Computation of the fraction of RNA sequences that fold sequentially into a unique free-energy minimum.
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Journal Article↗
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RNA sequence variants in live poliovirus vaccine and their relation to neurovirulence.
Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) was used to study sequence heterogeneity and stability in attenuated poliovirus type 3 at positions in which the vaccine virus differs from its wild-type progenitor. Of seven genomic positions tested, only two (positions 472 and 2493) show nucleotide heterogeneity. Propagation of the vaccine virus in cell cultures leads to rapid selection of virus with reversions at these two positions of the genome. The relative abundance of reversions at position 472 correlates with the results of monkey neurovirulence tests, while the mutation at position 2493 is not directly associated with neurovirulence of the virus in monkeys. Instead, the abundance of mutations at the latter position correlates with the source of the seed virus and its passage level. These results further indicate that MAPREC at position 472 can be used to assess the quality of poliovirus type 3 vaccine.
Animals↗
The intervening sequence RNA of Tetrahymena is an enzyme. 1986.
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Animals↗
Sequence-specific RNA recognition by the Xenopus Y-box proteins. An essential role for the cold shock domain.
The Xenopus Y-box protein FRGY2 has a role in the translational silencing of masked maternal mRNA. Here, we determine that FRGY2 will recognize specific RNA sequences. The evolutionarily conserved nucleic acid-binding cold shock domain is required for sequence-specific interactions with RNA. However, RNA binding by FRGY2 is facilitated by N- and C-terminal regions flanking the cold shock domain. The hydrophilic C-terminal tail domain of FRGY2 interacts with RNA independent of the cold shock domain but does not determine sequence specificity. Thus, both sequence-specific and nonspecific RNA recognition domains are contained within the FRGY2 protein.
Amino Acid Sequence↗
Binding of nuclear proteins to HTLV-II cis-acting repressive sequence (CRS) RNA correlates with CRS function.
The shift from viral regulatory to structural gene expression in human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) is mediated by Rex. We have previously shown that HTLV-II Rex acts through an element in R/U5 of the 5' long terminal repeat (LTR), the Rex-responsive element (RxRE), and that Rex protein binds to specific RNA sequences, the Rex binding element (RBE), contained within the RxRE (Black et al., J. Virol. 65, 6645-6653, 1991b). Rex action through the RBE (nt 405-520) overcomes the inhibition of expression conferred by a contiguous LTR RNA regulatory element, which contains cis-acting repressive sequences (CRS; nt 520-630) that are not bound by Rex protein (Black et al., Virology, 181, 433-444, 1991a). We now show by electrophoretic mobility shift assay (EMSA) that cellular proteins in a HeLa nuclear extract bind specifically to RNA transcripts containing the HTLV-II CRS. Using ultraviolet (uv) crosslinking of gel-retarded bands, we identified a major protein species of approximately 60 kDa, p60CRS, that binds to CRS RNA and, with weaker affinity, to RBE RNA. In addition, a distinct 40-kDa protein, p40CRS, binds to U5 RNA (nt 645-750) downstream from the CRS. Specific deletions within CRS RNA can reduce or abrogate binding to this 60-kDa protein. EMSA and uv crosslinking assays also suggest that both p60CRS and p40CRS interact with CRS RNA. CRS function in a 5' LTR-linked gene expression assay correlates with the ability of both p60CRS and p40CRS to interact with 5' LTR RNA in vitro.
Base Sequence↗
RNA editing in plant mitochondria and chloroplasts.
In the mitochondria and chloroplasts of higher plants there is an RNA editing activity responsible for specific C-to-U conversions and for a few U-to-C conversions leading to RNA sequences different from the corresponding DNA sequences. RNA editing is a post-transcriptional process which essentially affects the transcripts of protein coding genes, but has also been found to modify non-coding transcribed regions, structural RNAs and intron sequences. RNA editing is essential for correct gene expression: proteins translated from edited transcripts are different from the ones deduced from the genes sequences and usually present higher similarity to the corresponding non-plant homologues. Initiation and stop codons can also be created by RNA editing. RNA editing has also been shown to be required for the stabilization of the secondary structure of introns and tRNAs. The biochemistry of RNA editing in plant organelles is still largely unknown. In mitochondria, recent experiments indicate that RNA editing may be a deamination process. A plastid transformation technique showed to be a powerful tool for the study of RNA editing. The biochemistry as well as the evolutionary features of RNA editing in both organelles are compared in order to identify common as well as organelle-specific components.
Amino Acid Sequence↗
Beyond parental lines: multi-omics analyses reveal epigenetic and transcriptional mechanisms underlying heterosis in Oryza sativa × Oryza rufipogon hybrids.
Heterosis, or hybrid vigor, refers to the superior phenotypes of a hybrid compared with their parents and is widely exploited in agriculture. Interspecific hybrids within the Oryza genus demonstrate significant potential for the systematic improvement of rice varieties. Nevertheless, the mechanistic basis underlying heterosis in interspecific Oryza hybrids remains poorly understood. Here, we systematically performed phenotypic characterization, whole-genome bisulfite sequencing, RNA sequencing, and small RNA profiling using Oryza sativa L. ssp. japonica cv. Nipponbare (NIP), Oryza rufipogon Griff. acc. CWR, and their resulting F1 hybrid (named as NC). NIP and CWR showed distinct phenotypic and molecular differences. The interspecific hybrid, NC, exhibited significant yield heterosis. In the hybrid, most epigenetic and transcriptional features displayed additive inheritance patterns relative to parental lines. Analysis revealed that domestication-selected genes maintained relatively low DNA methylation coupled with high expression levels in both hybrid and parental lines. Additionally, we identified that non-additive miRNAs were potentially involved in regulating fertility, cell growth, and cell division processes in the hybrid. A significant negative correlation was observed between DNA methylation level and gene expression. Functional enrichment analysis revealed that hybrid-MPV DEGs were significantly associated with flowering time regulation, carbohydrate metabolism, photosynthesis, protein phosphorylation, seed development, and defense responses. Through weighted gene co-expression network analysis, we identified 102 functional gene modules, six of which were significantly associated with yield-related heterosis. Collectively, our results provide a multi-omics framework for understanding interspecific hybridization between elite cultivars and wild rice relatives, highlighting CWR as an untapped genetic reservoir for rice improvement.
Oryza↗
Finding the hairpin in the haystack: searching for RNA motifs.
A growing list of examples underscores the roles that regulatory RNA motifs play in controlling the genetic repertoire of cells and developing organisms. Once either an RNA-processing signal, a ribozyme, an element that controls translational or mRNA stability or an RNA localization signal has been identified, it is important to search for other RNA sequences that bear similar regulatory signals. While DNA regulatory elements can often be described by a consensus sequence, RNA signals are frequently composed of a combination of sequence and structure motifs. Here, we discuss the approaches that can be used to identify RNA motifs by searching databases.
Animals↗
Detection of Junin virus by the polymerase chain reaction.
Argentine hemorrhagic fever is an often fatal human disease caused by Junin virus, an RNA-containing virus and member of the Arenavirus family. This virus was detected in vitro by the polymerase chain reaction (PCR) procedure. A pair of Junin virus-specific PCR DNA oligonucleotide primers and an oligonucleotide probe were designed from a known portion of the viral RNA sequence. RNA was isolated from Junin virus-infected monkey kidney cells and used to produce complementary DNA (cDNA) by reverse transcription. A DNA segment, 151 +/- 24 bp long, was amplified from this cDNA and characterized by agarose gel electrophoresis and Southern blot hybridization with the Junin virus-specific DNA probe. Sensitivity experiments showed that Junin virus could be detected with nanogram quantities of RNA isolated from virus-infected cells. The rapid and sensitive assay described here may contribute towards the development of a procedure for the early diagnosis of Argentine hemorrhagic fever.
Arenaviruses, New World↗
Estimating the contributions of selection and self-organization in RNA secondary structure.
In addition to characteristic structural properties imposed by evolutionary modification, evolved, single-stranded RNAs also display characteristic structural properties imposed by intrinsic physical constraints on RNA polymer folding. The balance of intrinsic and functionally selected characters in the folded conformation of evolved secondary structures was determined by comparing the predicted secondary structures of evolved and unevolved (random) RNA sequences. Though evolved conformations are significantly more ordered than conformations of random-sequence RNA, this analysis demonstrates that the majority of conformational order within evolved structures results not from evolutionary optimization but from constraints imposed by rules intrinsic to RNA polymer folding.
Algorithms↗
Clinical and molecular prognostic factors in newly diagnosed pediatric T-cell lymphoblastic lymphoma: a prospective, multicenter, single-arm phase 2 clinical trial.
BACKGROUND: Poor early treatment response in T-cell lymphoblastic lymphoma (T-LBL) is associated with an unfavorable prognosis. This multicenter prospective study evaluated the efficacy of the response-adjusted Chinese Children's Cancer Group (CCCG-LBL-2016) protocol for pediatric T-LBL and examined clinical and molecular prognostic factors. METHODS: Clinical and laboratory data from seven pediatric oncology centers were analyzed. A sub-cohort of 23 patients underwent exploratory integrated genomic analysis, including targeted next-generation sequencing, RNA sequencing, and copy-number array analysis. Survival was evaluated using the Kaplan-Meier method, and prognostic factors were analyzed using multivariable Cox proportional hazards regression. RESULTS: A total of 163 patients (median age: 108 months; 116 males, 47 females) were enrolled, most with advanced disease (stage III: 81.0%; stage IV: 17.8%). Patients were stratified into the low-risk (R1, n = 2) and intermediate-risk groups (R2, n = 161); thirty one patients in the R2 group were escalated to the high-risk intensified regimen (R3) due to poor early response. The 3-year overall survival (OS) was 78.6% ± 3.3% and event-free survival (EFS) was 73.9% ± 3.5%. Outcomes differed by risk group (P < 0.05), with 3-year OS and EFS of 100% and 100% in R1, 82.6% ± 3.4% and 79.5% ± 3.4% in R2, and 58.6% ± 9.1% and 48.3% ± 9.1% in R3. Progression or recurrence occurred in 42 patients (median: 7 months; 3-year OS: 17.1% ± 6.3%). Clinical risk factors included R3 assignment and elevated lactate dehydrogenase. In the exploratory molecular sub-cohort, recurrent alterations included CDKN2A (39.1%), NOTCH1 (26.1%), FBXW7 (21.7%), and MTAP/PIK3R1/NRAS (13.0%). Exploratory multivariable Cox regression analysis identified that CDKN2A alteration was associated with an increased risk of progression or recurrence (hazard ratio = 35.89, 95% confidence interval: 3.07-419, P = 0.004). CONCLUSIONS: Adjusting the risk stratification based on treatment response significantly improved the overall prognosis of T-LBL. However, survival rates remain very low among patients who experience disease progression or recurrence. The preliminarily explored molecular genetic risk factors might contribute to further risk stratification and provide potential therapeutic targets.
Humans↗
A proflavin-induced frameshift hotspot in the thymidylate synthase gene of bacteriophage T4.
Twenty-one independent thymidylate synthase deficient (td) mutants were isolated after proflavin mutagenesis of T4D0 phage. A strikingly high proportion of these mutations (17 of 21; 80%) mapped in a small 122 nucleotide (nt) region which spans the 5' splice site of this intron-containing gene. This region comprises only 14% of the total td exon sequence. RNA sequence analysis of these mutants identified a series of frameshift insertion/deletion mutations and indicated a hotspot for proflavin-induced mutations in the 3' end of exon I of the td gene. The mutant sequences at the hotspot site fully support a previously proposed mutagenic mechanism for proflavin-induced mutations in which frameshifts are produced as a consequence of exonuclease or DNA polymerase activity at the 3' ends of nicks in the DNA produced by perturbation of the T4-encoded type II topoisomerase activity by the acridine. Sixteen of the seventeen DNA mutations in the hotspot region can be explained by the model as a consequence of enzymatic processing of nicks at two phosphodiester bonds staggered by 4 base pairs (bp) and located on opposite strands of the DNA. Thus, these mutants exhibit precisely the symmetry expected of topoisomerase-mediated mutagenesis. The DNA sequences of the td hotspot mutants, when considered with the sequences of proflavin-induced mutants in the T4 rIIB and lysozyme genes, confirm the view that proflavin-induced mutations in diverse bacteriophage T4 DNA sequences are all produced by the topoisomerase-dependent mechanisms and do not support the view that classical misalignments in DNA repeats are hotspots for proflavin-induced mutagenesis in T4.
Bacteriophage T4↗
Monoclonal origin of B cells producing k, lambda and k lambda immunoglobulin light chains in a patient with chronic lymphocytic leukemia.
Immunoglobulin gene rearrangements can be used as genetic markers of clonality in the study of B-cell populations [4]. We have therefore analysed the structure and expression of heavy and light chain immunoglobulin genes in lymphocytes of a patient with chronic lymphocytic leukemia, where we found both k and lambda producing B cells, but in most of the cells both k and lambda chains were co-expressed on the same surface membrane. Single rearrangements were observed in mu, JH, k and lambda DNA sequences, thus providing strong evidence for the monoclonal origin of the cells bearing different light chains. Moreover, the analysis of Ig sequence RNA showed, in addition to normal mu, k and lambda mRNA molecules, high levels of a small lambda related RNA sequence. These findings are discussed in relation to a model of B-lymphocyte differentiation which could be either an additional or an alternative hypothesis to the current one of isotypic exclusion.
Aged↗
Low-Grade Myofibroblastic Sarcoma Represents an Epigenetically Distinct Myofibroblastic Tumor With USP6 Upregulation and Stable Genome.
Low-grade myofibroblastic sarcoma (LGMS) is a rare, indolent mesenchymal neoplasm exhibiting myofibroblastic differentiation, with a propensity for local recurrence. The molecular basis of LGMS and its precise relationship with other histological mimics have remained largely undefined. To address this gap, we conducted the first comprehensive multiomics analysis of 6 LGMS cases, integrating whole-exome sequencing, RNA sequencing, and Illumina Methylation EPICv2 array profiling with comparative analysis against public sarcoma methylation cohorts and related fibroblastic tumors. Clinically, patients (median age 35.5 years) presented with small tumors (median size 1.45 cm), predominantly located in the head and neck, displaying classic histological features of diffusely infiltrative spindle cell fascicles with patchy mononuclear inflammation. Two of the 5 patients with follow-up developed local recurrence, and none metastasized (median follow-up duration 92.5 months). Genomically, all LGMS exhibited a low tumor mutational burden (median 2.31 mut/Mb) and a minimal fraction of genome altered, with TP53 and TSC2 deletions and NTRK1 and ERBB3 amplifications found in a subset of cases. No pathogenic fusions were detected. Transcriptomic profiling revealed a distinct signature featuring prominent USP6 overexpression and upregulation of inflammatory and immune-related genes, including CD274 (PD-L1), and enrichment of inflammatory and interferon-gamma response signatures. Epigenetically, LGMS formed a unique methylation cluster closest to inflammatory myofibroblastic tumor, with numerous differentially methylated regions and higher immune infiltration, particularly monocytes, compared with other fibroblastic tumors. These findings establish LGMS as a genomically stable, epigenetically distinct myofibroblastic sarcoma driven by USP6 overexpression and an inflammation-enriched transcriptome. They support its recognition as a standalone entity, facilitate integration into methylation-based sarcoma classifiers for improved diagnostic precision, and nominate USP6-associated pathways and immune checkpoint blockade as promising therapeutic strategies for recurrent or unresectable disease.
Humans↗
Multiomics Integration Identifies a Molecular Subtype of Intrahepatic Cholangiocarcinoma With Enhanced Benefit From Adjuvant Therapy.
Intrahepatic cholangiocarcinoma (iCCA) is a molecularly heterogeneous liver cancer with a poor prognosis. Improved stratification is needed to guide postoperative therapy. In this study, we applied integrative multiomics analysis to classify iCCA and identify biomarkers predictive of adjuvant treatment benefit. Using publicly available datasets (including whole exome sequencing, RNA sequencing, proteomics, and phosphoproteomics from FU-iCCA cohort and a transcriptomic cohort GSE244807), we defined 3 robust molecular subtypes of iCCA. These subtypes exhibited distinct genomic alterations, pathway activation, and immune microenvironments, with significant differences in overall survival (OS). Through protein-protein interaction network analysis and consensus feature selection using 10 clustering algorithms, we prioritized 8 marker genes distinguishing the subtypes. A Cox proportional-hazards model constructed from these markers stratified patients into high- and low-risk groups. High-risk iCCA, characterized by elevated expression of markers such as CLDN18, MUC1, and MUC5AC, had significantly worse OS in the absence of adjuvant therapy. Notably, in an independent validation of 174 patients with iCCA who underwent resection (single-center cohort), high expression of any of these 3 markers were associated with markedly prolonged OS in patients who received adjuvant chemotherapy or chemoembolization, compared with those who did not. In contrast, marker-negative patients showed no clear benefit from adjuvant therapy. In conclusion, our multiomics approach identified a high-risk, mucin-enriched subtype of iCCA. CLDN18, MUC1, and MUC5AC emerge as candidate predictive biomarkers for adjuvant chemotherapy benefit in iCCA, warranting prospective validation to improve personalized postoperative management.
Humans↗
Understanding tumor adaptations and resistance to MET inhibitors in MET-altered non-small cell lung cancer.
AIM: Type Ib MET inhibitors are clinically active in selected MET-altered non-small cell lung cancer, particularly tumors with MET exon 14 skipping or MET amplification, but acquired resistance remains incompletely understood. Here, we investigated resistance across biologically distinct MET-altered contexts, including MET exon 14 skipping, MET amplification, and MET overexpression. METHODS: Paired baseline and progression samples from seven patients treated with tepotinib or capmatinib were analyzed using spatial transcriptomics, whole-exome sequencing, RNA sequencing, CRISPR screening, and drug-combination assays. Patient-derived cultures and resistant cell-line models were used to explore resistance-associated changes. RESULTS: MET inhibitor resistance was heterogeneous, with persistence of the initial MET alteration in most evaluable cases and emergence of patient-specific genomic events. Three main resistance-associated, often overlapping, routes were identified: on-target MET evolution through kinase-domain alterations; extracellular matrix and tumor-microenvironment remodeling, including collagen and fibronectin upregulation, complement-related signaling, and partial EMT-associated programs; and bypass signaling involving EGFR/HER, MAPK, and PI3K/Akt pathways. In vitro models reproduced several tumor-cell-intrinsic features but only partially captured microenvironment-associated changes. CONCLUSIONS: MET inhibitor resistance in this cohort involved overlapping, context-dependent genomic, phenotypic, and signaling adaptations, supporting combination strategies for MET-altered lung cancer.
CRISPR screen↗
Pre-clinical safety and efficacy of human induced pluripotent stem cell-derived products for autologous cell therapy in Parkinson's disease.
Human induced pluripotent stem cell (hiPSC)-derived midbrain dopaminergic cells (mDACs) represent a promising source for autologous cell therapy in Parkinson's disease (PD), but standardized regulatory criteria are essential for clinical translation. In this pre-clinical study, we generated multiple clinical-grade hiPSC lines from freshly biopsied fibroblasts of four sporadic PD patients using episomal reprogramming and differentiated them into mDACs using a refined 21-day protocol. Rigorous evaluations included whole-genome/exome sequencing, RNA sequencing, and in vivo studies, including a 39-week Good Laboratory Practice-compliant mouse safety study. While mDACs from all lines met safety criteria, mDACs from one patient failed to improve rodent behavioral outcomes, underscoring inter-individual variability. Importantly, in vitro assessments did not reliably predict in vivo efficacy, identifying dopaminergic fiber density as a key efficacy criterion. These findings support comprehensive quality control guidelines for autologous cell therapy and pave the way for a clinical trial with eight sporadic PD patients, scheduled to commence in 2025.
Humans↗
Synthetic long peptide and DNA personalized cancer vaccines induce robust neoantigen-specific T cell responses in pancreatic cancer.
Pancreatic ductal adenocarcinoma (PDAC) is unresponsive to standard immunotherapies despite harboring cancer neoantigens capable of eliciting T cell responses. We completed two phase 1 clinical trials (NCT03956056 and NCT03122106) evaluating safety and immunogenicity of synthetic long peptide (SLP) and DNA personalized cancer vaccines (PCVs). PCVs were administered after resection and adjuvant chemotherapy. Tumor/normal whole-exome sequencing, RNA sequencing, and pVACtools were used to identify and prioritize candidate PCV neoantigens. PCVs were well tolerated without any grade ≥3 adverse events. Neoantigen-specific responses were demonstrated by interferon-γ enzyme-linked immunospot and intracellular cytokine staining. Expanded T cell receptor clonotypes were sequenced and transduced into autologous peripheral blood mononuclear cells to confirm neoantigen specificity. When compared with a contemporaneous institutional propensity-matched cohort, PCV patients demonstrated a trend toward prolonged median overall survival (4.4 versus 3.5 years, log-rank P = 0.23). Overall, PDAC PCVs are safe and feasible and elicit polyclonal T cell responses, linking prioritized cancer neoantigens to functional antitumor immunity.
Humans↗