Thermus thermophilus 70S ribosome along with the initiator transfer RNA N-formyl-methionyl-tRNA(i) (fMet-tRNA(i)(fMet)) and a short piece of messenger RNA (mRNA) at a resolution of 3.5 angstroms. EF-P binds to a site located between the binding site for the peptidyl tRNA (P site) and the exiting tRNA (E site). It spans both ribosomal subunits with its amino-terminal domain positioned adjacent to the aminoacyl acceptor stem and its carboxyl-terminal domain positioned next to the anticodon stem-loop of the P site-bound initiator tRNA. Domain II of EF-P interacts with the ribosomal protein L1, which results in the largest movement of the L1 stalk that has been observed in the absence of ratcheting of the ribosomal subunits. EF-P facilitates the proper positioning of the fMet-tRNA(i)(fMet) for the formation of the first peptide bond during translation initiation.

Characterization of a Mumps Virus Nucleocapsidlike Particle

The nucleocapsid protein (NP) of mumps virus (MuV), a paramyxovirus, was coexpressed with the phosphoprotein (P) in Escherichia coli. The NP and P proteins form a soluble complex containing RNA. Under a transmission electron microscope, the NP-RNA complex appears as a nucleocapsidlike ring that has a diameter of approximately 20 nm with 13 subunits. There is a piece of single-stranded RNA with a length of 78 nucleotides in the NP-RNA ring. Shorter RNA pieces are also visible. The MuV NP protein may provide weaker protection of the RNA than the NP protein of some other negative-strand RNA viruses, reflecting the degree of NP protein association.

RNA-Based Therapeutics: Ready for Delivery?

Delivery of RNA-based therapeutics to specific tissues for treating a variety of diseases faces many hurdles. But with several clinical trials and a slew of studies in animal models underway, the future of RNA-based therapeutics seems bright.

Slowing Down a Hammerhead Ribozyme Allows Glimpses of It in Action

Slowing Down a Hammerhead Ribozyme Allows Glimpses of It in Action

The origin of genes could be polyphyletic

The paradigm of the monophyletic origin of genes is deeply rooted in us all. For instance, this stems from the observation that the possibility of obtaining a good multiple alignment using the same protein from organisms from the three domains of life (Bacteria, Archaea and Eukarya) would seem to imply that the last universal common ancestor (LUCA) must have had that protein and, therefore, the origin of that gene must necessarily be monophyletic. The hypothesis of a polyphyletic origin of genes has to explain how it was possible to evolve highly conserved regions of multiple alignments of orthologous proteins from the three domains of life when these regions clearly seem to define a monophyletic origin of genes. If mRNAs were assembled at the stage of the LUCA through the trans-splicing of pieces of RNA representing mini-genes, and the translation of these mRNAs resulted in proteins whose genes (DNA) actually only evolved much later, i.e. only after the main domains of life were established, then this would explain why multiple alignments of orthologous proteins can be obtained from the three domains of life. Therefore, this makes these multiple alignments compatible with a polyphyletic origin of genes. I have analysed many multiple alignments of orthologous proteins from the three domains of life, reaching a conclusion that seems to suggest that these alignments are also compatible with a polyphyletic origin of genes because, for instance, they contain protein motifs characterising the domains of life. These motifs, and also genes, might have evolved late on, thus making their polyphyletic origin likely.

Neurons need miRNA to survive

Neurons missing their microRNA (miRNA) gradually deteriorate in structure and function, according to Schaefer et al. (page 1553). These bits of RNA prolong neuronal longevity and may keep neurodegenerative diseases at bay. Figure 1 Lack of Dicer causes neurons to degenerate (right). miRNA—small pieces of RNA generated by the RNA-chopping enzyme called Dicer—strategically shut off genes to allow embryonic cells to differentiate. Because mice that lack Dicer die as embryos, the role of miRNA in vivo has been studied by deleting Dicer only in specific cell types. These studies showed that Dicer—and thus miRNA—is required for both cell differentiation and the survival of some cells. Neurons need miRNA for differentiation, but whether they also need them for survival later was unknown. Schaefer et al. now find that inactivating Dicer in postmitotic neurons of adult mice eventually kills these cells. The neurons maintained some of their miRNA long after Dicer inactivation and looked and functioned normally. But the reduction in miRNA eventually took its toll. Neuronal signaling malfunctioned, and the cells died off. As cell death progressed, the mice developed symptoms reminiscent of those seen in humans with neurodegenerative disorders. Whether the human disorders are caused by changes in specific miRNA remains to be seen.

Resizing the Genomic Regulation of Restenosis

See related article, pages 1579–1588 With the introduction of drug eluting stents (DES) for percutaneous coronary interventions, restenosis appeared to be a problem of the past. However, the biology of arterial injury has returned to the limelight of clinical cardiology because of increased late-stent thrombosis associated with DES.1 As DES use initially increased, concerns were raised about altering the normal responses to arterial injury with antiproliferative drugs based on animal and human data.2 Since 2006, this concern has translated to dramatic decreases in DES deployment in catherization laboratories across the U.S. These events serve to underscore the importance of continuing to incorporate new clinical and basic experimental data for improving patient management. In a different realm, developmental biologists working on the primitive earthworm Caenorhabditis elegans discovered a novel regulatory mechanism involving short pieces of RNA (microRNA or miRNA) for which they were awarded the 2006 Nobel Prize in Physiology/Medicine. Over 500 different miRNAs have now been identified in the mouse and human genomes (miRBase: http://microrna.sanger.ac.uk/); a schematic overview of miRNA biogenesis is provided in the Figure (A). miRNAs generally act on their target messenger RNAs (mRNA) by promoting RNA degradation or inhibiting protein translation. They are expressed in a tissue- and condition-specific manner indicating their potential role in normal development and disease pathogenesis.3 They appear to regulate diverse processes such as cell proliferation, cell death, metabolism, hematopoiesis, angiogenesis, and tumorigenesis.3–6 Emerging genetic evidence also suggests an essential role of miRNAs in normal cardiogenesis and abnormal stress responses such as hypertrophy and arrhythmogenesis.7–9 There are also reports of miRNA regulation of skeletal muscle, but there are no studies on miRNA expression in proliferating vascular smooth muscle cells thought to …

Glossary of terms

Glossary of terms

Characterization of the structure, function, and mechanism of B2 RNA, an ncRNA repressor of RNA polymerase II transcription

We previously found that the SINE-encoded mouse B2 RNA binds RNA polymerase II and represses mRNA transcription during the cellular heat-shock response. In vitro B2 RNA assembles into preinitiation complexes on promoter DNA via its interaction with the polymerase, thus rendering the complexes inactive. With the goal of understanding which regions of B2 RNA are important for high-affinity binding to RNA polymerase II and repression of transcription, we performed a structural and deletion analysis of a 178 nucleotide (nt) B2 RNA. We describe an experimentally derived secondary structure model for B2 RNA, and show that RNA polymerase II protects a specific region from RNase digestion. Deletion studies revealed that a 51-nt region of B2 RNA is sufficient for high-affinity binding to RNA polymerase II, association with preinitiation complexes, and repression of transcription in vitro, the latter of which involves a large predominately single-stranded region within the RNA. In addition, this piece of B2 RNA blocked the polymerase from properly associating with template DNA during the assembly of elongation complexes. Further deletion revealed that a 33-nt piece of B2 RNA binds RNA polymerase II, associates with preinitiation complexes, but cannot repress transcription. These results support a model in which RNA polymerase II contains a high-affinity ncRNA docking site to which a distinct region of B2 RNA binds, thereby allowing another region of the RNA to repress transcription. Moreover, the mechanism of transcriptional repression by B2 RNA likely involves disrupting critical contacts between RNA polymerase II and the promoter DNA.

ANOTHER ROLE FOR RNA

IN ADDITION TO SILENCING gene expression, short pieces of double-stranded RNA may be able to activate gene expression. To activate gene expression, David R. Corey, Bethany A. Janowski, and coworkers at the University of Texas Southwestern Medical Center, Dallas, use RNA duplexes that target a portion of genomic DNA up-stream from the site where gene transcription begins ( Nat. Chem. Biol., DOI: 10.1038/nchem bio86O). The effect requires that the RNA duplex's sequence be complementary to that of the targeted genomic DNA. Although structurally similar to the RNA used in RNA interference, these RNA duplexes target DNA instead of messenger RNA. The researchers first saw inklings of the effect when they were studying similar DNA-targeting RNAs that reduce the expression of the progesterone receptor (C&EN, Aug. 8,2005, page 10). Some of those RNAs increased gene expression rather than silencing it, but the cell line they were using at the time produced so much progesterone receptor to begin ...

RNA Silencing Sheds Light on the RNA World

RNA silencing — also known as RNA interference — is an intriguing phenomenon in which short, double-stranded RNA “triggers” can prevent the expression of specific genes. First discovered in plants, RNA interference is now recognized as a widespread, if not ubiquitous, phenomenon, and it is causing great excitement as an experimental technique for selectively blocking gene expression. The mechanisms of RNA silencing have been intensively studied. One important step is the formation of single-stranded RNA pieces (called siRNAs) from the double-stranded triggers. In “lower” organisms—including plants, protozoa, fungi, and nematode worms—it also involves an enzyme—called RNA-dependent RNA polymerase—that can generate a strand of RNA using existing RNA as a template. This means that it can create double-stranded RNA from single-stranded pieces of RNA. By doing this, it generates more triggers and so amplifies the effect of RNA silencing. Paula Salgado and her colleagues have studied the structure of one such polymerase, called QDE-1, and found that it provides clues to the earliest stages of evolution. When a gene is transcribed and translated to generate a protein, the process begins with a DNA-dependent RNA polymerase. Like QDE-1, DNA-dependent RNA polymerases generate strands of RNA—the difference is that they use a DNA template to do it. The RNA they generate is called messenger RNA and is in turn used as the template for building a protein out of amino acids. The structures of DNA-dependent RNA polymerases have been described previously, so that the authors of this study could compare them with their new structure of QDE-1. What they found was a remarkable similarity. Both DNA-dependent RNA polymerases and QDE-1 have an active catalytic site—the working core of the enzyme—that is formed by two distinctive structural domains called double-psi β-barrels. This strong structural resemblance between QDE-1 and the DNA-dependent RNA polymerases points towards an evolutionary link between the two types of RNA polymerase. An influential theory on the origin of life proposes that RNA molecules were the first self-replicating molecules, forming a kind of precellular life in an “RNA world.” Initially, RNA molecules would have had to act as enzymes as well as genetic information so that they could replicate, but it is likely that an RNA-dependent RNA polymerase would have been one of the earliest protein-based enzymes to evolve. The similarity between QDE-1 and the DNA-dependent RNA polymerases suggests that both evolved from one ancestor, because otherwise, the resemblance between their active sites would be highly unlikely. This common ancestor might have been a primordial RNA polymerase in an RNA world. The authors suggest that this ancestor would have had just one double-psi β-barrel, and that gene duplication led to an enzyme with two barrels. From this early polymerase evolved both QDE-1–like RNA-dependent RNA polymerases and a diverse array of specialized DNA-dependent RNA polymerases. The diversification of DNA-dependent RNA polymerases would have been facilitated by the splitting of the two double-psi β-barrels onto separate subunits, rather than being borne on the same subunit as in QDE-1, so that different subunits could combine to create specialized polymerases. These findings provide a link between RNA silencing and the earliest mechanisms of RNA transcription—perhaps shedding light on both the origins of RNA replication (and therefore life) and the evolution of RNA silencing. They also provide insights into the mechanism of action of QDE-1 that might apply across the board to RNA-dependent RNA polymerases, and that will be built upon by further work.

RNA Interference: Big Applause for Silencing in Stockholm

Eight years ago, Craig Mello, Andrew Fire, and their coworkers provided the first demonstration that double-stranded RNA (dsRNA) triggers the gene-silencing technique that we now call RNA interference (RNAi). For this landmark discovery, Mello and Fire are honored with this year's Nobel Prize in Physiology or Medicine.

A Colorectal Catalog

BIOMEDICINE Global surveys, inaugurated by almost complete compendiums of the genes of various organisms, have been expanded to cover proteins and, more recently, microRNAs (miRNAs), which are roughly 25-nucleotide-long RNA molecules that function to block the production of proteins from mRNAs. Cummins et al. describe a protocol—the miRNA serial analysis of gene expression (miRAGE)—and its application to assessing the miRNA composition of human colorectal cancer cells. Their approach meets the technical challenge of recovering short RNA pieces, present in vanishingly small quantities; analyzing an enormous number of parallel amplification reactions resulted in the identification of 200 miRNAs known within these cells (with one-quarter differentially expressed in comparison to normal colonic epithelial cells) and of 168 candidate miRNAs, of which one-fifth were independently identified and deposited by other groups during the course of their study. — GJC Proc. Natl. Acad. Sci. U.S.A. 103 , 3687 (2006).

The RNA Silencing Pathway: The Bits and Pieces That Matter

Cellular pathways are generally proposed on the basis of available experimental knowledge. The proposed pathways, however, may be inadequate to describe the phenomena they are supposed to explain. For instance, by means of concise mathematical models we are able to reveal shortcomings in the current description of the pathway of RNA silencing. The silencing pathway operates by cleaving siRNAs from dsRNA. siRNAs can associate with RISC, leading to the degradation of the target mRNA. We propose and analyze a few small extensions to the pathway: a siRNA degrading RNase, primed amplification of aberrant RNA pieces, and cooperation between aberrant RNA to trigger amplification. These extensions allow for a consistent explanation for various types of silencing phenomena, such as virus induced silencing, transgene and transposon induced silencing, and avoidance of self-reactivity, as well as for differences found between species groups.

Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA

Splicing is a crucial step for human immunodeficiency virus, type 1 (HIV-1) multiplication; eight acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs. The effects of SR proteins have only been investigated on a limited number of HIV-1 splicing sites by using small HIV-1 RNA pieces. To understand how SR proteins influence the use of HIV-1 splicing sites, we tested the effects of overproduction of individual SR proteins in HeLa cells on the splicing pattern of an HIV-1 RNA that contained all the splicing sites. The steady state levels of the HIV-1 mRNAs produced were quantified by reverse transcriptase-PCR. For interpretation of the data, transcripts containing one or several of the HIV-1 acceptor sites were spliced in vitro in the presence or the absence of one of the tested SR proteins. Both in vivo and in vitro, acceptor sites A2 and A3 were found to be strongly and specifically regulated by SR proteins. ASF/SF2 strongly activates site A2 and to a lesser extent site A1. As a result, upon ASF/SF2 overexpression, the vpr mRNA steady state level is specifically increased. SC35 and SRp40, but not 9G8, strongly activate site A3, and their overexpression ex vivo induces a dramatic accumulation of the tat mRNA, to the detriment of most of the other viral mRNAs. Here we showed by Western blot analysis that the Nef protein synthesis is strongly decreased by overexpression of SC35, SRp40, and ASF/SF2. Finally, activation by ASF/SF2 and 9G8 was found to be independent of the RS domain. This is the first investigation of the effects of variations of individual SR protein concentrations that is performed ex vivo on an RNA containing a complex set of splicing sites.

Nonreplicative homologous RNA recombination: promiscuous joining of RNA pieces?

Biologically important joining of RNA pieces in cells, as exemplified by splicing and some classes of RNA editing, is posttranscriptional, whereas in RNA viruses it is generally believed to occur during viral RNA polymerase-dependent RNA synthesis. Here, we demonstrate the assembly of precise genome of an RNA virus (poliovirus) from its cotransfected fragments, which does not require specific RNA sequences, takes place before generation of the viral RNA polymerase, and occurs in different ways: Apparently unrestricted ligation of the terminal nucleotides, joining of any one of the two entire fragments with the relevant internal nucleotide of its partner, or internal crossovers within the overlapping sequence. Incorporation of the entire 5' or 3' partners into the recombinant RNA is activated by the presence of terminal 3'-phosphate and 5'-OH, respectively. Such postreplicative reactions, fundamentally differing from the known site-specific and structurally demanding cellular RNA rearrangements, might contribute to the origin and evolution of RNA viruses and could generate new RNA species during all stages of biological evolution.

RNA interference and Fragile X

RNA interference (RNAi) is an evolutionary conserved gene regulation mechanism found throughout the phyla. RNAi-induced gene silencing acts at the posttranscriptional stage and depends upon the introduction of double-stranded RNA. These dsRNAs are processed by’Dicer’ enzymes into short (∼21–22 nucleotide) pieces of double-stranded RNA called short interfering RNAs, or siRNAs. The siRNAs are incorporated into an RNAi effector complex called RISC (for’RNAi-induced silencing complex’), which uses them as a guide to target and destroy complementary mRNAs, and thereby prevent synthesis of the encoded protein. Although some components of the RNAi cellular machinery have been identified, the overall picture is far from clear. Recently however, two research groups demonstrated independently in Drosophila that one of the components of the RNAi pathway is dFMR1 (dFXR), the homologue of the human protein associated with fragile X mental retardation [ 1. Caudy A.A. et al. Fragile X-related protein and VIG associated with the RNA interference machinery. Genes Dev. 2002; 16: 2491-2496 Crossref PubMed Scopus (534) Google Scholar , 2. Ishizuka A. et al. A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev. 2002; 16: 2497-2508 Crossref PubMed Scopus (486) Google Scholar ]. Both groups identified an association between dFMR1, short dsRNAs and Argonaute2 (AGO2), a previously identified subunit of RISC. Ishizuka et al. [ 2. Ishizuka A. et al. A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev. 2002; 16: 2497-2508 Crossref PubMed Scopus (486) Google Scholar ] also found that dFMR1 associates with the Dicer processing enzyme. Together these findings suggest that dFMR1 functions in an RNAi-related process to regulate the expression of its target genes at the level of translation (protein synthesis).

RNA interference and fragile X

RNA interference (RNAi) is an evolutionarily conserved gene regulation mechanism that is found across phyla and that acts at the post-transcriptional level. RNAi-induced gene silencing depends on the introduction of double-stranded RNA into a cell, and subsequent processing by so called Dicer enzymes to give short (∼21–22 nucleotides in length) pieces of double-stranded RNA. These short interfering RNAs, or siRNAs as they are known, are incorporated into an RNAi effector complex (the RNAi-induced silencing complex, or RISC), which uses them as a guide to target and destroy complementary mRNAs, thereby preventing synthesis of the encoded protein. Although some components of the RNAi cellular machinery have been identified, the overall picture is far from clear. Recently however, two independent research groups have demonstrated in Drosophila that one of the components of the RNAi pathway is dFMR1 (also known as dFXR), the homologue of the human fragile-X mental-retardation protein [1,2]. Both groups identified an association between dFMR1, short double-stranded RNAs and Argonaute2 (AGO2), a previously identified subunit of RISC. One research team also found that dFMR1 associates with the Dicer processing enzymes [2]. Taken together, these findings suggest that dFMR1 could function in an RNAi-related process to regulate the expression of its target genes at the level of translation.

Youthful Restraint

SINGAPORE --Driving 70 kilometers per hour on the freeway annoys other motorists, but it adds to a car's life. Similarly, putting the brakes on cellular energy production helps animals persist, a hypothesis of aging posits. Research presented at the Asia Pacific Conference and Exhibition on Anti-Ageing Medicine 2002, held here from 23 to 26 June, suggests that restricting mileage on a biological energy generator early in life can prolong a worm's life. Some scientists say that the study throws a spanner in the hypothesis's works, but others argue that the worms aren't gaining meaningful life, they're just living in slow motion. The rate-of-living hypothesis says that the higher an animal's metabolic rate, the shorter its life, because toxic byproducts build up (see "The Two Faces of Oxygen" ). Several observations support the idea; for example, some mutations that reduce energy production let worms live longer, and keeping flies at low temperature, which slows chemical reactions, grants them extra time. As an antiaging strategy for humans, however, the idea holds little promise, because people wouldn't want to spend their extra years hibernating. New results hint that Caenorhabditis elegans with reduced metabolism only during their youth reap life-extending benefits in adulthood. Ao-Lin "Allen" Hsu and colleagues at the University of California, San Francisco, used short pieces of RNA to turn off C. elegans genes one by one. To get the RNA molecules into the worms, the researchers grew the animals on petri plates, each coated with bacteria carrying RNA molecules that target a different gene. The worms gulped the bacteria, including the small RNA molecules. After 24 days, most control worms had died, but worms on some test plates survived. This method enabled the researchers to identify four genes that influence longevity, each of which produces a different component of protein machines that help turn food into energy. The worms carried lower concentrations of the high-energy molecule ATP than did controls, indicating that thwarting the genes slowed metabolism. Then Hsu and colleagues probed whether the timing of gene inactivation influenced longevity. They grew one set of worms on bacteria with interfering RNA only after the animals had reached adulthood; they exposed another group to the engineered bacteria during larval stages and allowed the genes to turn back on once the worms had matured. Animals with the genes shut down during adulthood died when controls did, but those with genes turned off only as larvae kept wriggling. The results suggest that slowing metabolism during larval development dictates adult life-span, although the mechanism is unclear. Longevity didn't depend on the amount of time that energy production slowed, so the life-extending manipulation runs counter to the rate-of-living theory, the scientists reason. "It is elegant genetic work," says evolutionary biologist Michael Rose of the University of California, Irvine. The tenacious worms, however, move, defecate, and reproduce more slowly than do normal worms, and he suggests that the animals are not acquiring quality time. Nevertheless, the study hints that slowing your engine while young can have far-reaching effects. --R. John Davenport Further Reading Asia Pacific Conference and Exhibition on Anti-Ageing Medicine 2002 http://www.antiaging2002.org/index.shtml