Modification of the acid guanidinium thiocyanate-phenol-chloroform method for nuclear RNA isolation.
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3T3 Cells↗
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A contextual activity score (CAS) for inferring ADAR-associated transcriptional activity across RNA-seq, single-cell, and spatial transcriptomics.
BACKGROUND AND OBJECTIVE: Adenosine-to-inosine RNA editing, catalyzed by Adenosine Deaminases Acting on RNA (ADARs), is a widespread modification involved in neural function, immune regulation, and cancer. The Alu Editing Index (AEI) is the standard metric to estimate ADAR activity but requires raw sequencing reads and is poorly suited for single-cell and spatial transcriptomic data. This study aimed to develop an alternative framework for inferring ADAR-associated transcriptional activity from gene expression data across diverse transcriptomic technologies. METHODS: We developed the Contextual Activity Score (CAS), a framework based on transcriptional signatures from ADAR perturbation experiments. Context-specific signatures were generated for human neurons, mouse neurons, and cancer models to infer ADAR1 and ADAR2 activity. CAS was computed from normalized gene expression matrices using regulon-based enrichment analysis. Performance was evaluated by comparing with the Alu Editing Index across bulk RNA sequencing datasets, simulated sequencing depths, and library preparation protocols. RESULTS: CAS showed strong concordance with the Alu Editing Index across multiple datasets, while remaining robust to reduced sequencing depth and different library protocols. Unlike the Alu Editing Index, CAS can be applied to single-cell and spatial transcriptomic data and enables the independent assessment of ADAR2 activity. In cancer and neuronal contexts, CAS captured biologically meaningful variations in ADAR-associated transcriptional activity at sample, cell-type, and spatial levels. CONCLUSION: CAS provides a scalable approach applicable across multiple RNA-seq protocols for estimating ADAR-associated transcriptional activity using gene expression data. This method, implemented in an open-source R package for broad adoption, expands the ability to study ADAR-associated transcriptional activity across transcriptomic modalities where direct editing quantification is challenging, such as single-cell and spatial transcriptomics.
Adenosine Deaminase↗
Gene silencing activity of siRNAs with a ribo-difluorotoluyl nucleotide.
Recently, chemically synthesized short interfering RNA (siRNA) duplexes have been used with success for gene silencing. Chemical modification is desired for therapeutic applications to improve biostability and pharmacokinetic properties; chemical modification may also provide insight into the mechanism of silencing. siRNA duplexes containing the 2,4-difluorotoluyl ribonucleoside (rF) were synthesized to evaluate the effect of noncanonical nucleoside mimetics on RNA interference. 5'-Modification of the guide strand with rF did not alter silencing relative to unmodified control. Internal uridine to rF substitutions were well-tolerated. Thermal melting analysis showed that the base pair between rF and adenosine (A) was destabilizing relative to a uridine-adenosine pair, although it was slightly less destabilizing than other mismatches. The crystal structure of a duplex containing rFoA pairs showed local structural variations relative to a canonical RNA helix. As the fluorine atoms cannot act as hydrogen bond acceptors and are more hydrophobic than uridine, there was an absence of a well-ordered water structure around the rF residues in both grooves. siRNAs with the rF modification effectively silenced gene expression and offered improved nuclease resistance in serum; therefore, evaluation of this modification in therapeutic siRNAs is warranted.
Base Pairing↗
RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing.
Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in pre-siRNA transcription. We define a centromeric pre-siRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing.
Centromere↗
Synthesis and hybridization studies of oligonucleotides containing 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta-D-ribofuranosyl)thymine (2'-alpha-hm-dT).
We report the first investigation of oligoribonucleotides containing a few 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta-D-ribofuranosyl)thymine units (or 2'-hm-dT, abbreviated in this work as 'H'). Both the 2'-CH2O-phosphoramidite and 3'-O-phosphoramidite derivatives of H were synthesized and incorporated into both 2',5'-RNA and RNA chains. The hybridization properties of the modified oligonucleotides have been studied via thermal denaturation and circular dichroism studies. While 3',5'-linked H was shown previously to significantly destabilize DNA:RNA hybrids and DNA:DNA duplexes (modification in the DNA strand; DeltaT(m) approximately -3 degrees C/insert), we find that 2',5'-linked H have a smaller effect on 2',5'-RNA:RNA and RNA:RNA duplexes (DeltaT(m) = -0.3 degrees C and -1.2 degrees C, respectively). The incorporation of 3',5'-linked H into 2',5'-RNA:RNA and RNA:RNA duplexes was found to be more destabilizing (-0.7 degrees C and -3.6 degrees C, respectively). Significantly, however, the 2',5'-linked H units confer marked stability to RNA hairpins when they are incorporated into a 2',5'-linked tetraloop structure (DeltaT(m) = +1.5 degrees C/insert). These results are rationalized in terms of the compact and extended conformations of nucleotides.
Circular Dichroism↗
Primer on medical genomics part II: Background principles and methods in molecular genetics.
The nucleus of every human cell contains the full complement of the human genome, which consists of approximately 30,000 to 70,000 named and unnamed genes and many intergenic DNA sequences. The double-helical DNA molecule in a human cell, associated with special proteins, is highly compacted into 22 pairs of autosomal chromosomes and an additional pair of sex chromosomes. The entire cellular DNA consists of approximately 3 billion base pairs, of which only 1% is thought to encode a functional protein or a polypeptide. Genetic information is expressed and regulated through a complex system of DNA transcription, RNA processing, RNA translation, and posttranslational and cotranslational modification of proteins. Advances in molecular biology techniques have allowed accurate and rapid characterization of DNA sequences as well as identification and quantification of cellular RNA and protein. Global analytic methods and human genetic mapping are expected to accelerate the process of identification and localization of disease genes. In this second part of an educational series in medical genomics, selected principles and methods in molecular biology are recapped, with the intent to prepare the reader for forthcoming articles with a more direct focus on aspects of the subject matter.
Animals↗
Muscle channelopathies and critical points in functional and genetic studies.
Muscle channelopathies are caused by mutations in ion channel genes, by antibodies directed against ion channel proteins, or by changes of cell homeostasis leading to aberrant splicing of ion channel RNA or to disturbances of modification and localization of channel proteins. As ion channels constitute one of the only protein families that allow functional examination on the molecular level, expression studies of putative mutations have become standard in confirming that the mutations cause disease. Functional changes may not necessarily prove disease causality of a putative mutation but could be brought about by a polymorphism instead. These problems are addressed, and a more critical evaluation of the underlying genetic data is proposed.
Animals↗
Requirements for RNA heterodimerization of the human immunodeficiency virus type 1 (HIV-1) and HIV-2 genomes.
Retroviruses are prone to recombination because they package two copies of the RNA genome. Whereas recombination is a frequent event within the human immunodeficiency virus type 1 (HIV-1) and HIV-2 groups, no HIV-1/HIV-2 recombinants have been reported thus far. The possibility of forming HIV-1/HIV-2 RNA heterodimers was studied in vitro. In both viruses, the dimer initiation site (DIS) hairpin is used to form dimers, but these motifs appear too dissimilar to allow RNA heterodimer formation. Multiple mutations were introduced into the HIV-2 DIS element to gradually mimic the HIV-1 hairpin. First, the loop-exposed palindrome of HIV-1 was inserted. This self-complementary sequence motif forms the base pair interactions of the kissing-loop (KL) dimer complex, but such a modification is not sufficient to permit RNA heterodimer formation. Next, the HIV-2 DIS loop size was shortened from 11 to 9 nucleotides, as in the HIV-1 DIS motif. This modification also results in the presentation of the palindromes in the same position within the hairpin loop. The change yielded a modest level of RNA heterodimers, which was not significantly improved by additional sequence changes in the loop and top base pair. No isomerization of the KL dimer to the extended duplex dimer form was observed for the heterodimers. These combined results indicate that recombination between HIV-1 and HIV-2 is severely restricted at the level of RNA dimerization.
Base Pairing↗
Probing the structure of mouse Ehrlich ascites cell 5.8S, 18S and 28S ribosomal RNA in situ.
The secondary structure of mouse Ehrlich ascites 18S, 5.8S and 28S ribosomal RNA in situ was investigated by chemical modification using dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulphonate. These reagents specifically modify unpaired bases in the RNA. The reactive bases were localized by primer extension followed by gel electrophoresis. The three rRNA species were equally accessible for modification i.e. approximately 10% of the nucleotides were reactive. The experimental data support the theoretical secondary structure models proposed for 18S and 5.8/28S rRNA as almost all modified bases were located in putative single-strand regions of the rRNAs or in helical regions that could be expected to undergo dynamic breathing. However, deviations from the suggested models were found in both 18S and 28S rRNA. In 18S rRNA some putative helices in the 5'-domain were extensively modified by the single-strand specific reagents as was one of the suggested helices in domain III of 28S rRNA. Of the four eukaryote specific expansion segments present in mouse Ehrlich ascites cell 28S rRNA, segments I and III were only partly available for modification while segments II and IV showed average to high modification.
Animals↗
Site-specific crosslinks of yeast U6 snRNA to the pre-mRNA near the 5' splice site.
We have introduced a single photochemical crosslinking reagent into specific sites in the central domain of U6 to identify the sites that are in close proximity to the pre-mRNA substrate. Four distinct U6 snRNAs were synthesized with a single 4-thiouridine (4-thioU) at positions 46, 51, 54, and 57, respectively. Synthetic U6 RNA containing the 4-thioU modifications can functionally reconstitute splicing activity in cell-free yeast splicing extracts depleted of endogenous U6 snRNA. Upon photoactivation with UV (>300 nm), 4-thioU at position 46 forms crosslinks to pre-mRNA near the 5' splice site at nt +4, +5, +6, and +7 in the intron, whereas 4-thioU at position 51 crosslinks to the pre-mRNA at positions -2, -1, +1, +2, +3, and at the invariant G in the lariat intermediate. All crosslinks are dependent on the presence of ATP and the splicing substrate. The two crosslinks to the pre-mRNA from position 46 and 51 of U6 can also occur in prp2 heat-inactivated yeast splicing extracts blocked immediately prior to the first chemical step. Significantly, the crosslink from position 51 can undergo subsequent splicing when the mutant extract is complemented with functional Prp2 protein in a chase experiment, indicating that the crosslink reflects a functional interaction that is maintained during the first step. The crosslink to lariat intermediate appears when the mutant spliceosomes are complemented with functional Prp2 protein added exogenously. This experiment is a paradigm for future studies in which different mutant extracts are used to establish the stage in assembly at which particular RNA-RNA interactions defined by unique crosslinks occur.
Actins↗
Chemical nucleic acid synthesis, modification and labelling.
The chemical synthesis of RNA on solid phase has now become routine using labile protecting groups and mild deprotection methods. The great interest in antisense technology has sparked the development of P-chiral phosphorothioates and a large number of DNA analogues with modified sugars and/or backbones to increase resistance to nucleases, and with modifier groups attached to the sugar, nucleobase or internucleotide function to aid cellular uptake.
Nucleic Acids↗
The FtsJ/RrmJ heat shock protein of Escherichia coli is a 23 S ribosomal RNA methyltransferase.
Ribosomal RNAs undergo several nucleotide modifications including methylation. We identify FtsJ, the first encoded protein of the ftsJ-hflB heat shock operon, as an Escherichia coli methyltransferase of the 23 S rRNA. The methylation reaction requires S-adenosylmethionine as donor of methyl groups, purified FtsJ or a S(150) supernatant from an FtsJ-producing strain, and ribosomes from an FtsJ-deficient strain. In vitro, FtsJ does not efficiently methylate ribosomes purified from a strain producing FtsJ, suggesting that these ribosomes are already methylated in vivo by FtsJ. FtsJ is active on ribosomes and on the 50 S ribosomal subunit, but is inactive on free rRNA, suggesting that its natural substrate is ribosomes or a pre-ribosomal ribonucleoprotein particle. We identified the methylated nucleotide as 2'-O-methyluridine 2552, by reverse phase high performance liquid chromatography analysis, boronate affinity chromatography, and hybridization-protection experiments. In view of its newly established function, FtsJ is renamed RrmJ and its encoding gene, rrmJ.
Amino Acid Sequence↗
Specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer tRNA3Lys.
Initiation of RNA-dependent DNA synthesis by retroviral reverse transcriptases is generally considered as unspecific. In the case of human immunodeficiency virus type 1 (HIV-1), the natural primer is tRNA3Lys. We recently found evidence of complex interactions between tRNA3Lys and HIV-1 RNA that may be involved in the priming process. In this study, we compare the ability of natural and unmodified synthetic tRNA3Lys and 18mer oligoribo- and oligodeoxyribonucleotides complementary to the viral primer binding site to initiate replication of HIV-1 RNA using either homologous or heterologous reverse transcriptases. We show that HIV-1 RNA, HIV-1 reverse transcriptase and primer tRNA3Lys form a specific initiation complex that differs from the unspecific elongation complex formed when an oligodeoxyribonucleotide is used as primer. Modified nucleosides of tRNA3Lys are required for efficient initiation and transition to elongation. Transition from initiation to elongation, but not initiation of reverse transcription itself, is facilitated by extended primer-template interactions. Elongation, but not initiation of reverse transcription, is inhibited by Mn2+, which further differentiates these two different functional states of reverse transcriptase. These results define initiation of reverse transcription as a target to block viral replication.
Cell-Free System↗
Immunostimulating capacities of stabilized RNA molecules.
Since direct injection of naked mRNA induces an immune response, we tested the capacity of RNA to signal danger. We show here that mRNA molecules that are protected from immediate degradation either through interaction with cationic proteins (trans protection) or through chemical modification of the phosphodiester backbone (phosphorothioate RNA; cis protection) act as sequence-independent danger signals on mouse DC. As opposed to CpG DNA, the cis-stabilized RNA is degraded in a few minutes, does not activate B cells and, in contrast to double-stranded RNA, requires MyD88 for activation of the DC. We postulate that phosphorothioate RNA, which mimics trans-stabilized RNA, is a new PAMP.
Adaptor Proteins, Signal Transducing↗
Influence of the ionic environment on the in vitro transcription of the spinach plastid DNA by a selectively bound RNA-polymerase DNA complex.
The in vitro transcription of chloroplast DNA (ctDNA) is studied using a DNA-protein complex isolated from spinach plastids. The RNA products are compared to the in vivo synthesized ctRNA by competition for hybridization. At least 80% of the in vitro RNA sequences are present in vivo. Modifications of ionic strength or introduction of heparin in the reaction medium has an important effect on transcriptional activity of the complex. Furthermore, the length of the RNA chains increases ionic strength and amount of heparin. The RNA products are analysed by Southern hybridizations to EcoRI cTDNA fragments. Changes in the ionic strength or in the amount of heparin modify heterogeneously the transcription of the various DNA regions. The quantitative distribution of transcripts among the ctDNA fragments is used as evidence for the selectivity of the transcription. The activity of the DNA-protein complex is much more resistant to high ionic strength than an heterologous transcription system using Escherichia coli RNA polymerase and ctDNA. This latter system transcribes less ctDNA fragments.
Chloroplasts↗
Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins.
Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214-1218; Held et al., 1974, J Biol Chem 249:3103-3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J MoI Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, Sll, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 degrees C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II' (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles reconstituted from the recombinant proteins sediment at 30S in sucrose gradients, bind tRNA in a template-dependent manner, and associate with 50S subunits to form 70S ribosomes that are active in poly(U)-directed polyphenylalanine synthesis. Both the protein composition and the dimethyl sulfate modification pattern of 16S ribosomal RNA are similar for 30S subunits reconstituted with either recombinant proteins or proteins isolated as a mixture from ribosomal subunits as well as for natural 30S subunits.
Cloning, Molecular↗
NMDA receptors are not alone: dynamic regulation of NMDA receptor structure and function by neuregulins and transient cholesterol-rich membrane domains leads to disease-specific nuances of glutamate-signalling.
Glutamate receptors of the N-methyl-D-asparate (NMDA-) subtype are tetrameric allosteric and ligand-gated calcium channels. They are modulated by a variety of endogenous ligands and ions and play a pivotal role in memory-related signal transduction due to a voltage-dependent block by magnesium, which makes them Hebbian coincidence detectors. On the structural level NMDA receptors have an enormous flexibility due to seven genes (NR1, NR2A-D and NR3A-B), alternative splicing, RNA-editing and extensive posttranslational modifications, like phosphorylation and glycosylation. NMDA receptors are thought to be responsible for excitotoxicity and subsequent downstream events like neuroinflammation and apoptosis and thus have been implicated in many important human pathologies, ranging from amyotrophic lateral sclerosis, Alzheimer's and Parkinson' disease, depression, epilepsy, trauma and stroke to schizophrenia. This fundamental significance of NMDA receptor-related excitotoxicity is discussed in the context of the developing clinical success of Memantine, but moreover set into relation to various proteomic and genetic markers of said diseases. The very complex localisational and functional regulation of NMDA receptors appears to be dependent on neuregulins and receptor tyrosine kinases in cholesterol-rich membrane domains (lipid rafts), calcium-related mitochondrial feedback-loops and subsynaptic structural elements like PSD-95 (post-synaptic density protein of 95 kD). The flexibility and multitude of interaction partners and possibilities of these highly dynamic molecular systems are discussed in terms of drug development strategies, in particular comparing high affinity and sub-type specific ligands to currently successful or promising therapies.
Alternative Splicing↗
Verification of differential gene transcription using virtual northern blotting.
We introduce here an alternative to conventional northern blotting that requires only minute amounts of RNA. This has been achieved by modification of methods currently used for the mapping of mRNA 5'-terminal ends. The terminal desoxynucleotidyl transferase-mediated G-tailing, cap finder, ligation-anchored and RNA ligase-mediated approaches followed by polymerase chain reaction protocols all produced high quality cDNAs in large amounts. These cDNAs could be separated by electrophoresis to obtain virtual northern blots that could replace conventional northern blots. All the essential information, including transcript length and the expression pattern, are preserved in these cDNAs, even if the transcripts are long or GC-rich. In addition, minute amounts of material (less than 100 cells) are sufficient to produce more than 100 virtual northern blots, making this approach extremely versatile.
Animals↗