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PubMed2002

Distinct functions of two RNA ligases in active Trypanosoma brucei RNA editing complexes.

Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII. However, little is known about their catalytic functions, except that band IV and band V are RNA ligases and genetic analysis indicates that the former is important in U deletion. Here we establish biochemical approaches to distinguish the individual roles of these ligases, based on their distinctive ATP and pyrophosphate utilization. These in vitro analyses revealed that both ligases serve in RNA editing. Band V is the RNA editing ligase that functions very selectively to seal in U insertion (IREL), while band IV is the RNA editing ligase needed to seal in U deletion (DREL). In combination with our earlier findings about the cleavage and the U-addition/U-removal steps of U deletion and U insertion, these results show that all three steps of these editing pathways exhibit major differences and suggest that the editing complex could have physically separate regions for U deletion and U insertion.

Adenosine Triphosphate↗
PubMed2026

Tracking GAD-specific T-cell expansions in Type 1 diabetes by intradermal GAD-Alum challenge.

Identifying and monitoring autoreactive T cells that drive beta cell destruction remains a major obstacle to developing effective immunotherapies for type 1 diabetes (T1D). These cells are extremely rare in peripheral blood and cannot be accessed directly from the pancreas. We used intradermal injection of Glutamic Acid Decarboxylase (GAD)-Alum to recruit GAD-specific T cells to accessible sites in the skin and skin-draining lymph nodes (LNs), sampled by skin suction blisters and ultrasound-guided LN aspiration. Peripheral blood samples obtained before GAD injection were restimulated with GAD in vitro to detect reactive CD4+ T cells. Single-cell RNA sequencing (scRNAseq) followed by re-expression of selected T cell receptors (TCRs) confirmed antigen specificity. Up to 70% of T cells at the skin injection site were clonally-expanded and 4 of 14 (28%) re-expressed TCRs were GAD-reactive. In LNs 1 of 14 (4%) clonally-expanded TCRs was GAD-reactive, representing ~0.08% of all T-cells. GAD-reactive cells across compartments displayed Th1 and Th17-associated transcription signatures. These results demonstrate the intradermal autoantigen challenge and scRNAseq, enable direct identification and molecular profiling of autoreactive T cells in vivo. This minimally invasive approach provides a powerful platform for tracking antigen-specific T cells to monitor disease activity and evaluate immune interventions in T1D.

Autoimmunity↗
PubMed2000

Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes.

Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into the origin and functions of methylation guide small nucleolar RNAs and illuminate the still elusive role of rRNA ribose methylations.

Base Sequence↗
PubMed2026

Stepwise DNA-unwinding gates TnpB genome-editing activity.

TnpB is a compact RNA-guided endonuclease and an evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited, and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through a discrete, long-lived, partially unwound intermediate state before reaching a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, H4W-L304F-V305R (Ymu1-WFR), stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in plants. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.

Gene Editing↗
PubMed2026

SCMO: a deep learning model integrating the single-cell resolution TME ecosystem and multi-omics for survival prediction in CRC patients.

BACKGROUND: Colorectal cancer (CRC) remains a leading cause of global cancer mortality, highlighting the need for precise survival prediction to guide clinical decisions. Although tissue-level multi-omics is widely utilized for survival prediction, its limited resolution cannot capture tumor heterogeneity. Single-cell RNA sequencing (scRNA-seq) enables dissection of the tumor microenvironment (TME) at cellular resolution, supporting personalized prognostic assessment. METHODS: We collected 213 CRC scRNA-seq samples and established a CRC-specific TME atlas comprising 339,060 cells. Using this atlas as a reference, we deconvolved bulk RNA-seq data from TCGA-CRC cohort with the EcoTyper algorithm to reconstruct TME features. Clinical, genomic, and transcriptomic data were obtained from the Xena platform; microbial data were sourced from the BIC database. We integrated TME and multi-omics features through a self-normalizing neural network to construct a deep learning model (single-cell resolution TME ecosystem with multi-omics data [SCMO]) for survival prediction. To enhance interpretability, we utilized the Integrated Gradients algorithm and spatial transcriptomic data to analyze multi-omics and TME features. We performed anticancer drug screening with tumor necrosis factor receptor-associated protein 1 (TRAP1), a critical feature according to the Integrated Gradients algorithm, as a potential target. RESULTS: We identified 13 survival-related TME features from the CRC-specific atlas: 12 cell states and one multi-cellular ecosystem. SCMO, which combined TME and multi-omics features, improved survival prediction and outperformed existing methods, achieving a concordance index of 0.762. The SCMO demonstrated robust performance for long-term predictions, achieving areas under the curve (AUCs) of 0.752, 0.772, and 0.869 for 1-, 3-, and 5-year predictions in the training set, with corresponding test set AUCs of 0.639, 0.756, and 0.772. TME features from the SCMO model revealed that ecosystem density increased with CRC malignancy. Multi-omics features included TRAP1 as a potential drug target. Drug screening identified saikosaponin A as a novel TRAP1 inhibitor, and its anticancer activity was validated in vitro. We developed SCMO-Lite, a simplified model incorporating 12 high-attribution-weight multi-omics features, which demonstrated robust risk stratification. CONCLUSIONS: SCMO combines analytical precision with biological interpretability, offering novel insights for oncology survival prediction.

Humans↗
PubMed2003

[Novel approaches; improved diagnostics and therapeutics with DNA microarrays. II. Applications].

The most important (future) applications of DNA microarrays in medicine are based on three different molecular analyses: gene expression analysis, genotyping with a specific type of microarray (single nucleotide polymorphism chip (SNP chip)), and DNA sequencing to search for a well-known mutation in a certain gene. With gene expression analysis one can obtain a 'molecular signature' of a tissue by allowing tissue RNA to react with a DNA microarray. This information may help to refine disease classifications, to guide the choice of therapy and to find new therapeutic targets. Genotyping utilizes genomic DNA that, after digestion, reacts with a SNP chip to obtain an individual SNP pattern. An SNP is a change of a single base pair within a gene sequence that can sometimes influence the function of the gene product. SNPs are considered to be variations in the genome and should be distinguished from mutations. These variations can for instance provide information about the probability of a certain disease, or the effectiveness or side effect of a certain drug. Sequencing well characterized genes to search for mutations is used to screen for genetic abnormalities. This process can be accelerated with the aid of oligonucleotide arrays. With this tool an individual's risk of developing certain forms of cancer or monogenetic diseases can be estimated.

Diagnostic Techniques and Procedures↗
PubMed1995

Transcription, splicing and editing of plastid RNAs in the nonphotosynthetic plant Epifagus virginiana.

Expression of the vestigial plastid genome of the nonphotosynthetic, parasitic flowering plant Epifagus virginiana was examined by northern analysis and by characterization of cDNAs. Probes for each of 12 plastid genes tested hybridized to all lanes of northern blots containing total RNA prepared from stems and fruits of Epifagus and from leaves of tobacco. Certain transcript patterns in Epifagus plastids are highly complex and similar to those of tobacco operons. In contrast, genes such as rps2, which have become orphaned in Epifagus as a result of evolutionary loss of formerly cotranscribed genes, show simpler transcript patterns in Epifagus than in tobacco. Sizing and sequencing of cDNAs generated by reverse transcriptase-PCR for three genes, rps12, rpl2, and clpP, show that their transcripts are properly cis- and/or trans-spliced at the same five group II intron insertion sites used in photosynthetic plants. A single, conventional C-->U edit in rps12 was found among the total of 1401 nucleotides of cDNA sequence that was determined for the three genes. An octanucleotide sequence identical to a putative guide RNA of plant organelles and perfectly complementary to the rps12 edit site itself was identified just 200 bp upstream of the edit site. These data, together with previous results from the complete sequencing of the Epifagus plastid genome, provide compelling evidence that this degenerate genome is nonetheless expressed and functional. Analysis of the putative maturase MatK, encoded by the group II intron of trnK in photosynthetic land plants but by a freestanding gene in Epifagus, leads us to hypothesize that it acts 'in trans' to assist the splicing of group II introns other than the one in which it is normally encoded.

Adenosine Triphosphatases↗
PubMed2005

Expression of complementary RNA from chloroplast transgenes affects editing efficiency of transgene and endogenous chloroplast transcripts.

The expression of angiosperm chloroplast genes is modified by C-to-U RNA editing. The mechanism for recognition of the approximately 30 C targets of editing is not understood. There is no single consensus sequence surrounding editing sites, though sites can be grouped into small 'clusters' of two to five sites exhibiting some sequence similarity. While complementary RNA that guides nucleotides for alteration has been detected in other RNA modification systems, it is not known whether complementary RNA is involved in chloroplast editing site recognition. We investigated the effect of expressing RNA antisense to the sequences -20 to +6 surrounding the RpoB-2 C target of editing, which is a member of a cluster that includes the PsbL-1 and Rps14-1 sites. Previous experiments had shown that chloroplast rpoB transgene transcripts carrying only these 27 nt were edited in vivo at the proper C. Though transcripts carrying sequences -31 to +60 surrounding the RpoB-2 sites were edited in chloroplast transgenic plants, transcripts carrying the -31 to +62 region followed by the 27 nt complementary region were not edited at all. In contrast, a similar construct, in which the C target as well as the preceding and subsequent nucleotides were mismatched within the 27 nt region, was efficiently edited. The presence of any of the four transgenes carrying RpoB-2 sequences in sense and/or antisense orientation resulted in reduced editing at the PsbL-1 site. Chloroplast transgenic plants expressing the three different antisense RNA constructs exhibited abnormal growth and development, though plants expressing the 92 nt sense transcripts were phenotypically normal.

Chloroplasts↗
PubMed2002

Age at infection affects the long-term outcome of transfusion-associated chronic hepatitis C.

Before the introduction of hepatitis C virus (HCV) screening for blood donors, the risk of acquiring HCV infection as a result of a transfusion was about 10%. The aim of this study was to assess the frequency and rate of progression to cirrhosis in patients with transfusion-associated chronic HCV infection and identify possibly negative prognostic factors. Of 2477 consecutive patients with clinical or laboratory evidence of liver disease, 392 (16%) were anti-HCV- and HCV-RNA-positive, had anamnestic evidence of a single and precisely dated transfusion event, and showed no other causes of chronic liver disease; 268 (68%) underwent ultrasound-guided liver biopsy and were enrolled in the study. After a mean interval of 18.4 years, 54 patients (20.1%) had cirrhosis, which multivariate analysis showed to be independently associated with the duration of follow-up, age at infection and at the time of liver biopsy, and serum alanine aminotransferase levels at biopsy. The time necessary to have a 50% probability of developing cirrhosis in patients aged 21-30, 31-40, and more than 40 years was 33, 23, and 16 years, respectively. In comparison with those aged 20 years or less at infection, the risk ratio of developing cirrhosis over a period of 30 years for patients aged 21-30 and at least 31 years at infection was, respectively, 4.51 (95% confidence interval, 1.03-19.76) and 12.29 (95% confidence interval, 3.06-49.40). In patients with transfusion-associated chronic hepatitis C, the risk of cirrhosis is related to age at infection and disease activity. Our findings suggest that an aggressive therapeutic approach should be adopted in patients infected by HCV at an older age to prevent the progression to end-stage liver disease.

Adolescent↗
PubMed2004

A novel 4-base-recognizing RNA cutter that can remove the single 3' terminal nucleotides from RNA molecules.

Mammalian tRNase ZL shows versatility in substrate recognition. This enzyme can not only process pre-tRNAs by cleaving off their 3' trailer sequences, but also recognize and cleave pre-tRNA-like complexes and micro-pre-tRNAs. Here we demonstrate that 24-27 nt hairpin RNAs (hook RNAs) can guide cleavages of separate target RNAs by tRNase ZL through the micro-pre-tRNA-like complexes between the targets and the hook RNAs and that tRNase ZL together with hook RNA works as 4-7-base-recognizing RNA cutters. The cleavage sites were located only after the nucleotide corresponding to the discriminator nucleotide. Cleavage assays for various substrate/hooker complexes showed that the cleavage efficiency changes depending on the maximum number of substrate/hooker recognition base pairings and the stem length of hook RNA and that a 5 nt recognition sequence and a hook RNA containing a 6 or 7 bp stem are the best combination for the optimal target cleavage. We also show that a 4-base RNA cutter can remove the single 3' terminal nucleotides from RNA molecules. These results indicate that this new type of RNA cutter can be utilized to homogenize at their 3' termini RNA transcripts synthesized in vitro with a bacteriophage RNA polymerase.

Base Pairing↗
PubMed2003

Sequence and structural requirements for optimal guide RNA-directed insertional editing within Leishmania tarentolae.

The coding sequence of several mitochondrial mRNAs of the trypanosomatid family of protozoa is created by the guide RNA-directed insertion and deletion of uridylates (Us). Selection-amplification was used to explore the sequence and structure of the guide RNA and mRNA required for efficient insertional editing within a mitochondrial extract prepared from Leishmania tarentolae. This study identifies several novel features of the editing reaction in addition to several that are consistent with the previous mutagenesis and phylogenetic analysis of the reaction in Trypanosoma brucei, a distantly related trypanosomatid. Specifically, there is a strong bias against cytidines 5' of the editing sites and guanosines immediately 3' of guiding nucleotides. U insertions are directed both 5' and 3' of a genomically encoded U, which was previously assumed not to occur. Base pairing immediately flanking an editing site can significantly stimulate the editing reaction and affect the reaction fidelity but is not essential. Likewise, single-stranded RNA in the region upstream of the editing site, not necessarily immediately adjacent, can facilitate editing but is also not essential. The editing of an RNA containing many of the optimal features is linear with increasing quantities of extract permitting specific activity measurements to be made that are not possible with previously described T. brucei and L. tarentolae assays. The reaction catalyzed by the L. tarentolae extract can be highly accurate, which does not support a proposed model for editing that was based largely on the inaccuracy of an earlier in vitro reaction.

Animals↗
PubMed1998

Short oligonucleotides as external guide sequences for site-specific cleavage of RNA molecules with human RNase P.

Human RNase P recognizes a small model substrate consisting of only the 5' leader sequence, aminoacyl acceptor stem, and T stem and loop of a tRNA precursor. It was demonstrated here that a bimolecular construct in which the T loop is opened between G57 and A58 (tRNA numbering system) is still processed by RNase P. The strand that is cleaved can be considered the target RNA, whereas the other strand serves as an external guide sequence (EGS). The nucleotides corresponding to nt 58-60 in the T loop could be deleted without affecting cleavage of the substrate. Thus, the complete T loop can be replaced by the single-stranded sequence UUCG or UUCA (nt 55-57 in the T loop). The four nucleotides UUCR possibly form a structure that resembles the uridine turn in the T loop of tRNA. Because recognition by RNase P is independent of the helical sequence, this motif can be used for targeting RNA molecules for EGS-directed cleavage by human RNase P. Chemically modified EGSs with 2'-O-methyl groups also showed activity in inducing RNase P cleavage. Several 13-mer EGSs targeted to the 2.1-kb surface antigen mRNA of hepatitis B virus (HBV) were designed and tested using a co-transcriptional cleavage assay with a 2.1-kb HBV transcript. Some of the new EGSs were capable of inducing cleavage of the HBV RNA by RNase P.

Base Sequence↗
PubMed2003

A biochemical framework for RNA silencing in plants.

RNA silencing phenomena were first discovered in plants, yet only the RNA interference pathway in animals has been subject to biochemical analysis. Here, we extend biochemical analysis to plant RNA silencing. We find that standard wheat germ extract contains Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs, as well as an RNA-dependent RNA polymerase activity that can convert exogenous single-stranded RNA into dsRNA. In this plant embryo extract, an endogenous microRNA (miRNA) that lacks perfect complementarity to its RNA targets nonetheless acts as a small interfering RNA. The miRNA guides an endonuclease to cleave efficiently wild-type Arabidopsis PHAVOLUTA mRNA, but not a dominant mutant previously shown to perturb leaf development. This finding supports the view that plant miRNAs direct RNAi and that miRNA-specified mRNA destruction is important for proper plant development. Thus, endonuclease complexes guided by small RNAs are a common feature of RNA silencing in both animals and plants.

Arabidopsis↗
PubMed2025

CROPseq-multi: a universal solution for multiplexed perturbation in high-content pooled CRISPR screens.

Forward genetic screens seek to dissect complex biological systems by systematically perturbing genetic elements and observing the resulting phenotypes. While standard screening methodologies introduce individual perturbations, multiplexing perturbations improves the performance of single-target screens and enables combinatorial screens for the study of genetic interactions. Current tools for multiplexing perturbations are limited by technical challenges and do not offer compatibility across diverse screening methodologies, including enrichment, single-cell sequencing, and optical pooled screens. Here, we report the development of CROPseq-multi (CSM), a CROPseq1-inspired lentiviral system to multiplex Streptococcus pyogenes (Sp) Cas9-based perturbations with versatile readout compatibility and high performance for both perturbation and barcode identification. CSM has equivalent per-guide activity to CROPseq and low lentiviral recombination frequencies. Dual-guide CSM libraries are constructed in a single, facile molecular cloning step that facilitates the use of unique molecular identifiers. CSM is compatible with enrichment screening methodologies, single-cell RNA-sequencing readouts, and optical pooled screens. For optical pooled screens, an optimized and multiplexed in situ detection protocol improves barcode counts 10-fold (for mRNA detection), enables detection of recombination events, and reduces the number of sequencing cycles required for decoding by 3-fold relative to CROPseq. CROPseq-multi-v2 (CSMv2) adds compatibility for detection methods based on T7 RNA polymerase in vitro transcription2-5. CSM provides a single system for CRISPR screens that is compatible with individual and combinatorial perturbations, diverse SpCas9-based perturbation technologies, and multiple high-content, single-cell phenotypic readouts.

CRISPR Cas9↗
PubMed1980

An examination of the relationship between experimentally altered rates of epidermal proliferations and rates of epidermal metabolism assayed in vitro.

Continuously regenerating stratified squamous epithelia form an interesting model for examining mechanisms controlling the balance between rates of cell formation and cell maturation and death. Previous investigations of epidermal metabolism have been mainly based on single enzyme assays which may not form a reliable guide to changing rates of flux through metabolic pathways. Methods for in vitro assays of rates of glycolysis, protein synthesis and RNA synthesis of epidermal sheets free from dermal contamination were developed and used to examine rates of epidermal metabolism after experimental alteration of rates of epidermal proliferation. Starvation resulted in a 45-53% reduction in the in vivo epidermal labeling index and a 49-56% reduction in glycolysis and incorporation of amino acids assayed in vitro. Induction of epidermal hyperplasia with hexadecane resulted in a 4-fold increase in labeling index, a 6-fold increase in vitro glycolysis and a 3 to 4-fold increase in in vitro assays of incorporation of amino acids and uridine. Hyperplastic epidermis also showed an increased rate of incorporation of histidine (a marker for keratokyalin synthesis) relative to leucine (a marker for basal cell protein synthesis) indicating a change in maturation. The results suggest mechanisms linking rates of cell proliferation and death and indicate the possible value of such assays investigating these mechanisms.

Animals↗
PubMed2003

Generalized RNA-directed recombination of RNA.

RNA strand exchange through phosphor-nucleotidyl transfer reactions is an intrinsic chemistry promoted by group I intron ribozymes. We show here that Tetrahymena and Azoarcus ribozymes can promote RNA oligonucleotide recombination in either two-pot or one-pot schemes. These ribozymes bind one oligonucleotide, cleave following a guide sequence, transfer the 3' portion of the oligo to their own 3' end, bind a second oligo, and catalyze another transfer reaction to generate recombinant oligos. Recombination is most effective with the Azoarcus ribozyme in a single reaction vessel in which over 75% of the second oligo can be rapidly converted to recombinant product. The Azoarcus ribozyme can also create a new functional RNA, a hammerhead ribozyme, which can be constructed via recombination and then immediately promote its own catalysis in a homogeneous milieu, mimicking events in a prebiotic soup.

Animals↗
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↗