Molecular biology. A self-help guide for a trim genome.
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Animals↗
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The high diversity of snoRNAs in plants: identification and comparative study of 120 snoRNA genes from Oryza sativa.
Using a powerful computer-assisted analysis strategy, a large-scale search of small nucleolar RNA (snoRNA) genes in the recently released draft sequence of the rice genome was carried out. This analysis identified 120 different box C/D snoRNA genes with a total of 346 gene variants, which were predicted to guide 135 2'-O-ribose methylation sites in rice rRNAs. Though not exhaustive, this analysis has revealed that rice has the highest number of known box C/D snoRNAs among eukaryotes. Interestingly, although many snoRNA genes are conserved between rice and Arabidopsis, almost half of the identified snoRNA genes are rice specific, which may highlight further the differences in rRNA methylation patterns between monocotyledons and dicotyledons. In addition to 76 singletons, 70 clusters involving 270 snoRNA genes were also found in rice. The large number of the novel snoRNA polycistrons found in the introns of rice protein-coding genes is in contrast to the one-snoRNA-per-intron organization of vertebrates and yeast, and of Arabidopsis in which only a few intronic snoRNA gene clusters were identified. Furthermore, due to a high degree of gene duplication, rice snoRNA genes are clearly redundant and exhibit great sequence variation among isoforms, allowing generation of new snoRNAs for selection. Thus, the large snoRNA gene family in plants can serve as an excellent model for a rapid and functional evolution.
Base Sequence↗
Structure of Aquifex aeolicus argonaute highlights conformational flexibility of the PAZ domain as a potential regulator of RNA-induced silencing complex function.
Gene silencing mediated by RNA interference requires the sequence-specific recognition of target mRNA by the endonuclease Argonaute, the primary enzymatic component of the RNA-induced silencing complex. We report the crystal structure of Aquifex aeolicus Argonaute, refined at 3.2A resolution. Relative to recent Argonaute structures, a 24 degrees reorientation of the PAZ domain in our structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. This rearrangement leads to a branched, Y-shaped system of grooves that extends through the molecule and merges in a central channel containing the catalytic residues. A 5.5-ns molecular dynamics simulation of Argonaute shows a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute monitored by total internal reflection fluorescence spectroscopy shows biphasic kinetics, also indicative of domain rearrangement upon DNA binding. Conformational rearrangement of the PAZ domain may therefore be critical for the catalytic cycle of Argonaute and the RNA-induced silencing complex.
Bacteria↗
Laser capture microdissection of epithelial cancers guided by antibodies against fibroblast activation protein and endosialin.
Transcriptional profiling of cancer biopsies is used extensively to identify expression signatures for specific cancer types, diagnostic and prognostic subgroups, and novel molecular targets for therapy. To broaden these applications, several challenges remain. For example, the integrity of RNA extracted even from small tissue samples has to be insured and monitored. Moreover, total tumor RNA may hide the marked histologic heterogeneity of human cancers. A principle approach to this heterogeneity has been provided by laser capture microdissection performed on antibody-stained tissue sections (immuno-LCM; iLCM). In this study, we have established a procedure to assess the quality of RNA obtained from tissue sections, coupled with immunostaining using antibodies to different tumor stromal markers, and subsequent iLCM to selectively capture the cancer stroma compartments. The procedure was applied to 53 frozen specimens of human epithelial cancers. Sections were stained for histopathological evaluation, and RNA was isolated from adjacent serial sections. RNA quality was assessed by the Agilent-Bioanalyzer (Agilent, Palo Alto, CA) and by multiplex RT-PCR. Two thirds of the specimens were found to yield good to excellent RNA quality. For microdissection of the tumor stroma with reactive fibroblasts and tumor blood vessels, a rapid incubation protocol with antibodies against fibroblast activation protein (FAP) and against endosialin was developed to ensure RNA integrity for subsequent iLCM. Using these procedures, RNA from distinct tumor compartments can be isolated, analyzed, amplified, and used for transcription profiling.
Antibodies, Neoplasm↗
The P9.1-P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme.
We have previously proposed a hierarchical model for the folding mechanism of the Tetrahymena ribozyme that may illustrate general features of the folding pathways of large RNAs. While the role of elements in the conserved catalytic core of this ribozyme during the folding process is beginning to emerge, the participation of non-conserved peripheral extensions in the kinetic folding mechanism has not yet been addressed. We now show that the 3'-terminal P9.1-P9.2 extension of the Tetrahymena ribozyme plays an important role during the folding process and appears to guide formation of the catalytic core.
Animals↗
Reconstruction of metabolic networks using incomplete information.
This paper describes an approach that uses methods for automated sequence analysis (Gaasterland et al. August 1994) and multiple databases accessed through an object+attribute view of the data (Baehr et al. 1992), together with metabolic pathways, reaction equations, and compounds parsed into a logical representation from the Enzyme and Metabolic Pathway Database (Selkov, Yunus, & et.al. 1994), as the sources of data for automatically reconstructing a weighted partial metabolic network for a prokaryotic organism. Additional information can be provided interactively by the expert user to guide reconstruction.
Algorithms↗
Genotypic resistance tests for the management of the patient failing highly active antiretroviral therapy: the resistance pattern in different biological compartments.
Single witness: HIV-1 drug resistance assays have been shown to be of value for guiding antiretroviral therapy (ART) decisions. Different tissues or body fluids in which HIV-1 can reside may contain viruses with distinct characteristics. HIV-1 variants with genotypic resistance markers are present in the male genital tract and evolve over time on incompletely suppressive ART. The magnitude of decline in the semen HIV-RNA level in with therapy is usually similar to the effect of treatment on the blood viral burden. Not all men on ART have complete suppression of HIV-1 replication in genital tract; thus, they may shed resistant HIV-1 strains. Failure of treatment to suppress HIV-RNA levels in the blood is common, resulting in the selection of resistant HIV-1 variants. Sexual transmission of resistant variants may have a negative impact on treatment outcome in newly infected individuals and on the spread of the diseases within a population. The use of rapid assessment of HIV resistance in plasma and semen may be useful in some situations, e.g. HIV transmitted infection by sexual contact from a viraemic patient. The recognition of resistant HIV isolates in the blood and semen of patients receiving ART leads to the question of the potential role of resistance testing. In many but not all infectious diseases, the choice of therapy is guided by sensitivity testing. Recent data suggest that testing the drug sensitivity of viral isolates from the blood can benefit patient management and response to therapy, albeit at considerable cost. However, since drug resistance may evolve independently in blood and semen, the public health benefits of such testing are unproven.
Antiretroviral Therapy, Highly Active↗
The development of West Nile virus safety policies by Canadian blood services: guiding principles and a comparison between Canada and the United States.
To address the emerging threat of West Nile virus (WNV) to the blood supply, Canadian Blood Services (CBS) made a series of policy decisions that were either similar to those adopted in the United States or more stringent than policies formulated in the United States at the same time. More stringent Canadian policies included the development of an in-house WNV RNA assay, the stockpiling of frozen plasma components in the winter for transfusion in WNV-affected areas in the summer, a special recruitment campaign for red blood cell collections before the start of the 2003 WNV season, and an inventory exchange (ie, WNV-tested for untested red blood cells) initiated 2 weeks after the onset of WNV screening, as well as the implementation of targeted individual-donation WNV testing on August 2, 2004, in the absence of any positive donors or clinical cases of WNV infection in Canada. The general principles that guided CBS decision making with regard to WNV safety included application of the precautionary principle, harmonization with policies in the United States, a consideration of logistic issues, compliance with Health Canada requests, responsiveness to public expectations about transfusion safety, and transparency in decision making with timely communication to stakeholders. Before implementing WNV blood safety policies, CBS assessed their impact on blood availability. When policies were implemented, data were obtained quickly to ensure that the prior impact assessments were accurate. This review discusses the guiding principles affecting CBS policy development and compares CBS WNV safety policies to policies adopted in the United States.
Antibodies, Viral↗
Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences.
To generate an extensive set of subgenomic (sg) mRNAs, nidoviruses (arteriviruses and coronaviruses) use a mechanism of discontinuous transcription. During this process, mRNAs are generated that represent the genomic 5' sequence, the so-called leader RNA, fused at specific positions to different 3' regions of the genome. The fusion of the leader to the mRNA bodies occurs at a short, conserved sequence element, the transcription-regulating sequence (TRS), which precedes every transcription unit in the genome and is also present at the 3' end of the leader sequence. Here, we have used site-directed mutagenesis of the infectious cDNA clone of the arterivirus equine arteritis virus to show that sg mRNA synthesis requires a base-pairing interaction between the leader TRS and the complement of a body TRS in the viral negative strand. Mutagenesis of the body TRS of equine arteritis virus RNA7 reduced sg RNA7 transcription severely or abolished it completely. Mutations in the leader TRS dramatically influenced the synthesis of all sg mRNAs. The construction of double mutants in which a mutant leader TRS was combined with the corresponding mutant RNA7 body TRS resulted in the specific restoration of mRNA7 synthesis. The analysis of the mRNA leader-body junctions of a number of mutants with partial transcriptional activity provided support for a mechanism of discontinuous minus-strand transcription that resembles similarity-assisted, copy-choice RNA recombination.
5' Untranslated Regions↗
The mRNA capping apparatus as drug target and guide to eukaryotic phylogeny.
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Acid Anhydride Hydrolases↗
Dissection of the role of the conserved G.U pair in group I RNA self-splicing.
Phylogenetic conservation among > 100 group I introns and previous in vitro studies have implicated a G.U pair as defining the 5'-splice site for exon ligation. The U residue defines the 3' end of the 5' exon, and the complementary G residue is part of the internal guide sequence (IGS) that base pairs to the 5' exon. We now quantitate the effect of this pair on individual reaction steps using the L-21ScaI ribozyme, which is derived from the group I intron of Tetrahymena thermophila pre-rRNA. The following results indicate that interactions with this G.U pair contribute to the binding of the 5'-exon, the positioning of the 5'-splice site with respect to the catalytic site, and the chemical step. The oligonucleotide, CCCUCU, binds to the ribozyme approximately 20-fold stronger than CCCUCC despite the fact that the U-containing oligonucleotide forms an approximately 5-fold less stable duplex with an oligonucleotide analog of the IGS, GGAGGG. This and two independent experimental observations indicate that the G.U pair contributes approximately 100-fold (3 kcal/mol, 50 degrees C) to tertiary interactions that allow the P1 duplex, which is formed between the 5'-exon and the IGS, to dock into the ribozyme's core. The approximately 50-80-fold increase in miscleavage of 5'-exon analogs upon replacement of the 3'-terminal U of CCCUCU with C or upon removal of the 3'-terminal U suggests that the tertiary interactions with the G.U pair not only contribute to docking but also ensure correct positioning of the 5'-splice site with respect to the catalytic site, thereby minimizing the selection of incorrect splice sites. Comparison of the rates of the chemical cleavage step with G.U vs G.C suggests that the G.U pair contributes approximately 10-fold to the chemical step. It was previously suggested that the 2'-hydroxyl of this U residue helps stabilize the 3'-oxyanion leaving group in the chemical transition state via an intramolecular hydrogen bond. Relative reactivities of oligonucleotide substrates with ribose and deoxyribose U and C are consistent with a model based on a recent X-ray crystallographic structure in which the exocyclic amino group of G helps orient the 2'-hydroxyl of U via a bridging water molecule, thereby strengthening the hydrogen bond donated from the 2'-hydroxyl group to the neighboring incipient 3'-oxyanion. Finally, kinetic and thermodynamic evidence for the formation of a G.C+ wobble pair is presented.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos.
BACKGROUND: RNA interference (RNAi) is a phenomenon in which introduced double-stranded RNAs (dsRNAs) silence gene expression through specific degradation of their cognate mRNAs. Recent analyses in vitro suggest that dsRNAs may be copied, or converted, into 21-23 nucleotide (nt) guide RNAs that direct the nucleases responsible for RNAi to their homologous mRNA targets. Such small RNAs are also associated with gene silencing in plants. RESULTS: We developed a quantitative single-embryo assay to examine the mechanism of RNAi in vivo. We found that dsRNA rapidly induced mRNA degradation. A fraction of dsRNAs were converted into 21-23 nt RNAs, and their time of appearance and persistence correlated precisely with inhibition of expression. The strength of RNAi increased disproportionately with increasing dsRNA length, but an 80bp dsRNA was capable of effective gene silencing. RNAi was saturated at low dsRNA concentration and inhibited by excess unrelated dsRNA. The antisense strand of the dsRNA determined target specificity, and excess complementary sense or antisense single-stranded RNAs (ssRNAs) competed with the RNAi reaction. CONCLUSIONS: Processed dsRNAs can act directly to mediate RNAi, with the antisense strand determining mRNA target specificity. The involvement of 21-23 nt RNAs is supported by the kinetics of the processing reaction and the observed size dependence. RNAi depends on a limiting factor, possibly the nuclease that generates the 21-23 mer species. The active moiety appears to contain both sense and antisense RNA strands.
Animals↗
Long-read transcriptomics corrects Trichomonas vaginalis intron annotations and refines transcript-end features.
BACKGROUND: Trichomonas vaginalis causes the most prevalent non-viral sexually transmitted infection worldwide. Despite its large genome (181.5 Mb; 36,310 predicted protein-coding genes in NYU_TvagG3_2), intron annotations remain limited and inconsistently validated. A recent short-read RNA-seq study reported 63 putative active introns, but short reads can misassign splice boundaries and cannot resolve complete transcript structures. METHODS: We integrated Oxford Nanopore direct RNA sequencing (DRS), ONT cDNA long-read sequencing, and Illumina RNA-seq to refine intron annotations, transcript-end features, and UTR boundaries in T. vaginalis. Candidate introns were validated by targeted PCR and Sanger sequencing, and representative splicing events were further assessed using public SRA datasets. RESULTS: Starting from 31 historically annotated introns, motif-guided long-read screening and orthogonal validation identified 17 additional validated introns, increasing the curated set to 48 confirmed introns. Among these 17 events, three were previously unrecognized in the current NYU_TvagG3_2 reference annotation. We also corrected five reported loci, including two false-positive introns, two splice-coordinate misannotations, and one gene-sequence error. DRS further supported transcript termination site mapping, UAAA polyadenylation-signal profiling relative to poly(A) addition sites, and single-molecule poly(A)-tail estimation. StringTie mixed-mode assemblies provided updated UTR boundaries for intron-bearing transcripts and transcripts without curated introns. CONCLUSIONS: This study provides a rigorously validated, long-read-refined resource of intron annotations, UTR boundaries, and UAAA-guided transcript-end features for T. vaginalis, together with a reproducible workflow for non-model protists. These refinements improve the current reference annotation and support future studies of functional genomics, parasite biology, pathogenesis, and diagnostic development.
Trichomonas vaginalis↗
Multiplexed RT-LAMP Assays in Handheld Devices for In-Situ Detection of Chikungunya, Dengue, Mayaro, and Zika Viruses.
Mosquito-borne viruses pose a significant global health challenge, particularly in resource-limited settings where multiple viruses often cause illnesses with similar symptoms that require different treatment. We introduce the first 7-plex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in a hand-held device capable of detecting the presence of Chikungunya virus (CHIKV), dengue virus serotypes (DENV 1-4), Mayaro virus (MAYV), and Zika virus (ZIKV) in a single test. If the result is positive from the single-plex device for the 7-plex assay, 3-plex and 4-plex devices are then used to identify the exact virus within a specimen. In-situ detection is achieved by integrating valve-enabled, paper-based sample preparation with fluorescence detection using a blue LED flashlight as a light source and a yellow plastic film as a filter, allowing visual discrimination between positive and negative samples by the naked eye or by recording images using a smart phone. The detection limits ranged from 2 genome equivalents (GE)/reaction (for ZIKV) to 92 GE/reaction (for DENV-3) across 7 types of viruses when 1 μL of viral RNA was used. We observed 90% overall agreement between the point-of-care (POC) device and lab-based reverse transcription polymerase chain reactions (RT-PCR) when blinded clinical specimens were tested. This assay and device have a potential to address critical surveillance gaps in endemic regions, enabling timely detection of multiple mosquito-borne viruses to guide appropriate clinical management and public health countermeasures in settings where laboratory resources are scarce.
Nucleic Acid Amplification Techniques↗
Combined depletion of cell cycle and transcriptional cyclin-dependent kinase activities induces apoptosis in cancer cells.
Selective cyclin-dependent kinase (cdk) 2 inhibition is readily compensated. However, reduced cdk2 activity may have antiproliferative effects in concert with other family members. Here, inducible RNA interference was used to codeplete cdk2 and cdk1 from NCI-H1299 non-small cell lung cancer and U2OS osteosarcoma cells, and effects were compared with those mediated by depletion of either cdk alone. Depletion of cdk2 slowed G1 progression of NCI-H1299 cells and depletion of cdk1 slowed G2-M progression in both cell lines, with associated endoreduplication in U2OS cells. However, compared with the incomplete cell cycle blocks produced by individual depletion, combined depletion had substantial consequences, with G2-M arrest predominating in NCI-H1299 cells and apoptosis the primary outcome in U2OS cells. In U2OS cells, combined depletion affected RNA polymerase II expression and phosphorylation, causing decreased expression of the antiapoptotic proteins Mcl-1 and X-linked inhibitor of apoptosis (XIAP), effects usually mediated by inhibition of the transcriptional cdk9. These events do not occur after individual depletion of cdk2 and cdk1, suggesting that reduction of cdk2, cdk1, and RNA polymerase II activities all contribute to apoptosis in U2OS cells. The limited cell death induced by combined depletion in NCI-H1299 cells was significantly increased by codepletion of cdk9 or XIAP or by simultaneous treatment with the cdk9 inhibitor flavopiridol. These results show the potency of concomitant compromise of cell cycle and transcriptional cdk activities and may guide the selection of clinical drug candidates.
Apoptosis↗
Identification and Analysis of Small Nucleolar RNAs by Real-Time Quantitative PCR.
One of the greatest scientific achievements of the twenty-first century is the completion of The Human Genome Project (HGP). Thereafter, we came to know that the human genome codes nearly 2% for making proteins and thus named as coding genes, suggesting the rest of the genome as noncoding or junk. However, research in the past two decades has shown and established that noncoding RNAs are major contributors of regulating and modulating the various function of cells as well as tissues. Noncoding RNAs can be classified as basis of their sizes in two categories, long noncoding RNAs (>200 nt) and small noncoding RNAs (<200 nt). Small nucleolar RNAs (snoRNAs) are part of the small noncoding RNA family and primarily reside inside the nucleus of eukaryotes. Sno RNAs can be divided into two major categories based on their distinguished structure and function; these are C/D box and HACA box snoRNAs. They participate in the posttranscriptional modifications on ribosomal RNAs (r-RNAs), transfer RNAs (t-RNAs), messenger RNAs (m-RNAs), and small nuclear RNAs (snRNAs). Sno RNAs act as guide RNAs to modify other noncoding RNAs by pseudouridylation or 2'O ribomethylation. We discussed in this protocol about one of the widely used techniques for detection and analysis of snoRNAs, i.e., real-time quantitative PCR (RT-qPCR).
RNA, Small Nucleolar↗
Microbial allies in a cotton pest: A descriptive account of associated microbiota dynamics in Dysdercus cingulatus across development.
BACKGROUND: Hemipteran insects harbour several symbiotic partners, mainly bacteria, which play pivotal roles for hosts like dietary provision, support overall physiology, xenobiotic degradation and manipulate/regulate behaviour. Most of these symbionts usually reside and operate from the digestive tracts of the animals. Cotton is one of the major cash crops in India and Dysdercus cingulatus (D. cingulatus) though a secondary pest, is causing significant destruction of cotton bolls, poor lint quality and reduce oil content of seeds. Premature opening of cotton bolls often leads to bacterial and fungal infections, thus resulting in extensive economic loss worldwide. D. cingulatus is a hemimetabolous insect that comprises of developmental stages like egg, nymph (5 instar stages), and adult. The present work explored the ontogeny specific diversity in the associated microbiota and predicted their probable functional inputs in D. cingulatus. RESULTS: The data obtained using 16S rRNA gene sequencing (NovaSeq 6000) revealed presence of members of Proteobacteria (65.83%), Firmicutes (24%), Actinobacteria (10%) phyla throughout the ontogeny of D. cingulatus. Highest alpha diversity of these symbiotic bacteria was recorded in the third instar nymphs in contrast to rest of the developmental stages. Among all the observed genera, Stenotrophomonas, Hungatella and Glutamicibacter were predominant from egg to adult stages. MicFunPred, a tool used for predicting the probable functional inputs of these symbionts, hinted at their probable stage specific contribution in crucial biochemical pathways such as polyketide biosynthesis, ascorbate/aldarate metabolism, pentose phosphate and glyoxylate cycles, steroid hormone and peptidoglycan biosynthesis, and glycolysis/pyruvate metabolism. CONCLUSIONS: The primary investigations on the ontogenetic composition and diversity of associated microbiota, suggest dynamic shifts in D. cingulatus, concurrent with their probable functions/roles in the host development and metabolism. To the best of our knowledge, this is the first report on symbiotic microbiota variation across the developmental stages of D. cingulatus that provides preliminary descriptive observations that may guide future functional and experimental investigations into microbiota-based pest management.
Animals↗
Molecular and cellular aspects of nerve regeneration.
Injury of an axon leads to at least four independent events, summarized in Figure 1: first, deprivation of the nerve cell body from target-derived or mediated substances, which leads to a derepressed or a permissive state; second, disruption of anterograde transport, with a resultant accumulation of anterogradely transported molecules; third, environmental response with possible consequent changes in constituents of the extracellular matrix and substances secreted from the surrounding cells; and fourth, appearance of growth inhibitors and modified protease activity. It seems that the first three of these events are obligatory, but not sufficient, i.e., they lead to a growth state only if the cell body is able to respond to the injury-induced signals from the environment (a and b). The regenerative state is characterized by alterations in protein synthesis and axonal transport and by sprouting activity. The subsequent elongation of the growing fibers depends on a continuous supply of appropriate growth factors. These factors are presumably anchored to the appropriate extracellular matrix that serves as a substratum for elongating fibers. It should be mentioned that the proliferating nonneuronal cells have a conducive effect on regeneration by forming a scaffold for the growing fibers. Accordingly, the lack of regeneration may stem from a deficiency in the ability of glial cells to provide the appropriate soluble components or from insufficient formation of extracellular matrix. In this respect, one may consider regeneration of an injured axon as a process which involves regeneration of both the nonneuronal cells and the supported axons. The regeneration of glial cells may fulfill the rules which are applied to regeneration of any other proliferating tissue. Furthermore, the processes of regeneration in the axon and the glial cells are mutually dependent. Perhaps the triggering factors provided by the nonneuronal cells affect the nonneuronal cells themselves by modulating their postlesion gliosis and thereby inducing their appropriate activation. In such a case, regeneration of nonneuronal cells may resemble an autocrine type of regulation that exists also during ontogeny. The growth regulation is shifted back to the paracrine type upon neuronal maturation or cessation of axonal growth. When the elongating fibers reach the vicinity of the target organ, they are under the influence of the target-derived factors, which guide the fibers and eventually cease their elongation.
Animals↗