12 research outputs found
Effect of Loops and G‑Quartets on the Stability of RNA G‑Quadruplexes
The
loop length, loop composition, salt concentration, and number of G-quartets
are major determinants of G-quadruplex stability. We examined the
effect of each of these factors on the thermal stability and folding
topology of a library of RNA quadruplexes. The thermal stability of
G2 and G3 RNA quadruplexes was investigated upon varying the loop
length (from 1-1-1 to 15-15-15) and salt concentration (from 1 to
100 mM KCl), while the effect of loop composition was explored using
18 naturally occurring potential RNA quadruplexes predicted in untranslated
regions (UTRs). We found loop length and quadruplex stability to be
inversely related for G2 RNA quadruplexes and G3 RNA quadruplexes
with shorter loops. However, melting temperature saturates for G3
RNA quadruplexes with longer loops. RNA G-quadruplexes with longer
loops (G3 15-15-15) displayed <i>T</i><sub>m</sub> values
significantly higher than the physiological temperature. This study
thus highlights the need to modify the consensus motif presently used
by quadruplex prediction tools. An increase in the loop size from
7 bases to 15 bases in the consensus motif will add to its predictive
value for the discovery of potential RNA quadruplexes across transcriptomes
Human Telomeric RNA G‑Quadruplex Response to Point Mutation in the G‑Quartets
Many
putative G-quadruplex forming sequences have been predicted
to exist in the human genome and transcriptome. As these sequences
are subject to point mutations or SNPs (single nucleotide polymorphisms)
during the course of evolution, we attempt to understand impact of
these mutations in context of RNA G-quadruplex formation using human
telomeric RNA (TERRA) as a model sequence. Our studies suggest that
G-quadruplex stability is sensitive to substitution of the guanines
comprising G-quartets. While central G-quartet plays a crucial role
in maintaining the DNA G-quadruplex stability as evident in literature,
there is equal importance of three G-quartets in the stability of
RNA quadruplex structure. The work here highlights the alterations
in the G-quartet are detrimental to the integrity of overall RNA G-quadruplex
structure. Furthermore, TmPyP4 molecules are shown to exhibit similar
binding behavior toward telomeric RNA G-quadruplex harboring base
substitutions employing CD titrations and isothermal titration calorimetry;
well indicating that mutation does not influence TmPyP4 recognition
ability as it affects the stability of RNA G-quadruplex. Thus, our
study implicates that mutation in G-quartets causes destabilization
of RNA G-quadruplex without affecting its trans factor binding ability
The G‑Quadruplex Augments Translation in the 5′ Untranslated Region of Transforming Growth Factor β2
Transforming growth factor β2
(TGFβ2) is a versatile cytokine with a prominent role in cell
migration, invasion, cellular development, and immunomodulation. TGFβ2
promotes the malignancy of tumors by inducing epithelial–mesenchymal
transition, angiogenesis, and immunosuppression. As it is well-documented
that nucleic acid secondary structure can regulate gene expression,
we assessed whether any secondary motif regulates its expression at
the post-transcriptional level. Bioinformatics analysis predicts an
existence of a 23-nucleotide putative G-quadruplex sequence (PG4)
in the 5′ untranslated region (UTR) of TGFβ2 mRNA. The
ability of this stretch of sequence to form a highly stable, intramolecular
parallel quadruplex was demonstrated using ultraviolet and circular
dichroism spectroscopy. Footprinting studies further validated its
existence in the presence of a neighboring nucleotide sequence. Following
structural characterization, we evaluated the biological relevance
of this secondary motif using a dual luciferase assay. Although PG4
inhibits the expression of the reporter gene, its presence in the
context of the entire 5′ UTR sequence interestingly enhances
gene expression. Mutation or removal of the G-quadruplex sequence
from the 5′ UTR of the gene diminished the level of expression
of this gene at the translational level. Thus, here we highlight an
activating role of the G-quadruplex in modulating gene expression
of TGFβ2 at the translational level and its potential to be
used as a target for the development of therapeutics against cancer
The RNA Stem–Loop to G‑Quadruplex Equilibrium Controls Mature MicroRNA Production inside the Cell
The
biological role of the existence of overlapping structures
in RNA is possible yet remains very unexplored. G-Rich tracts of RNA
form G-quadruplexes, while GC-rich sequences prefer stem–loop
structures. The equilibrium between alternate structures within RNA
may occur and influence its functionality. We tested the equilibrium
between G-quadruplex and stem–loop structure in RNA and its
effect on biological processes using pre-miRNA as a model system.
Dicer enzyme recognizes canonical stem–loop structures in pre-miRNA
to produce mature miRNAs. Deviation from stem–loop leads to
deregulated mature miRNA levels, providing readout of the existence
of an alternate structure per se G-quadruplex-mediated structural
interference in miRNA maturation. <i>In vitro</i> analysis
using beacon and Dicer cleavage assays indicated that mature miRNA
levels depend on relative amounts of K<sup>+</sup> and Mg<sup>2+</sup> ions, suggesting an ion-dependent structural shift. Further <i>in cellulo</i> studies with and without TmPyP<sub>4</sub> (RNA
G-quadruplex destabilizer) demonstrated that miRNA biogenesis is modulated
by G-quadruplex to stem–loop equilibrium in a subset of pre-miRNAs.
Our combined analysis thus provides evidence of the formation of noncanonical
G-quadruplexes in competition with canonical stem–loop structure
inside the cell and its effect on miRNA maturation in a comprehensive
manner
Optical bias with optical wedges in point sampling
Supplementary Tables and Figures. (PDF 454Ă‚Â kb
Additional file 3: of RNA secondary structure profiling in zebrafish reveals unique regulatory features
Multi-conformation position counts in transcripts with overlapping peaks. (TXT 712Ă‚Â kb
Additional file 4: of RNA secondary structure profiling in zebrafish reveals unique regulatory features
Number of read starts for every position covered in RNase V1 sample. (TXT 32051Ă‚Â kb
Additional file 9: of RNA secondary structure profiling in zebrafish reveals unique regulatory features
PARS scores of 54,083 transcripts and non-coding RNAs. (TXT 35418Ă‚Â kb
Additional file 5: of RNA secondary structure profiling in zebrafish reveals unique regulatory features
Number of read starts for every position covered in S1 nuclease sample. (TXT 45980Ă‚Â kb
Additional file 2: of RNA secondary structure profiling in zebrafish reveals unique regulatory features
Load score and percentage coverage of 54,083 transcripts. (TXT 3635Ă‚Â kb