scholarly journals Molecular determinants of metazoan tricRNA biogenesis

2018 ◽  
Author(s):  
Casey A. Schmidt ◽  
Joseph D. Giusto ◽  
A. Gregory Matera
Keyword(s):  
Base Pair ◽  
Circular Rnas ◽  
Trna Genes ◽  
Weak Base ◽  
Base Pairs ◽  
New Class ◽  
Molecular Determinants ◽  
Intron Removal ◽  
Processing Steps ◽  
Processing Factors

ABSTRACTMature tRNAs are generated by multiple post-transcriptional processing steps, which can include intron removal. Recently, our laboratory discovered a new class of metazoan circular RNAs formed by ligation of excised tRNA introns; we termed these molecules tRNA intronic circular (tric)RNAs. To investigate the mechanism of tricRNA biogenesis, we generated constructs that replace the native introns of two Drosophila tRNA genes with the Broccoli fluorescent RNA aptamer. Using these reporters, we identified cis-acting elements required for tricRNA formation in both human and fly cells. We observed that disrupting the conserved anticodon-intron base pair dramatically reduces tricRNA levels. Although the integrity of this base pair is necessary for proper splicing, it is not sufficient. Furthermore, we found that strengthening weak base pairs in the pre-tRNA also impairs tricRNA production. We also used the reporters to identify trans-acting tricRNA processing factors. We found that several known tRNA processing factors, such as RtcB ligase and components of the TSEN endonuclease complex, are involved in tricRNA biogenesis. Depletion of these factors inhibits tRNA intron circularization. Furthermore, we observed that depletion of Clipper endonuclease results in increased tricRNA levels. In summary, our work characterizes the major players in Drosophila tricRNA biogenesis.

scholarly journals Molecular determinants of metazoan tricRNA biogenesis

2019 ◽  
Vol 47 (12) ◽  
pp. 6452-6465 ◽  
Author(s):  
Casey A Schmidt ◽  
Joseph D Giusto ◽  
Alicia Bao ◽  
Anita K Hopper ◽  
A Gregory Matera
Keyword(s):  
Base Pair ◽  
Fruit Fly ◽  
Trna Genes ◽  
Cis Acting ◽  
Processing Enzymes ◽  
New Class ◽  
Weak Bases ◽  
Molecular Determinants ◽  
Intron Removal

Abstract Mature tRNAs are generated by multiple post-transcriptional processing steps, which can include intron removal. Recently, we discovered a new class of circular non-coding RNAs in metazoans, called tRNA intronic circular (tric)RNAs. To investigate the mechanism of tricRNA biogenesis, we generated constructs that replace native introns of human and fruit fly tRNA genes with the Broccoli fluorescent RNA aptamer. Using these reporters, we identified cis-acting elements required for tricRNA formation in vivo. Disrupting a conserved base pair in the anticodon-intron helix dramatically reduces tricRNA levels. Although the integrity of this base pair is necessary for proper splicing, it is not sufficient. In contrast, strengthening weak bases in the helix also interferes with splicing and tricRNA production. Furthermore, we identified trans-acting factors important for tricRNA biogenesis, including several known tRNA processing enzymes such as the RtcB ligase and components of the TSEN endonuclease complex. Depletion of these factors inhibits Drosophila tRNA intron circularization. Notably, RtcB is missing from fungal genomes and these organisms normally produce linear tRNA introns. Here, we show that in the presence of ectopic RtcB, yeast lacking the tRNA ligase Rlg1/Trl1 are converted into producing tricRNAs. In summary, our work characterizes the major players in eukaryotic tricRNA biogenesis.

scholarly journals Identification of weak non-canonical base pairs around riboswitch-ligand recognition sites by solid-state NMR exchange spectroscopy

2021 ◽  
Author(s):  
Sha Zhao ◽  
Ziyang Wen ◽  
Xinming Li ◽  
Mengbing zou ◽  
Ge Yu ◽  
...  
Keyword(s):  
Solid State ◽  
Base Pair ◽  
Solid State Nmr ◽  
Chemical Exchange ◽  
Critical Role ◽  
Building Blocks ◽  
Weak Base ◽  
Base Pairs ◽  
Canonical Base ◽  
Exchange Spectroscopy

Abstract Base pairs are fundamental building blocks of RNA structures, and their stability and open-close equilibrium constitutes the dynamic picture. Weak base pairs, which feature the characteristics of low stability and rapid base pair opening, often play a critical role in RNA functions. However, site-specific identification of weak base pairs in RNA is challenging. Here, we report a solid-state NMR (SSNMR)-based two-dimensional proton-detected water–RNA exchange spectroscopy (WaterREXSY) to address this challenge. The approach uses the chemical exchange between hydrogen-bonded imino protons within the base pair and excited water molecules to polarize the imino protons for SSNMR observation. This process takes advantages that the imino protons within weak pairs undergo fast exchange rates with water, enabling a quick build-up and efficient detection. This method is used to characterize the weak pair in the riboA71–adenine complex (i.e., the 71nt-aptamer domain of the add adenine riboswitch from Vibrio vulnificus). We identify U47•U51, a weak non-canonical base pair that constitutes the U47•U51•(adenine-U74) base tetrad around the ligand-binding pocket. This result suggests that the breakage of U47•U51 may be the early stage in the process of ligand release.

Detection of the Glanzmann's Thrombasthenia Mutations in Arab and Iraqi-Jewish Patients by Polymerase Chain Reaction and Restriction Analysis of Blood or Urine Samples

1991 ◽  
Vol 66 (04) ◽  
pp. 500-504 ◽  
Author(s):  
H Peretz ◽  
U Seligsohn ◽  
E Zwang ◽  
B S Coller ◽  
P J Newman
Keyword(s):  
Polymerase Chain Reaction ◽  
Base Pair ◽  
Restriction Analysis ◽  
Urine Samples ◽  
Chain Reaction ◽  
Base Pairs ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Base Pair Deletion ◽  
Polymerase Chain

SummarySevere Glanzmann's thrombasthenia is relatively frequent in Iraqi-Jews and Arabs residing in Israel. We have recently described the mutations responsible for the disease in Iraqi-Jews – an 11 base pair deletion in exon 12 of the glycoprotein IIIa gene, and in Arabs – a 13 base pair deletion at the AG acceptor splice site of exon 4 on the glycoprotein IIb gene. In this communication we show that the Iraqi-Jewish mutation can be identified directly by polymerase chain reaction and gel electrophoresis. With specially designed oligonucleotide primers encompassing the mutation site, an 80 base pair segment amplified in healthy controls was clearly distinguished from the 69 base pair segment produced in patients. Patients from 11 unrelated Iraqi-Jewish families had the same mutation. The Arab mutation was identified by first amplifying a DNA segment consisting of 312 base pairs in controls and of 299 base pairs in patients, and then digestion by a restriction enzyme Stu-1, which recognizes a site that is absent in the mutant gene. In controls the 312 bp segment was digested into 235 and 77 bp fragments, while in patients there was no change in the size of the amplified 299 bp segment. The mutation was found in patients from 3 out of 5 unrelated Arab families. Both Iraqi-Jewish and Arab mutations were detectable in DNA extracted from blood and urine samples. The described simple methods of identifying the mutations should be useful for detection of the numerous potential carriers among the affected kindreds and for prenatal diagnosis using DNA extracted from chorionic villi samples.

scholarly journals Comprehensive landscape and future perspectives of circular RNAs in colorectal cancer

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Fei Long ◽  
Zhi Lin ◽  
Liang Li ◽  
Min Ma ◽  
Zhixing Lu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Circular Rnas ◽  
Future Perspectives ◽  
Cis Acting ◽  
New Class ◽  
Functional Peptides ◽  
Mirna Sponges

AbstractColorectal cancer (CRC) is a common hereditary tumor that is often fatal. Its pathogenesis involves multiple genes, including circular RNAs (circRNAs). Notably, circRNAs constitute a new class of noncoding RNAs (ncRNAs) with a covalently closed loop structure and have been characterized as stable, conserved molecules that are abundantly expressed in tissue/development-specific patterns in eukaryotes. Based on accumulating evidence, circRNAs are aberrantly expressed in CRC tissues, cells, exosomes, and blood from patients with CRC. Moreover, numerous circRNAs have been identified as either oncogenes or tumor suppressors that mediate tumorigenesis, metastasis and chemoradiation resistance in CRC. Although the regulatory mechanisms of circRNA biogenesis and functions remain fairly elusive, interesting results have been obtained in studies investigating CRC. In particular, the expression of circRNAs in CRC is comprehensively modulated by multiple factors, such as splicing factors, transcription factors, specific enzymes and cis-acting elements. More importantly, circRNAs exert pivotal effects on CRC through various mechanisms, including acting as miRNA sponges or decoys, interacting with RNA binding proteins, and even translating functional peptides. Finally, circRNAs may serve as promising diagnostic and prognostic biomarkers and potential therapeutic targets in the clinical practice of CRC. In this review, we discuss the dysregulation, functions and clinical significance of circRNAs in CRC and further discuss the molecular mechanisms by which circRNAs exert their functions and how their expression is regulated. Based on this review, we hope to reveal the functions of circRNAs in the initiation and progression of cancer and highlight the future perspectives on strategies targeting circRNAs in cancer research.

Repair of heteroduplex plasmid DNA after transformation into Saccharomyces cerevisiae

1986 ◽  
Vol 6 (10) ◽  
pp. 3401-3409
Author(s):  
D K Bishop ◽  
R D Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasmid Dna ◽  
Base Pair ◽  
Base Pairs ◽  
Plasmid Dnas ◽  
Repair Efficiency ◽  
Single Insertion ◽  
Base Pair Insertion

Purified heteroduplex plasmid DNAs containing 8- or 12-base-pair insertion mismatches or AC or CT substitution mismatches were used to transform Saccharomyces cerevisiae. Two insertion mismatches, separated by 943 base pairs, were repaired independently of each other at least 55% of the time. This suggested that repair tracts were frequently shorter than 1 kilobase. The two insertion mismatches were repaired with different efficiencies. Comparison of the repair efficiency of one mismatched site with or without an adjacent mismatch suggests that mismatches promote their own repair and can influence the repair of neighboring mismatches. When two different plasmids containing single-insertion mismatches were transformed into S. cerevisiae cells, a slight preference towards insertion was detected among repair products of one of the two plasmids, while no repair preference was detected among transformants with the second plasmid.

scholarly journals Spo0A-Dependent Activation of an Extended −10 Region Promoter in Bacillus subtilis

2006 ◽  
Vol 188 (4) ◽  
pp. 1411-1418 ◽  
Author(s):  
Guangnan Chen ◽  
Amrita Kumar ◽  
Travis H. Wyman ◽  
Charles P. Moran
Keyword(s):  
Bacillus Subtilis ◽  
Base Pair ◽  
Promoter Activity ◽  
Mutational Analysis ◽  
Dna Binding Protein ◽  
Base Pairs ◽  
Dnase I Footprinting ◽  
Level Of Activity ◽  
High Level ◽  
Activity Dependent

ABSTRACT At the onset of endospore formation in Bacillus subtilis the DNA-binding protein Spo0A directly activates transcription from promoters of about 40 genes. One of these promoters, Pskf, controls expression of an operon encoding a killing factor that acts on sibling cells. AbrB-mediated repression of Pskf provides one level of security ensuring that this promoter is not activated prematurely. However, Spo0A also appears to activate the promoter directly, since Spo0A is required for Pskf activity in a ΔabrB strain. Here we investigate the mechanism of Pskf activation. DNase I footprinting was used to determine the locations at which Spo0A bound to the promoter, and mutations in these sites were found to significantly reduce promoter activity. The sequence near the −10 region of the promoter was found to be similar to those of extended −10 region promoters, which contain a TRTGn motif. Mutational analysis showed that this extended −10 region, as well as other base pairs in the −10 region, is required for Spo0A-dependent activation of the promoter. We found that a substitution of the consensus base pair for the nonconsensus base pair at position −9 of Pskf produced a promoter that was active constitutively in both ΔabrB and Δspo0A ΔabrB strains. Therefore, the base pair at position −9 of Pskf makes its activity dependent on Spo0A binding, and the extended −10 region motif of the promoter contributes to its high level of activity.

Three-centre C—H...O hydrogen bonds in the DNA minor groove: analysis of oligonucleotide crystal structures

1999 ◽  
Vol 55 (12) ◽  
pp. 2005-2012 ◽  
Author(s):  
Anirban Ghosh ◽  
Manju Bansal
Keyword(s):  
Hydrogen Bonds ◽  
Crystal Structures ◽  
Base Pair ◽  
Minor Groove ◽  
Local Geometry ◽  
Data Sets ◽  
Base Pairs ◽  
Dna Minor Groove ◽  
Close Proximity ◽  
Using Data

AA·TT and GA·TC dinucleotide steps in B-DNA-type oligomeric crystal structures and in protein-bound DNA fragments (solved using data with resolution <2.6 Å) show very small variations in their local dinucleotide geometries. A detailed analysis of these crystal structures reveals that in AA·TT and GA·TC steps the electropositive C2—H2 group of adenine is in very close proximity to the keto O atoms of both the pyrimidine bases in the antiparallel strand of the duplex structure, suggesting the possibility of intra-base pair as well as cross-strand inter-base pair C—H...O hydrogen bonds in the DNA minor groove. The C2—H2...O2 hydrogen bonds in the A·T base pairs could be a natural consequence of Watson–Crick pairing. However, the cross-strand interactions between the bases at the 3′-end of the AA·TT and GA·TC steps obviously arise owing to specific local geometry of these steps, since a majority of the H2...O2 distances in both data sets are considerably shorter than their values in the uniform fibre model (3.3 Å) and many are even smaller than the sum of the van der Waals radii. The analysis suggests that in addition to already documented features such as the large propeller twist of A·T base pairs and the hydration of the minor groove, these C2—H2...O2 cross-strand interactions may also play a role in the narrowing of the minor groove in A-tract regions of DNA and help explain the high structural rigidity and stability observed for poly(dA)·poly(dT).

scholarly journals Theoretical modelling of epigenetically modified DNA sequences

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 52 ◽  
Author(s):  
Alexandra Teresa Pires Carvalho ◽  
Maria Leonor Gouveia ◽  
Charan Raju Kanna ◽  
Sebastian K. T. S. Wärmländer ◽  
Jamie Platts ◽  
...  
Keyword(s):  
Molecular Mechanics ◽  
Dna Sequences ◽  
Base Pair ◽  
Density Functional ◽  
Epigenetic Modifications ◽  
Theoretical Modelling ◽  
Sugar Phosphate ◽  
Base Pairs ◽  
Phosphate Backbone ◽  
Modified Dna

We report herein a set of calculations designed to examine the effects of epigenetic modifications on the structure of DNA. The incorporation of methyl, hydroxymethyl, formyl and carboxy substituents at the 5-position of cytosine is shown to hardly affect the geometry of CG base pairs, but to result in rather larger changes to hydrogen-bond and stacking binding energies, as predicted by dispersion-corrected density functional theory (DFT) methods. The same modifications within double-stranded GCG and ACA trimers exhibit rather larger structural effects, when including the sugar-phosphate backbone as well as sodium counterions and implicit aqueous solvation. In particular, changes are observed in the buckle and propeller angles within base pairs and the slide and roll values of base pair steps, but these leave the overall helical shape of DNA essentially intact. The structures so obtained are useful as a benchmark of faster methods, including molecular mechanics (MM) and hybrid quantum mechanics/molecular mechanics (QM/MM) methods. We show that previously developed MM parameters satisfactorily reproduce the trimer structures, as do QM/MM calculations which treat bases with dispersion-corrected DFT and the sugar-phosphate backbone with AMBER. The latter are improved by inclusion of all six bases in the QM region, since a truncated model including only the central CG base pair in the QM region is considerably further from the DFT structure. This QM/MM method is then applied to a set of double-stranded DNA heptamers derived from a recent X-ray crystallographic study, whose size puts a DFT study beyond our current computational resources. These data show that still larger structural changes are observed than in base pairs or trimers, leading us to conclude that it is important to model epigenetic modifications within realistic molecular contexts.

scholarly journals Supercoiling DNA locates mismatches

2017 ◽  
Author(s):  
Andrew Dittmore ◽  
Sumitabha Brahmachari ◽  
Yasuhara Takagi ◽  
John F. Marko ◽  
Keir C. Neuman
Keyword(s):  
Dna Sequence ◽  
Base Pair ◽  
Dna Supercoiling ◽  
Magnetic Tweezers ◽  
Relative Probability ◽  
Base Pairs ◽  
Damage Sensing ◽  
Mismatched Base Pair ◽  
Monovalent Salt

We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.5 pN force and show that, under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.

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