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PubMed2003

A novel mode of RBD-protein recognition in the Y14-Mago complex.

Y14 and Mago are conserved eukaryotic proteins that associate with spliced mRNAs in the nucleus and remain associated at exon junctions during and after nuclear export. In the cytoplasm, Y14 is involved in mRNA quality control via the nonsense-mediated mRNA decay (NMD) pathway and, together with Mago, is involved in localization of osk (oskar) mRNA. We have determined the crystal structure of the complex between Drosophila melanogaster Y14 and Mago at a resolution of 2.5 A. The structure reveals an atypical mode of protein-protein recognition mediated by an RNA-binding domain (RBD). Instead of binding RNA, the RBD of Y14 engages its RNP1 and RNP2 motifs to bind Mago. Using structure-guided mutagenesis, we show that Mago is also a component of the NMD pathway, and that its association with Y14 is essential for function. Heterodimerization creates a single structural platform that interacts with the NMD machinery via phylogenetically conserved residues.

Amino Acid Sequence↗
PubMed1998

Improving the efficiency of antisense and EGS methods.

Hammerhead ribozymes and stable stem loop structures function as inhibitors of 3'-5'-exonuclease degradation of external guide sequences (EGSs) when covalently linked to the 3'-end of EGS RNAs. This observation may be of use in improving the efficiency of gene inactivation techniques that use single-stranded RNA (e.g., antisense RNA, EGS RNA) in vivo.

Base Sequence↗
PubMed2025

Genome editing with programmable base editors in human cells.

Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.

Humans↗
PubMed2006

PCR-suppression effect: kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples.

In the present study, we analyzed the kinetics of the PCR-suppression effect (PS-effect) and firstly demonstrate that lower annealing temperature enhances PS-effect. Furthermore, we controlled the average size of complex PCR products over a wide range, and simultaneously amplified targets from 0.25 to 10kb by regulating the degree of suppression in a single-primer PCR. In addition, we describe an improved template-switching full-length cDNA synthesis method that greatly reduces truncated cDNA. This study provides a general guide for the design of PS-effect related PCR and is useful for representative or long-transcript enriched cDNA library construction, especially when only a small amount of total RNA is available.

Base Sequence↗
PubMed2003

Dyskinetoplastic Trypanosoma brucei contains functional editing complexes.

Mitochondrial pre-mRNAs undergo posttranscriptional RNA editing as directed by small guide RNAs (gRNAs) to produce functional mRNAs in kinetoplastid protozoa. The pre-mRNAs and gRNAs are encoded in the maxicircle and minicircle components, respectively, of the kinetoplastid mitochondrial DNA (kDNA), and editing is catalyzed by a multienzyme protein complex. Trypanosoma evansi AnTat3/3, which lacks maxicircles but retains a single class of minicircles, and a dyskinetoplastic mutant of Trypanosoma brucei EATRO164, which is devoid of kDNA, were both shown to retain genes and proteins for the editing complex. The proteins are present in complexes that immunoprecipitate and sediment indistinguishably from wild-type complexes. The complexes catalyze precleaved insertion and deletion editing as well as full-round deletion editing in vitro. Thus, mutants which lack the natural substrates for RNA editing and all or most gRNAs retain editing complexes that contain the four primary catalytic activities of editing and function in editing, at least in vitro. Therefore neither pre-mRNA nor gRNA is required to form functional RNA-editing complexes.

Animals↗
PubMed2026

Enhancing pan-cancer spatial transcriptomics at single-cell resolution with stPainter.

Subcellular spatial transcriptomics can resolve tissue architecture at cellular scale, but sparse gene panels and limited detection sensitivity constrain downstream analysis. Existing enhancement methods often require tissue-matched single-cell RNA sequencing (scRNA-seq) references and dataset-specific retraining. Here we show that stPainter, a conditional generative model pretrained on a pan-cancer scRNA-seq atlas, can enhance spatial transcriptomics data without matched references or retraining. Using a latent diffusion architecture guided by Stochastic Differential Equations (SDE), stPainter reconstructs expanded expression profiles from sparse measurements and produces latent representations for clustering and cell-state analysis. When we apply stPainter upon 6 spatial transcriptomics datasets of different cancer types, we demonstrate that our model empowers downstream biological analyses, including fine-grained subpopulation clustering and pathway enrichment. Comparison with spatially resolved proteomics (CODEX) provided independent support for regional agreement between imputed cellular compositions and protein-level tissue organization. These results establish stPainter as a scalable approach for analyzing tumor microenvironments without auxiliary sequencing data.

Spatial Transcriptomics↗
PubMed2005

Complex management: RNA editing in trypanosomes.

Most mitochondrial mRNAs in kinetoplastids require editing, that is, the posttranscriptional insertion and deletion of uridine nucleotides that are specified by guide RNAs and catalyzed by multiprotein complexes. Recent studies have identified many of the proteins in these complexes, in addition to some of their functions and interactions. Although much remains unknown, a picture of highly organized complexes is emerging that shows that the complex that catalyzes the central steps of editing is partitioned into distinct insertion and deletion editing subcomplexes. These subcomplexes coordinate hundreds of ordered catalytic steps that function to produce a single mature mRNA. The dynamic processes, which might entail interactions among multiprotein complexes and changes in their composition and conformation, remain to be elucidated.

Animals↗
PubMed2000

Novel trypanosomatid small nucleolar RNAs that guide methylation: their genome organization, expression and potential use to direct specific methylation on target RNA molecules.

Trypanosomatids are the causative agent of several major parasitic diseases including African trypanosomiasis, American trypanosomiasis, and leishmaniasis. These parasites possess unique RNA-processing mechanisms including trans-splicing of pre-mRNA and RNA editing of mitochondrial transcripts. In this study, we identified a trypanosomatid novel group of small nucleolar RNAs that belong to the box C/D snoRNA, which were shown to guide ribose methylation on rRNA. Three snoRNA genes were identified; snoRNA-2 carrying a single snoRNA and g2 and b2 coding for a single or multiple snoRNAs, respectively. Mapping of the methylation sites guided by snoRNA-2 using two different approaches suggest that snoRNA-2 has the potential to guide methylation on both 5.8S and 18S rRNAs. The trypanosomes follow the same guide-methylation rule established for yeast and for mammals. As a first attempt to change the methylation pattern of target RNAs, we generated transgenic parasites carrying the B2 and snoRNA-2, which were engineered to shift the methylation site on rRNA. Despite efficient expression of these tagged snoRNAs, the novel methylation site was not generated. However, efficient expression of tagged snoRNAs in transgenic parasites opens the possibility of engineering novel methylation sites on different target RNAs in vivo.

Animals↗
PubMed2003

Small nucleolar RNA clusters in trypanosomatid Leptomonas collosoma. Genome organization, expression studies, and the potential role of sequences present upstream from the first repeated cluster.

Trypanosomatid small nucleolar RNA (snoRNA) genes are clustered in the genome. snoRNAs are transcribed polycistronically and processed into mature RNAs. In this study, we characterized four snoRNA clusters in Leptomonas collosoma. All of the clusters analyzed carry both C/D and H/ACA RNAs. The H/ACA RNAs are composed of a single hairpin, a structure typical to trypanosome and archaea guide RNAs. Using deletion and mutational analysis of a tagged C/D snoRNA situated within the snoRNA cluster, we identified 10-nucleotide flanking sequences that are essential for processing snoRNA from its precursor. Chromosome walk was performed on a snoRNA cluster, and a sequence of 700 bp was identified between the first repeat and the upstream open reading frame. Cloning of this sequence in an episome vector enhanced the expression of a tagged snoRNA gene in an orientation-dependent manner. However, continuous transcript spanning of this region was detected in steady-state RNA, suggesting that snoRNA transcription also originates from an upstream-long polycistronic transcriptional unit. The 700-bp fragment may therefore represent an example of many more elements to be discovered that enhance transcription along the chromosome, especially when transcription from the upstream gene is reduced or when enhanced transcription is needed.

Amino Acid Sequence↗
PubMed2024

Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus.

Spatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with in situ reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX. We developed computational pipelines to register data from two distinct modalities. Performing both DBiT-seq and CODEX on the same tissue slide enables accurate cell typing in each spatial transcriptome spot and subsequently image-guided decomposition to generate single-cell resolved spatial transcriptome atlases. DBiTplus was applied to mouse embryos with limited protein markers but still demonstrated excellent integration for single-cell transcriptome decomposition, to normal human lymph nodes with high-plex protein profiling to yield a single-cell spatial transcriptome map, and to human lymphoma FFPE tissue to explore the mechanisms of lymphomagenesis and progression. DBiTplusCODEX is a unified workflow including integrative experimental procedure and computational innovation for spatially resolved single-cell atlasing and exploration of biological pathways cell-by-cell at genome-scale.

Journal Article↗
PubMed2024

Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus.

Spatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with in situ reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX. We developed computational pipelines to register data from two distinct modalities. Performing both DBiT-seq and CODEX on the same tissue slide enables accurate cell typing in each spatial transcriptome spot and subsequently image-guided decomposition to generate single-cell resolved spatial transcriptome atlases. DBiTplus was applied to mouse embryos with limited protein markers but still demonstrated excellent integration for single-cell transcriptome decomposition, to normal human lymph nodes with high-plex protein profiling to yield a single-cell spatial transcriptome map, and to human lymphoma FFPE tissue to explore the mechanisms of lymphomagenesis and progression. DBiTplusCODEX is a unified workflow including integrative experimental procedure and computational innovation for spatially resolved single-cell atlasing and exploration of biological pathways cell-by-cell at genome-scale.

Journal Article↗
PubMed1997

Inter-domain cross-linking and molecular modelling of the hairpin ribozyme.

The hairpin ribozyme is a small catalytic RNA composed of two helical domains containing a small and a large internal loop and, thus, constitutes a valuable paradigm for the study of RNA structure and catalysis. We have carried out molecular modelling of the hairpin ribozyme to learn how the two domains (A and B) might fold and approach each other. To help distinguish alternative inter-domain orientations, we have chemically synthesized hairpin ribozymes containing 2'-2' disulphide linkages of known spacing (12 or 16 A) between defined ribose residues in the internal loop regions of each domain. The abilities of cross-linked ribozymes to carry out RNA cleavage under single turnover conditions were compared to the corresponding disulphide-reduced, untethered ribozymes. Ribozymes were classed in three categories according to whether their cleavage rates were marginally, moderately, or strongly affected by cross-linking. This rank order of activity guided the docking of the two domains in the molecular modelling process. The proposed three-dimensional model of the hairpin ribozyme incorporates three different crystallographically determined structural motifs: in domain A, the 5'-GAR-3'-motif of the hammerhead ribozyme, in domain B, the J4/5 motif of group I ribozymes, and connecting the two domains, a "ribose zipper", another group I ribozyme feature, formed between the hydroxyl groups of residues A10, G11 of domain A and C25, A24 of domain B. This latter feature might be key to the selection and precise orientation of the inter-domain docking necessary for the specific phosphodiester cleavage. The model provides an important basis for further studies of hairpin ribozyme structure and function.

Computer Simulation↗
PubMed2006

UTP-bound and Apo structures of a minimal RNA uridylyltransferase.

3'-Uridylylation of RNA is emerging as a phylogenetically widespread phenomenon involved in processing events as diverse as uridine insertion/deletion RNA editing in mitochondria of trypanosomes and small nuclear RNA (snRNA) maturation in humans. This reaction is catalyzed by terminal uridylyltransferases (TUTases), which are template-independent RNA nucleotidyltransferases that specifically recognize UTP and belong to a large enzyme superfamily typified by DNA polymerase beta. Multiple TUTases, recently identified in trypanosomes, as well as a U6 snRNA-specific TUTase enzyme in humans, are highly divergent at the protein sequence level. However, they all possess conserved catalytic and UTP recognition domains, often accompanied by various auxiliary modules present at the termini or between conserved domains. Here we report identification, structural and biochemical analyses of a novel trypanosomal TUTase, TbTUT4, which represents a minimal catalytically active RNA uridylyltransferase. The TbTUT4 consists of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The 2.0 Angstroms crystal structure reveals two significantly different conformations of this TUTase: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase-UTP complex. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTP's base, ribose and phosphate moieties. The structure-guided mutagenesis and cross-linking studies define the amino acids essential for catalysis, uracil base recognition, ribose binding and phosphate coordination by uridylyltransferases. In addition, the cluster of positively charged residues involved in RNA binding is identified. We also report a 2.4 Angstroms crystal structure of TbTUT4 with the bound 2' deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides.

Amino Acid Sequence↗
PubMed2025

RAPID-DASH: Fast and Efficient Assembly of Guide RNA Arrays for Multiplexed CRISPR-Cas9 Applications.

Guide RNA (gRNA) arrays can enable targeting multiple genomic loci simultaneously using CRISPR-Cas9. In this study, we present a streamlined and efficient method to rapidly construct gRNA arrays with up to 10 gRNA units in a single day. We demonstrate that gRNA arrays maintain robust functional activity across all positions, and can incorporate libraries of gRNAs, combining scalability and multiplexing. Our approach will streamline combinatorial perturbation research by enabling the economical and rapid construction, testing, and iteration of gRNA arrays. To facilitate the adaptation of this approach, we have made a web tool to design oligo sequences necessary to assemble gRNA arrays.

CRISPR-Cas9↗
PubMed1972

Action of DNA polymerase I of Escherichia coli with DNA-RNA hybrids as templates.

Experiments indicating the ability of the ribo strand of a DNA-RNA template to guide polydeoxynucleotide synthesis by highly purified DNA polymerase I of E. coli (EC 2.7.7.7) are presented. With poly(rA).poly(dT) as template, poly(dT) is formed with a high efficiency, but almost no poly(dA). The specific activity of the enzyme, when tested with this template under suitable conditions, is eight times greater than that found for the poly(dA-dT) template. Single-stranded DNA fractions, with no template activity for DNA polymerase, are converted to efficient templates after their transcription by RNA polymerase. A concerted polymerization reaction, in which the action of DNA polymerase is dependent on that of RNA polymerase, can also be demonstrated with synthetic polydeoxynucleotides and single-stranded fractions of denatured DNA as templates.

Adenine Nucleotides↗
PubMed2025

Next-generation sequencing in breast cancer: current clinical applications and future directions.

INTRODUCTION: Breast cancer is a heterogeneous disease that claims 670,000 lives by 2022. Omic technologies, particularly next generation sequencing (NGS) offers promising avenues for precision medicine. American Society of Clinical Oncology (ASCO) outlines genomic testing's utility, emphasizing prognostic and diagnostic potential. OBJECTIVES: This review succinctly explores NGS's evolution and clinical applications of NGS in breast cancer, thereby guiding future research to enhance patient care. METHODS: Comprehensive literature searches were conducted using databases such as PubMed, Google Scholar, and ResearchGate, focusing on keywords including breast cancer, HER-2 low breast cancer, circulating tumour DNA, single-cell RNA sequencing, and next-generation sequencing. Peer-reviewed, high-quality articles published in English were selected for inclusion. RESULTS: Previous studies have explored the evolution of NGS technology and its clinical applications in breast cancer, including genomic and transcriptomic characterization, treatment guidance, and resistance prediction. Molecular profiling of challenging entities such as early-onset breast cancer and HER-2 low tumours was summarized, with key findings highlighted. This review also discusses emerging technologies, including circulating DNA and single-cell sequencing, as promising avenues for discovery. CONCLUSION: NGS has revealed the genomic and transcriptomic diversity of breast cancer, identifying actionable alterations associated with chemotherapy response and resistance to therapies such as trastuzumab, TKIs, and CDK4/6 inhibitors. Circulating tumour DNA (ctDNA) shows potential for diagnosis, prediction, prognosis, and monitoring, despite tumour heterogeneity. Single-cell analysis enables exploration of individual cell transcriptomes, though high costs and low throughput remain barriers to widespread adoption. HER2-low tumours continue to pose significant research challenges.

Humans↗
PubMed1996

[Primary non-Hodgkin's lymphomas of the liver in human immunodeficiency virus infected patients. Three cases].

The occurrence of primary non Hodgkin's lymphoma of the liver has rarely been reported in patients with the human immunodeficiency virus infection. We report 3 cases in these patients, presenting with a single liver tumor in 2 cases and multiple tumors in one case. Diagnosis was made by guided-biopsy. Histologically, there was B diffuse large cell lymphoma in 2 cases, and immunoblastic lymphoma in one case. In one case, RNA of Epstein-Barr virus was found in the nucleus of tumor cells by in situ hybridization. No extrahepatic localization was found in any of the 3 cases. One patient died one week after diagnosis, but the 2 other patients were treated by chemotherapy and were still alive 6 and 30 months later. In patients infected by human immunodeficiency virus, primary non Hodgkin's lymphoma should be investigated in case of liver tumor in order to provide early treatment.

AIDS-Related Opportunistic Infections↗