Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides.
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ABSTRACT:
SUBMITTER: Soller KJ
PROVIDER: S-EPMC4543939 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport response to β-adrenergic stimulation, thus modulating cardiac output during the fight-or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), keeping this enzyme's function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLN's cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzyme's active site, augmenting Ca2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally modified bitopic membrane proteins.
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Project description:The membrane protein complex between the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLN) controls Ca(2+) transport in cardiomyocytes, thereby modulating cardiac contractility. ?-Adrenergic-stimulated phosphorylation of PLN at Ser-16 enhances SERCA activity via an unknown mechanism. Using solid-state nuclear magnetic resonance spectroscopy, we mapped the physical interactions between SERCA and both unphosphorylated and phosphorylated PLN in membrane bilayers. We found that the allosteric regulation of SERCA depends on the conformational equilibrium of PLN, whose cytoplasmic regulatory domain interconverts between three different states: a ground T state (helical and membrane associated), an excited R state (unfolded and membrane detached), and a B state (extended and enzyme-bound), which is noninhibitory. Phosphorylation at Ser-16 of PLN shifts the populations toward the B state, increasing SERCA activity. We conclude that PLN's conformational equilibrium is central to maintain SERCA's apparent Ca(2+) affinity within a physiological window. This model represents a paradigm shift in our understanding of SERCA regulation by posttranslational phosphorylation and suggests strategies for designing innovative therapeutic approaches to enhance cardiac muscle contractility.
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Project description:Circularized single-stranded DNA oligonucleotides, or coligos, show promise as promoter-independent RNA polymerase III (Pol III) transcription templates for generating small RNA in human cells. Using a modified small RNA-seq method, we studied the sequence and secondary structure characteristics that determine Pol III initiation and termination on six coligo templates. The coligos each consisted of an imperfectly base-paired stem flanked by one larger and one smaller loop and were unrelated in sequence. Small RNA-seq data from Pol III coligo transcripts revealed a strong preference for initiating transcription within a 5-nucleotide (nt) window spanning the stem-larger loop junction (loop size 11-24 nt). Transcription in all cases proceeded into the stem rather than into the larger loop, indicating the junction is a site-specific, secondary structure-based Pol III transcription initiator. On average, 81% of sequencing reads showed initiation within this 5 nt junction region, with a template start site nucleotide preference of C > T >> A > G, and a requirement for a template purine at Tss-1. Termination was less precise than initiation and occurred in the larger loop at the same end of the stem where transcription initiated. Termination efficiency was on average 82% and was distributed among the first 11 single-stranded larger loop nt following the stem. The size heterogeneity of Pol III coligo transcripts is thus mainly due to 3' end heterogeneity, whereas the RNA 5' ends were more predictable and homogeneous. Transcription termination did not require an oligo dA template sequence, indicating that termination in this context may be mechanistically different than Pol III's normal gene-context termination. A stepwise model for coligo transcription by Pol III is proposed.
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Project description:Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Förster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between release of the oligonucleotides from endosomes, degradation by RNases and subsequent depletion from the cells.
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Project description:Single-stranded DNA (ssDNA) is characterized by high conformational flexibility that allows these molecules to adopt a variety of conformations. Here we used native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy to show that cytosine methylation at CpG sites affects the conformational flexibility of short ssDNA molecules. The CpG containing 37-nucleotide PDYN (prodynorphin) fragments were used as model molecules. The presence of secondary DNA structures was evident from differences in oligonucleotide mobilities on PAGE, from CD spectra, and from formation of A-T, G-C, and non-canonical G-T base pairs observed by NMR spectroscopy. The oligonucleotides displayed secondary structures at 4°C, and some also at 37°C. Methylation at CpG sites prompted sequence-dependent formation of novel conformations, or shifted the equilibrium between different existing ssDNA conformations. The effects of methylation on gel mobility and base pairing were comparable in strength to the effects induced by point mutations in the DNA sequences. The conformational effects of methylation may be relevant for epigenetic regulatory events in a chromatin context, including DNA-protein or DNA-DNA recognition in the course of gene transcription, and DNA replication and recombination when double-stranded DNA is unwinded to ssDNA.
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Project description:Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.
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Project description:The ability to precisely alter the genome holds immense potential for molecular biology, medicine and biotechnology. The development of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) into a genomic editing tool has vastly simplified genome engineering. Here, we explored the use of chemically synthesized chimeric oligonucleotides encoding a target-specific crRNA (CRISPR RNA) fused to a single-stranded DNA repair template for RNP-mediated precision genome editing. By generating three clinically relevant oncogenic driver mutations, two non-stop extension mutations, an FGFRi resistance mutation and a single nucleotide change, we demonstrate the ability of chimeric oligos to form RNPs and direct Cas9 to effectively induce genome editing. Further, we demonstrate that the polarity of the chimeric oligos is crucial: only chimeric oligos with the single-stranded DNA repair template fused to the 3'-end of the crRNA are functional for accurate editing, while templates fused to the 5'-end are ineffective. We also find that chimeras can perform editing with both symmetric and asymmetric single-stranded DNA repair templates. Depending on the target locus, the editing efficiency using chimeric RNPs is similar to or less than the efficiency of editing using the bipartite standard RNPs. Our results indicate that chimeric RNPs comprising RNA-DNA oligos formed from fusing the crRNA and DNA repair templates can successfully induce precise edits. While chimeric RNPs do not display an advantage over standard RNPs, they nonetheless represent a viable approach for one-molecule precision genome editing.
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Project description:BackgroundSingle-stranded nucleic acids (ssNAs) have important biological roles and a high biotechnological potential linked to their ability to bind to numerous molecular targets. This depends on the different spatial conformations they can assume. The first level of ssNAs spatial organisation corresponds to their base pairs pattern, i.e. their secondary structure. Many computational tools have been developed to predict the ssNAs secondary structures, making the choice of the appropriate tool difficult, and an up-to-date guide on the limits and applicability of current secondary structure prediction tools is missing. Therefore, we performed a comparative study of the performances of 9 freely available tools (mfold, RNAfold, CentroidFold, CONTRAfold, MC-Fold, LinearFold, UFold, SPOT-RNA, and MXfold2) on a dataset of 538 ssNAs with known experimental secondary structure.ResultsThe minimum free energy-based tools, namely mfold and RNAfold, and some tools based on artificial intelligence, namely CONTRAfold and MXfold2, provided the best results, with [Formula: see text] of exact predictions, whilst MC-fold seemed to be the worst performing tool, with only [Formula: see text] of exact predictions. In addition, UFold and SPOT-RNA are the only options for pseudoknots prediction. Including in the analysis of mfold and RNAfold results 5-10 suboptimal solutions further improved the performances of these tools. Nevertheless, we could observe issues in predicting particular motifs, such as multiple-ways junctions and mini-dumbbells, or the ssNAs whose structure has been determined in complex with a protein. In addition, our benchmark shows that some effort has to be paid for ssDNA secondary structure predictions.ConclusionsIn general, Mfold, RNAfold, and MXfold2 seem to currently be the best choice for the ssNAs secondary structure prediction, although they still show some limits linked to specific structural motifs. Nevertheless, actual trends suggest that artificial intelligence has a high potential to overcome these remaining issues, for example the recently developed UFold and SPOT-RNA have a high success rate in predicting pseudoknots.
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Project description:Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested.
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