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Decrypting noncoding RNA interactions, structures, and functional networks.

The world of noncoding RNAs (ncRNAs) is composed of an enormous and growing number of transcripts, ranging in length from tens of bases to tens of kilobases, involved in all biological processes and altered in expression and/or function in many types of human disorders. The premise of this review is the concept that ncRNAs, like many large proteins, have a multidomain architecture that organizes them spatially and functionally. As ncRNAs are beginning to be imprecisely classified into functional families, we review here how their structural properties might inform their functions with focus on structural architecture-function relationships. We will describe the properties of "interactor elements" (IEs) involved in direct physical interaction with nucleic acids, proteins, or lipids and of "structural elements" (SEs) directing their wiring within the "ncRNA interactor networks" through the emergence of secondary and/or tertiary structures. We suggest that spectrums of "letters" (ncRNA elements) are assembled into "words" (ncRNA domains) that are further organized into "phrases" (complete ncRNA structures) with functional meaning (signaling output) through complex "sentences" (the ncRNA interactor networks). This semiotic analogy can guide the exploitation of ncRNAs as new therapeutic targets through the development of IE-blockers and/or SE-lockers that will change the interactor partners' spectrum of proteins, RNAs, DNAs, or lipids and consequently influence disease phenotypes.

DNA↗

Mapping 2'-O-methyl groups in ribosomal RNA.

Ribosomal RNAs (rRNAs) from all sources contain modified nucleosides, whose numbers range from a few in mitochondrial rRNA to more than 200 in the complete rRNAs of some higher eukaryotes. In eukaryotic rRNA the great majority of modified nucleosides are 2'-O-methylated nucleosides or pseudouridines. The locations of most of the 2'-O-methylated nucleosides in rRNA from some representative eukaryotes are known from studies whose aim was full characterization of rRNA methylation. More recently, and particularly in connection with the discovery of methylation guide RNAs, it is often required to check for the presence or absence of 2'-O-methyl nucleosides at specified locations within rRNA. Three methods that can be applied for such "local" objectives are reviewed. Two of the methods are based on primer extension by reverse transcriptase. They exploit, respectively, a tendency of 2'-O-methyl groups to impede reverse transcriptase at low dNTP concentrations, or the resistance of phosphodiester bonds adjacent to 2'-O-methyl groups to alkaline hydrolysis. Examples of these methods are summarized. Although the two methods are relatively straightforward, they suffer from various experimental limitations, as discussed. The third method is technically more sophisticated but is capable of overcoming the limitations of the first two methods. It is based on the resistance of a target 2'-O-methylated site to cleavage by RNase H when the site is hybridized to an appropriate chimeric oligonucleotide. An overview of the approaches and methods now available for the complete mapping of 2'-O-methyl groups in rRNA is presented.

Binding Sites↗

Activation of a cryptic 5' splice site in the upstream exon of the phage T4 td transcript: exon context, missplicing, and mRNA deletion in a fidelity mutant.

A collection of 100 td mutants defective in phage T4 thymidylate synthase (TS) production was screened for splicing impairments. Splicing-defective mutants were identified by a rapid assay developed to detect imbalances in the td protein products (TS, the exon ligation product, and NH2TS, encoded by the pre-mRNA). Thirteen selected mutants, confirmed to be splicing defective by an RNA-oligodeoxynucleotide hybridization assay, were all shown to be inhibited in the first step of the group I splicing pathway, cleavage at the 5' splice site. Of these, only one, SC99, appeared to be a specificity mutant. Whereas the 12 other mutants had sequence changes within the functionally important 5' and 3' domains of the intron, SC99 was shown to be an exon mutant. The G----A change at residue -3 of the upstream exon of SC99 resulted in loss of normal 5' splice site recognition. Furthermore, activation of a remote cryptic splice site at residue -29 of the upstream exon and missplicing of mRNA that is deleted for 29 nucleotides of the 5' exon are characteristic for this mutant. These results underscore the role of exon sequences in guiding the fidelity of the splicing reaction and they raise provocative questions about the alignment of introns within exon contexts that are consistent with accurate splicing and synthesis of an intact gene product.

Base Sequence↗

Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not.

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.

Actins↗

GABA(A) receptor subunit expression within hypophysiotropic CRH neurons: a dual hybridization histochemical study.

Dual hybridization histochemical studies were conducted to investigate the extent of colocalization of mRNA transcripts encoding the alpha1-2 and beta1-3 subunits of the gamma aminobutyric acid (GABA)(A) receptor with those for corticotropin-releasing hormone (CRH) within the rat hypothalamic paraventricular nucleus (PVN). A vast majority of CRH neurons (>94.5%) were found to express transcripts specific for the the alpha2, beta1 and beta3 subunits; mRNAs for the alpha1 and beta2 subunits of the GABA(A) receptor were detected within 53.3% and 65.7% of PVN CRH neurons, respectively. The results may have important implications for studies aimed at understanding GABAergic influences upon the hypothalamic-pituitary-adrenocortical (HPA) axis. Hypophysiotropic CRH neurons serve as the origin of the final common pathway for glucocorticoid secretion in response to stressful stimuli, and GABAergic afferents have been implicated in afferent control of these neurons. The subunit composition of GABA(A) receptors at this key regulatory locus may affect the efficacy of a major inhibitory input, and thus the magnitude and/or duration of stress-induced glucocorticoid secretion. The present findings reveal basal expression patterns of transcripts encoding several subunits of the GABA(A) receptor within stress-integrative CRH neurons, data which may be used to guide regulatory studies of GABAergic influences on the HPA axis under a variety of conditions.

Animals↗

Novel insights into retinoblastoma: From oncogenic circuitry to precision diagnosis and eye-preserving therapies.

Retinoblastoma (RB) represents the most common primary intraocular malignancy in childhood and stands as a paradigm for translating molecular oncology into precision clinical management. This review synthesizes the comprehensive evolution in the understanding and treatment of RB. First, we deconstruct the intricate oncogenic circuitry that extends far beyond Knudson's classic "two-hit" RB1 inactivation model, describing non-classical MYCN-driven pathogenesis, multi-layered epigenetic reprogramming (including chromatin, RNA and histone changes), and distinct histological subtypes with defined clinical correlates, such as the favorable-prognosis cavitary RB. Single-cell genomics has elucidated the cellular origin from cone precursor cells and intratumoral heterogeneity. Risk stratification has been refined through well-defined classification systems, from the therapy-guiding International Intraocular Retinoblastoma Classification (IIRC) to the comprehensive American Joint Committee on Cancer Tumor-Node-metastasis (AJCC TNM) staging. Furthermore, the diagnostic paradigm has advanced from conventional anatomical imaging to liquid biopsies, enabling non-invasive molecular staging and monitoring via tumor-derived cell-free DNA analysis. Concurrently, the therapeutic landscape has undergone a radical shift, moving from enucleation and external-beam radiotherapy to an era dominated by local sight-preserving strategies. We provide a critical synthesis of the evidence for intravenous chemotherapy and the transformative role of super-selective intra-arterial chemotherapy (IAC), and describe essential randomized controlled trials, technical innovations, and optimized drug regimens. Finally, we explore emerging targeted molecular therapies and future directions. By integrating cutting-edge molecular insights with robust, high-level clinical evidence, this review offers the framework for achieving patient and eye survival as well as vision preservation in children with Retinoblastoma.

Intra-arterial chemotherapy↗

Tissue distribution profiles of the human TRPM cation channel family.

Eight members of the TRP-melastatin (TRPM) subfamily have been identified, whose physiological functions and distribution are poorly characterized. Although tissue expression and distribution patterns have been reported for individual TRPM channels, comparisons between individual studies are not possible because of variations in analysis techniques and tissue selection. We report here a comparative analysis of the expression patterns of all of the human TRPM channels in selected peripheral tissues and the central nervous system (CNS) using two distinct but complimentary approaches: TaqMan and SYBR Green real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). These techniques generated comparative distribution profiles and demonstrated tissue-specific co-expression of TRPM mRNA species, indicating significant potential for the formation of heteromeric channels. TRPM channels 2, 4, 5, 6, and 7 in contrast to 1, 3, and 8 are widely distributed in the CNS and periphery. The tissues demonstrating highest expression for individual family members were brain (TRPM1), brain and bone marrow (TRPM2), brain and pituitary (TRPM3), intestine and prostate (TRPM4), intestine, pancreas, and prostate (TRPM5), intestine and brain (TRPM6), heart, pituitary, bone, and adipose tissue (TRPM7), and prostate and liver (TRPM8). The data reported here will guide the elucidation of TRPM channel physiological functions.

Base Sequence↗

Use of genotypic resistance testing to guide hiv therapy: clinical impact and cost-effectiveness.

BACKGROUND: Genotypic sequencing for drug-resistant strains of HIV can guide the choice of antiretroviral therapy. OBJECTIVE: To assess the cost-effectiveness of genotypic resistance testing for patients acquiring drug resistance through failed treatment (secondary resistance) and those infected with resistant virus (primary resistance). DESIGN: Cost-effectiveness analysis with an HIV simulation model incorporating CD4 cell count and HIV RNA level as predictors of disease progression. DATA SOURCES: Published randomized trials and data from the Multicenter AIDS Cohort Study, the national AIDS Cost and Services Utilization Survey, the Red Book, and an institutional cost-accounting system. TARGET POPULATION: HIV-infected patients in the United States with baseline CD4 counts of 0.250 x 10(9) cells/L. TIME HORIZON: Lifetime. PERSPECTIVE: Societal. INTERVENTIONS: Genotypic resistance testing and clinical judgment, compared with clinical judgment alone, in two contexts: after initial treatment failure (secondary resistance testing) and before initiation of antiretroviral therapy (primary resistance testing). OUTCOME MEASURES: Life expectancy, quality-adjusted life expectancy, and cost-effectiveness in dollars per quality-adjusted life-year (QALY) gained. RESULTS OF BASE-CASE ANALYSIS: Secondary resistance testing increased life expectancy by 3 months, at a cost of $17 900 per QALY gained. The cost-effectiveness of primary resistance testing was $22 300 per QALY gained with a 20% prevalence of primary resistance but increased to $69 000 per QALY gained with 4% prevalence. RESULTS OF SENSITIVITY ANALYSIS: The cost-effectiveness ratio for secondary resistance testing remained under $25 000 per QALY gained, even when effectiveness and cost of testing and antiretroviral therapy, quality-of-life weights, and discount rate were varied. CONCLUSIONS: Genotypic antiretroviral resistance testing following antiretroviral failure is cost-effective. Primary resistance testing also seems to be reasonably cost-effective and will become more so as the prevalence of primary resistance increases.

Anti-HIV Agents↗

New reactions of the ribosomal RNA precursor of Tetrahymena and the mechanism of self-splicing.

The availability of Tetrahymena pre-rRNA of discrete size, produced by transcription of recombinant plasmids with bacteriophage SP6 RNA polymerase, has permitted a more detailed investigation of the self-splicing reaction. The predicted splicing intermediate, the product of cleavage by guanosine at the 5' splice site, was identified. This intermediate was tested in the intermolecular exon ligation reaction and found to be competent to undergo the second step of splicing. These results and others that evaluated the reactivity of the 5' and 3' splice sites independently show that splicing occurs in two separable steps. The 3' splice site was found to be susceptible to site-specific hydrolysis leaving a hydroxyl terminus. This is interpreted as an indication that the 3' splice site is activated for nucleophilic attack in general and for exon ligation in particular. Preliminary evidence for specific hydrolysis at the 5' splice site was also obtained. All of the newly characterized intervening sequence RNA-mediated reactions as well as those found previously are divided into three categories: transesterification by guanosine at sites following two or three pyrimidine nucleotides (and, as a minor reaction, at sites following other guanosine residues); transesterification by oligopyrimidines or by the 5' exon (which terminates with C-U-C-U-C-UOH) at the site following the 3'-terminal guanosine residue of the intervening sequence; and specific hydrolysis at the splice sites. One of the products of the reactions at the 3' splice site is a molecule that contains the 5' exon still attached to the intervening sequence. It has a 3'-terminal GOH and undergoes cyclization both at the normal cyclization site within the intervening sequence and at the 5' splice site. The finding that the splice site can act as a cyclization site, combined with the earlier observation that the normal cyclization site is subject to attack by guanosine mononucleotide, leads us to propose that all these reactions may be occurring in the same active site. Translocation (a conformational change) would then bring different oligopyrimidine sequences into the active site for attack by guanosine. On the basis of the experimental results, a model for the local structure at the active site is described. A key feature of the model is the interaction between the U at the end of the oligopyrimidine sequence, a G residue within the internal guide sequence in the intervening sequence, and another G residue that can be either the attacking group for transesterification or the 3'-terminal G of the intervening sequence.

Animals↗

A determination of the identity elements in yeast 18 S ribosomal RNA for the recognition of ribosomal protein YS11: the role of the kink-turn motif in helix 11.

A description of the site of interaction of YS11, the yeast homolog of eubacterial S17, with 18 S rRNA was obtained by assessing the binding of the ribosomal protein, in a filter retention assay, to oligoribonucleotides that reproduce regions of 18 S rRNA. YS11 binds predominantly to domain I; the Kd value is 113 nM. The dimensions of the YS11 binding site were refined, guided by chemical protection data and by the atomic structure of the Thermus thermophilus 30 S subunit, which has the S17 recognition site in 16 S rRNA. An oligoribonucleotide that mimics helix 11, a phylogenetically conserved region in domain I, binds YS11 with a Kd value of 230 nM; a second oligoribonucleotide that contains only the kink-turn motif in helix 11 binds YS11 with a Kd value of 528 nM. Thus, helix 11 has most of the nucleotides required for the recognition of YS11. To identify those nucleotides a set of 27 transversion mutations in H11 was constructed and their contribution to the binding of YS11 determined. Mutations of nine nucleotides (U313, C314, A316, G337, C338, G347, U348, U350, and C351) increased the Kd value for YS11 binding by at least eightfold; G325U and U349A mutations increased the Kd value fivefold. Eight of the 11 mutations are in the kink-turn in H11, confirming the critical importance of the motif for YS11 recognition. The other three nucleotides are in the lower stem and the terminal loop of H11, which makes a lesser, but still important, contribution to YS11 binding. The identity elements for YS11 recognition are: A316, G325, G337, G347, U348, U349, U350, and C351. The effect of the other nucleotides that decrease binding is probably indirect, presumably they affect the conformation of the binding site but do not have contacts to YS11 amino acid residues. The eight identity element nucleotides are in regions of H11 that deviate from A-form geometry and the contacts are predominantly, if not exclusively, to backbone phosphate and sugar oxygen atoms, indicating that YS11 recognizes the shape of the rRNA binding site rather than reading the sequence of nucleotides.

Amino Acid Sequence↗

ADP-ribose-1"-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture.

Replication of the approximately 30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

Amino Acid Sequence↗

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↗

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↗

Heterologous protection of mice from a lethal human H1N1 influenza A virus infection by H3N8 equine defective interfering virus: comparison of defective RNA sequences isolated from the DI inoculum and mouse lung.

We have examined the RNAs involved in the heterologous protection of adult mice from otherwise lethal intranasal infection with mouse-adapted human A/WSN (H1N1) by defective interfering (DI) equine A/equine/Newmarket/7339/79 (H3N8: EQV) influenza virus, as well as the RNAs involved in the protection of WSN- or EQV-infected mice by their homologous DI viruses. The aim of this study was to describe the types of defective RNAs present in protected mice in order to guide the design of potentially protective DI RNAs. The interfering and mouse-protecting activity of DI virus was destroyed by prolonged UV irradiation (iDI virus) demonstrating that protection correlated with an active DI genome, and not viral antigen. Protected mice were all infected but suffered a lower degree of morbidity than those given iDI virus. The DI EQV inoculum contained defective segment 1-8 RNAs while DI WSN inoculum contained only defective segment 1-6 RNAs. However lungs of mice given EQV + DI EQV contained only defective segments 1-4 or 1-6 RNAs (mouse-to-mouse variation), while control mice given EQV or EQV + iDI EQV contained few very defective RNAs. Thus prevention of death was the result of quantitative and/or qualitative differences in defective RNAs administered to the mice. Only defective segments 1-3 RNAs were isolated from the lungs of mice given WSN + DI WSN, confirming the earlier report of Noble and Dimmock (1995). A detailed analysis showed that most defective RNAs isolated from the lungs of mice protected from a lethal WSN infection by DI EQV were EQV in origin. Thus, as no infectious EQV was present, these defective RNAs from the DI EQV inoculum must have been heterologously replicated in mouse lung by WSN. All defective segment 3-6 RNAs isolated were of EQV origin, indicating that they were replicated by WSN in preference to its own. Defective segments 1 and 2 were a mixture of EQV and WSN RNAs. Of 17 defective EQV segment 1-3 sequences from mouse lung, all but three differed in their primary central deletion from 20 defective RNAs isolated from the inoculum. No bias in the break points was evident. A number of minor deletions of 2 or more nts were also present in defective EQV and WSN RNAs in segments 1 and 2, but none in segment 3. Their 5', but not 3', breakpoints were heterogeneous, suggesting that defective RNAs were generated during positive strand synthesis. Two cloned EQV-defective segment 3 RNAs were chimeras containing a 30 nt insert from segment 1. Most defective RNAs possessed at least 178 nts from the 5' end of vRNA. The amount of 5' sequence present in those RNAs correlated with the segment of origin, suggesting that this was the minimum required for propagation of viral RNA in mouse lung and hence possibly for protection also.

Animals↗

Molecular Characterisation of Treacher Collins Syndrome in a South African Cohort: Novel Disease-Causing Variants in TCOF1 and POLR1D.

BACKGROUND: Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterised by variable expressivity. It is caused by pathogenic variants in the TCOF1, POLR1D, POLR1C, or POLR1B genes. Common clinical features include hypoplasia of the zygomatic complex and mandible, downward-slanting palpebral fissures, lower eyelid anomalies, microtia, and hearing loss. Owing to its phenotypic overlap with other craniofacial syndromes, molecular testing is essential for establishing an accurate diagnosis and guiding effective clinical management. METHODS: Ten South African patients with a suspected clinical diagnosis of TCS underwent targeted next-generation sequencing (NGS) using a custom gene panel including TCOF1, POLR1C, and POLR1D genes. Variants were classified according to ACMG/AMP guidelines, with validation by Sanger sequencing where necessary. RESULTS: Disease-causing variants were identified in six of the ten patients (60%). These included five heterozygous variants in TCOF1 and one homozygous variant in POLR1D. Notably, five of the six variants were identified for the first time in this study. Additionally, a recurrent TCOF1 deletion was identified for the first time in an African family. CONCLUSION: This study expands the mutational spectrum of TCS in general and provides African data in particular. Findings support the use of panel-based NGS for diagnosis in resource-limited settings and highlight the need for population-specific variant data to improve diagnostic accuracy, guide clinical care, and support genetic counselling for affected individuals and their families.

Humans↗

Databases of mRNA untranslated regions for metazoa.

The important role that untranslated regions of mRNAs (UTR) may play in gene regulation and expression is now widely accepted. For this reason we developed UTRDB, a specialized database of 5'- and 3'-UTR of eukaryotic mRNAs cleaned of redundancy. This paper describes the composition and the general feature of UTRDB. The analysis of UTRDB by using suitable statistical methods could provide useful information for guiding the experimental work aimed at delucidating the role of UTR sequences in gene regulation and expression.

Algorithms↗

Uracil content of 16S rRNA of thermophilic and psychrophilic prokaryotes correlates inversely with their optimal growth temperatures.

We report here the finding of a highly significant inverse correlation of the uracil content of 16S rRNA and the optimum growth temperature (T(opt)) of cultured thermophilic and psychrophilic prokaryotes. This correlation was significantly different from the weaker correlations between the contents of other nucleotides and T(opt). Analysis of the 16S rRNA secondary structure regions revealed a fall in the A:U base-pair content in step with the increase in T(opt) that was much steeper than that of mismatched base-pairs, which are thermodynamically less stable. These findings indicate that the 16S rRNA sequences of thermophiles and psychrophiles are under a strong thermo-adaptive pressure, and that structure-function constraints play a crucial role in determining their 16S rRNA nucleotide composition. The derived relationship between uracil content and T(opt) was used to develop an algorithm to predict the T(opt) values of uncultured prokaryotes lacking cultured close relatives and belonging to the phyla predominantly containing thermophiles. This algorithm may be useful in guiding the design of cultivation conditions for hitherto uncultured microbes.

Adaptation, Physiological↗