93 research outputs found
Tracing Translational Footprint by Ribo-Seq: Principle, Workflow, and Applications to Understand the Mechanism of Human Diseases
No full textRNA-seq has been widely used as a high-throughput method to characterize transcript dynamic changes in a broad context, such as development and diseases. However, whether RNA-seq-estimated transcriptional dynamics can be translated into protein level changes is largely unknown. Ribo-seq (Ribosome profiling) is an emerging technology that allows for the investigation of the translational footprint via profiling ribosome-bounded mRNA fragments. Ribo-seq coupled with RNA-seq will allow us to understand the transcriptional and translational control of the fundamental biological process and human diseases. This review focuses on discussing the principle, workflow, and applications of Ribo-seq to study human diseases
PK-PD modeling of 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine and the enhanced antitumor effect of its phospholipid derivatives in long-circulating liposomes
No full textThe efficacy of an antitumor nucleoside, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (3'-ethynylcytidine, ECyd), was analyzed in vitro and in vivo. The in vivo antitumor effect of ECyd encapsulated into long-circulating liposomes was also examined. Based on pharmacokinetic (PK) and pharmacodynamic (PD) analyses, a model that quantitatively explains the in vivo effects of ECyd was proposed, using the concept of minimum effective concentration. The model suggests that ECyd followed a time-dependent mechanism of action in vivo, and that availability of ECyd in tumor tissue was highly important. To improve the availability of ECyd, its phospholipid derivatives were synthesized and encapsulated into long-circulating liposomes, which increased the antitumor effect. These results indicate that it is very important to design carriers of antitumor drugs based on PK-PD modeling
A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing
No full textAbstractTo compare the two RNA-sequencing protocols, ribo-minus RNA-sequencing (rmRNA-seq) and polyA-selected RNA-sequencing (mRNA-seq), we acquired transcriptomic data–52 and 32 million alignable reads of 35 bases in length–from the mouse cerebrum, respectively. We found that a higher proportion, 44% and 25%, of the uniquely alignable rmRNA-seq reads, is in intergenic and intronic regions, respectively, as compared to 23% and 15% from the mRNA-seq dataset. Further analysis made an additional discovery of transcripts of protein-coding genes (such as Histone, Heg1, and Dux), ncRNAs, snoRNAs, snRNAs, and novel ncRNAs as well as repeat elements in rmRNA-seq dataset. This result suggests that rmRNA-seq method should detect more polyA- or bimorphic transcripts. Finally, through comparative analyses of gene expression profiles among multiple datasets, we demonstrated that different RNA sample preparations may result in significant variations in gene expression profiles
Developing variational Bayesian inference for applications to gene expression data
No full textBayesian hierarchical generalized linear models are intuitively appealing for applications to gene sequencing data. However, they can be computationally costly to fit in high-dimensional settings using standard Markov-chain Monte Carlo methods. Here we explore the use of variational inference techniques to approximate the posterior of Bayesian hierarchical GLMMs for detecting differential expression in RNA-Seq data or differential translational efficiency in Ribo-Seq data. We find that in simulation studies the variational approach is comparable to two common methods for detecting differential expression, and that the variational posterior is close to the Markov-chain Monte Carlo posterior.</p
RNase protection assay on mitochondrial (−)strand transcripts mapping around the cyt b/ND1-binding site in DmTTF-depleted cells
No full text<p><b>Copyright information:</b></p><p>Taken from "The termination factor DmTTF regulates mitochondrial transcription"</p><p>Nucleic Acids Research 2006;34(7):2109-2116.</p><p>Published online 28 Apr 2006</p><p>PMCID:PMC1450328.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Schematic representation of digestion products using probes Ribo-1 (295 nt) and Ribo-2 (218 nt). Riboprobes (grey bold arrows), mature transcripts (continuous arrows) and read-through transcripts (dotted arrows) are indicated above the cyt b/ND1 region map. Dashed regions indicate non-coding sequences. () Total cellular RNA (50 µg), extracted from untreated (control) and DmTTF-dsRNA treated (RNAi) D.Mel-2 cells, was hybridized with about 1.5 × 10 c.p.m. of Ribo-1 and Ribo-2 probes and digested with RNase A and T1. Digestion products were denatured and run on a 10% polyacrylamide/7 M urea gel. Y, sample containing 50 µg of yeast total RNA. M, Decade RNA marker (Ambion)
A DNA polymerase ϵ inhibitor activates the ribo and deoxyribo modes of primase expression and induces a unique phenomenon of primer accumulation11The author dedicates this piece of work in memory of his teacher, Professor F. Bruno Straub.
No full textAbstractCarbonyldiphosphonate (COMDP), a selective inhibitor of DNA polymerase (pol) ϵ, strongly stimulates expression of the ribo and deoxyribo modes of primase (Pr) activities of the Pr–DNA pol α enzyme complex associated with special cytoplasmic nucleoprotein complexes of chicken leukemic myeloblasts [J. Řı́man and A. Šulová, Acta Virol. 41 (1997) 181–214]. Besides stimulation, COMDP uncouples the Pr activities from those of DNA pol α, inducing in this way a unique phenomenon of accumulation of primers of basic length. In the presence of dNTPs, the COMDP effect is counteracted by excess of mimosine. The mutually exclusive effects of these agents are discussed
Variational inference for detecting differential translation in ribosome profiling studies
No full textTranslational efficiency change is an important mechanism for regulating protein synthesis. Experiments with paired ribosome profiling (Ribo-seq) and mRNA-sequencing (RNA-seq) allow the study of translational efficiency by simultaneously quantifying the abundances of total transcripts and those that are being actively translated. Existing methods for Ribo-seq data analysis either ignore the pairing structure in the experimental design or treat the paired samples as fixed effects instead of random effects. To address these issues, we propose a hierarchical Bayesian generalized linear mixed effects model which incorporates a random effect for the paired samples according to the experimental design. We provide an analytical software tool, “riboVI,” that uses a novel variational Bayesian algorithm to fit our model in an efficient way. Simulation studies demonstrate that “riboVI” outperforms existing methods in terms of both ranking differentially translated genes and controlling false discovery rate. We also analyzed data from a real ribosome profiling experiment, which provided new biological insight into virus-host interactions by revealing changes in hormone signaling and regulation of signal transduction not detected by other Ribo-seq data analysis tools.This article is published as Walker DC, Lozier ZR, Bi R, Kanodia P, Miller WA and Liu P (2023) Variational inference for detecting differential translation in ribosome profiling studies. Front. Genet. 14:1178508. doi: 10.3389/fgene.2023.1178508. Posted with permission. © 2023 Walker, Lozier, Bi, Kanodia, Miller and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms
The first salen-type ligands derived from 3',5'-diamino-3',5'-dideoxythymidine and -dideoxyxylothymidine and their corresponding copper(II) complexes
No full textBackground There are many nucleoside metal complexes known. According to observations made, only very few of them reveal their central ion to be co-ordinated by the sugar part of their molecules. The regio- and stereospecific exchange of the hydroxyl groups at the sugar moiety by chelating units improves its complexation ability and should give access to a new class of chiral ligands. Results In this paper we present the synthesis of 3',5'-diamino substituted thymidines with ribo- as well as xylo-configuration and the preparation of copper(II) complexes derived from their corresponding Schiff bases. Starting from thymidine, the amino derivatives were prepared in a three and four step reaction sequence respectively. The absolute configuration of the ligands was proved by the three-bond 1H-1H spin spin coupling constants 3J obtained by NMR-studies. Condensation of the amino derivatives with salicylic aldehydes resulted in the corresponding diimines, which represent a new class of chiral salen-type ligands. All ligands formed uncharged stable copper(II) complexes. The structure of 3',5'-bis(3,5-di-tert-butylsalicylaldiminato)-3',5'-dideoxyxylothymidine-copper(II) could be determined by single crystal X-ray structure analysis. The copper centre in this complex has distorted tetrahedral coordination geometry. Conclusion For the synthesis of 3',5'-diamino-3',5'-dideoxy thymidines with xylo- as well as ribo-configuration an effective synthesis pathway has been developed. Their corresponding salicylidene imines form stable coordination compounds with copper(II) ions. They represent the first salen type complexes of nucleosides with this substitution pattern.Applied Science
Genome-wide identification and differential analysis of translational initiation
No full textAbstractTranslation is principally regulated at the initiation stage. The development of the translation initiation (TI) sequencing (TI-seq) technique has enabled the global mapping of TIs and revealed unanticipated complex translational landscapes in metazoans. Despite the wide adoption of TI-seq, there is no computational tool currently available for analyzing TI-seq data. To fill this gap, we develop a comprehensive toolkit named Ribo-TISH, which allows for detecting and quantitatively comparing TIs across conditions from TI-seq data. Ribo-TISH can also predict novel open reading frames (ORFs) from regular ribosome profiling (rRibo-seq) data and outperform several established methods in both computational efficiency and prediction accuracy. Applied to published TI-seq/rRibo-seq data sets, Ribo-TISH uncovers a novel signature of elevated mitochondrial translation during amino-acid deprivation and predicts novel ORFs in 5′UTRs, long noncoding RNAs, and introns. These successful applications demonstrate the power of Ribo-TISH in extracting biological insights from TI-seq/rRibo-seq data.</jats:p
Global effects of plant virus infection, viral noncoding RNAs, and unfolded protein response on plant gene expression
No full textPlants are subject to a variety of abiotic and biotic stresses, including virus infection. This leads to enormous losses in crop yield and quality worldwide. Understanding how plants respond to these stresses can enable researchers to develop more healthy and robust plant varieties. The main objective of my research is to explore (i) the transcriptional and translational control of cellular gene expression in response to virus infection and (ii) the role(s) of viral noncoding subgenomic (ncsg)RNAs during infection. For this, I used red clover necrotic mosaic virus (RCNMV) as a model for economically important Tombusvirids. RCNMV generates a 3’ coterminal viral ncsgRNA, called SR1f, that belongs to the class of viral subgenomic (sg)RNAs that are functional in human flavivirus pathogenesis but which are still understudied in plant virus infection. Additionally, I also explored how translation is regulated in plants during unfolded protein response (UPR), which is elicited by many viruses and abiotic stresses.
A prerequisite for investigating viral sgRNAs is an RNA detection method that can distinguish between the coterminal genomic and sgRNAs. Using RCNMV SR1f and the analogous ncsgRNA from Zika virus (sfRNA), I developed a novel RT-PCR-based method, called DeSCo-PCR (Detection of smaller coterminal RNAs by PCR), for simple, quick, quantitative, and specific detection of viral sgRNAs. I demonstrate its advantages over the traditionally-used northern blot hybridization for detecting viral sgRNAs. This is the first RT-PCR method that distinguishes genomic from sgRNAs in most positive-sense RNA viruses.
Next, I wanted to assess the role(s) of RCNMV SR1f during infection. RCNMV SR1f belongs to the class of exoribonuclease-resistant (xr)RNA-derived viral ncsgRNAs. In plants, viral ncsgRNAs play a role in determining the severity of symptoms and the success of infection. Therefore, to explore the functions and effects of SR1f, I (i) used RNA sequencing (RNA-seq) to compare how infection with RCNMV constructs, which can or cannot produce SR1f, affect the transcriptomes of Nicotiana benthamiana and RCNMV, (ii) assessed the role of SR1f in counteracting the antiviral RNA silencing response in Arabidopsis thaliana, and (iii) determined the requirement of XRN4 for generating RCNMV SR1f in A. thaliana.
Next, I used ribosome profiling (Ribo-seq) to assess how host and viral genes are translationally regulated in RCNMV-infected plants. Most genome-wide host-virus interaction studies have used RNA-seq, which does not provide any information on translational control. Translational control is a tightly-regulated process that provides a more rapid change in gene expression than a transcriptional response. Furthermore, viruses rely completely on cellular translation machinery for viral protein synthesis. However, translational control during plant virus-host interaction has rarely been studied at the genome-wide level. Therefore, I used Ribo-seq to (i) assess the effects of RCNMV infection on the transcriptome and the translatome of A. thaliana at early and late stages of infection, (ii) identify cellular genes that are transcriptionally and translationally-regulated in response to virus infection, and (iii) assess the translational landscape of RCNMV mRNAs in infected cells.
Finally, I also used Ribo-seq to assess the translational control in roots of Zea mays seedlings during UPR. The PKR-like ER kinase (PERK)-mediated UPR pathway, which results in phosphorylation of eIF2α and subsequent inhibition of global translation in mammalian cells, is absent in the plant system. Therefore, I wanted to determine if translational control is as important in plants as it is in mammalian cells during UPR. I used Ribo-seq and other molecular assays to (i) determine if there is global inhibition of translation in plants during UPR, (ii) calculate the translational efficiencies of several UPR-responsive mRNAs, and (iii) determine the fate of the UPR-responsive mRNAs that were transcriptionally upregulated during UPR
- …
