scholarly journals Stem–loop formation drives RNA folding in mechanical unzipping experiments

2022 ◽  
Vol 119 (3) ◽  
pp. e2025575119
Author(s):  
Paolo Rissone ◽  
Cristiano V. Bizarro ◽  
Felix Ritort
Keyword(s):  
Optical Tweezers ◽  
Rna Structure ◽  
Nearest Neighbor ◽  
Rna Folding ◽  
General Mechanism ◽  
Loop Formation ◽  
Stem Loop ◽  
Wide Range ◽  
Irreversible Effects ◽  
And Function

Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force–distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping–rezipping process, we introduce a barrier energy landscape of the stem–loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem–loops that kinetically trap the long-lived intermediates observed in the FDC. Stem–loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.

scholarly journals Accurate Detection of RNA Stem-Loops in Structurome Data Reveals Widespread Association with Protein Binding Sites

2021 ◽  
Author(s):  
Pierce Radecki ◽  
Rahul Uppuluri ◽  
Kaustubh Deshpande ◽  
Sharon Aviran
Keyword(s):  
Rna Structure ◽  
Nearest Neighbor ◽  
Rna Binding ◽  
Learning Algorithm ◽  
Accurate Method ◽  
Stem Loop ◽  
Rna Molecules ◽  
Computational Overhead ◽  
Local Sequence ◽  
Functional Relevance

ABSTRACTRNA molecules are known to fold into specific structures which often play a central role in their functions and regulation. In silico folding of RNA transcripts, especially when assisted with structure profiling (SP) data, is capable of accurately elucidating relevant structural conformations. However, such methods scale poorly to the swaths of SP data generated by transcriptome-wide experiments, which are becoming more commonplace and advancing our understanding of RNA structure and its regulation at global and local levels. This has created a need for tools capable of rapidly deriving structural assessments from SP data in a scalable manner. One such tool we previously introduced that aims to process such data is patteRNA, a statistical learning algorithm capable of rapidly mining big SP datasets for structural elements. Here, we present a reformulation of patteRNA’s pattern recognition scheme that sees significantly improved precision without major compromises to computational overhead. Specifically, we developed a data-driven logistic classifier which interprets patteRNA’s statistical characterizations of SP data in addition to local sequence properties as measured with a nearest neighbor thermodynamic model. Application of the classifier to human structurome data reveals a marked association between detected stem-loops and RNA binding protein (RBP) footprints. The results of our application demonstrate that upwards of 30% of RBP footprints occur within loops of stable stem-loop elements. Overall, our work arrives at a rapid and accurate method for automatically detecting families of RNA structure motifs and demonstrates the functional relevance of identifying them transcriptome-wide.

scholarly journals Self-regulation of single-stranded DNA wrapping dynamics by E. coli SSB promotes both stable binding and rapid dissociation

2019 ◽  
Author(s):  
M. Nabuan Naufer ◽  
Michael Morse ◽  
Guðfríður Björg Möller ◽  
James McIsaac ◽  
Ioulia Rouzina ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Nearest Neighbor ◽  
Self Regulation ◽  
Bound State ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
E Coli ◽  
Wide Range ◽  
Multiple Conformations ◽  
Protein Density

AbstractE. coli SSB (EcSSB) is a model protein for studying functions of single-stranded DNA (ssDNA) binding proteins (SSBs), which are critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations in order to protect these transiently formed regions during processes such as replication and repair. Using optical tweezers, we measure the binding and wrapping of a single long ssDNA substrate under various conditions and free protein concentrations. We show that EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as protein density on the substrate passes a critical saturation. Increasing free EcSSB concentrations increase the fraction of EcSSBs in less-wrapped conformations, including a previously uncharacterized EcSSB8 bound state in which ∼8 nucleotides of ssDNA are bound by a single domain of the tetramer with minimal substrate deformation. When the ssDNA is over-saturated with EcSSB, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped EcSSB-ssDNA complexes. We develop a multi-step kinetic model in which EcSSB tetramers transition through multiple wrapped conformations which are regulated through nearest neighbor interactions and ssDNA occupancy. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers can be rapidly removed from an ssDNA substrate to allow for DNA maintenance and replication functions while still fully protecting ssDNA over a wide range of protein concentrations.

scholarly journals The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA

2019 ◽  
Vol 48 (2) ◽  
pp. 830-846 ◽  
Author(s):  
Rita Pinto ◽  
Cathrine B Vågbø ◽  
Magnus E Jakobsson ◽  
Yeji Kim ◽  
Marijke P Baltissen ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Rna Structure ◽  
Mrna Translation ◽  
28S Rrna ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Reversible Modification ◽  
And Shifts ◽  
And Function

Abstract RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem–loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

scholarly journals Major alteration in coxsackievirus B3 genomic RNA structure distinguishes a virulent strain from an avirulent strain

2014 ◽  
Vol 42 (15) ◽  
pp. 10112-10121 ◽  
Author(s):  
Jerome Prusa ◽  
Johanna Missak ◽  
Jeff Kittrell ◽  
John J. Evans ◽  
William E. Tapprich
Keyword(s):  
Rna Structure ◽  
Virulent Strain ◽  
Structural Difference ◽  
Coxsackievirus B3 ◽  
Comparative Sequence Analysis ◽  
Avirulent Strain ◽  
Stem Loop ◽  
Chemical Probing ◽  
Major Alteration ◽  
And Function

AbstractCoxsackievirus B3 (CV-B3) is a cardiovirulent enterovirus that utilizes a 5′ untranslated region (5′UTR) to complete critical viral processes. Here, we directly compared the structure of a 5′UTR from a virulent strain with that of a naturally occurring avirulent strain. Using chemical probing analysis, we identified a structural difference between the two 5′UTRs in the highly substituted stem-loop II region (SLII). For the remainder of the 5′UTR, we observed conserved structure. Comparative sequence analysis of 170 closely related enteroviruses revealed that the SLII region lacks conservation. To investigate independent folding and function, two chimeric CV-B3 strains were created by exchanging nucleotides 104–184 and repeating the 5′UTR structural analysis. Neither the parent SLII nor the remaining domains of the background 5′UTR were structurally altered by the exchange, supporting an independent mechanism of folding and function. We show that the attenuated 5′UTR lacks structure in the SLII cardiovirulence determinant.

scholarly journals Detailed mapping of RNA secondary structures in core and NS5B-encoding region sequences of hepatitis C virus by RNase cleavage and novel bioinformatic prediction methods

2004 ◽  
Vol 85 (10) ◽  
pp. 3037-3047 ◽  
Author(s):  
A. Tuplin ◽  
D. J. Evans ◽  
P. Simmonds
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Structure ◽  
Rna Folding ◽  
Rna Structures ◽  
Stem Loop ◽  
Hcv Genotypes ◽  
Functional Studies ◽  
The Core ◽  
Synonymous Sites

There is accumulating evidence from bioinformatic studies that hepatitis C virus (HCV) possesses extensive RNA secondary structure in the core and NS5B-encoding regions of the genome. Recent functional studies have defined one such stem–loop structure in the NS5B region as an essential cis-acting replication element (CRE). A program was developed (structur_dist) that analyses multiple rna-folding patterns predicted by mfold to determine the evolutionary conservation of predicted stem–loop structures and, by a new method, to analyse frequencies of covariant sites in predicted RNA folding between HCV genotypes. These novel bioinformatic methods have been combined with enzymic mapping of RNA transcripts from the core and NS5B regions to precisely delineate the RNA structures that are present in these genomic regions. Together, these methods predict the existence of multiple, often juxtaposed stem–loops that are found in all HCV genotypes throughout both regions, as well as several strikingly conserved single-stranded regions, one of which coincides with a region of the genome to which ribosomal access is required for translation initiation. Despite the existence of marked sequence conservation between genotypes in the HCV CRE and single-stranded regions, there was no evidence for comparable suppression of variability at either synonymous or non-synonymous sites in the other predicted stem–loop structures. The configuration and genetic variability of many of these other NS5B and core structures is perhaps more consistent with their involvement in genome-scale ordered RNA structure, a structural configuration of the genomes of many positive-stranded RNA viruses that is associated with host persistence.

scholarly journals The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

2020 ◽  
Author(s):  
Ryota Kurimoto ◽  
Tomoki Chiba ◽  
Yoshiaki Ito ◽  
Takahide Matsushima ◽  
Yuki Yano ◽  
...  
Keyword(s):  
Binding Proteins ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Luciferase Reporter ◽  
Transcriptional Gene Silencing ◽  
Stem Loop ◽  
Pseudouridine Synthase ◽  
Wide Range ◽  
And Function

AbstractLet-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter, and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, HITS-CLIP (High throughput sequencing crosslinking immunoprecipitation) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR-8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation and function.

scholarly journals Bridging the gap betweenin vitroandin vivoRNA folding

2016 ◽  
Vol 49 ◽  
Author(s):  
Kathleen A. Leamy ◽  
Sarah M. Assmann ◽  
David H. Mathews ◽  
Philip C. Bevilacqua
Keyword(s):  
Rna Structure ◽  
Rna Folding ◽  
Three Dimensional ◽  
Molecular Crowding ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Folding Pathways ◽  
And Function

AbstractDeciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functionsin vivo. Within the last few years, methods have been developed to probe RNA structuresin vivoand genome-wide. These studies reveal that RNA often adopts very different structuresin vivoandin vitro, and provide profound insights into RNA biology. Nonetheless, bothin vitroandin vivoapproaches have limitations: studies in the complex and uncontrolled cellular environment make it difficult to obtain insight into RNA folding pathways and thermodynamics, and studiesin vitrooften lack direct cellular relevance, leaving a gap in our knowledge of RNA foldingin vivo. This gap is being bridged by biophysical and mechanistic studies of RNA structure and function under conditions that mimic the cellular environment. To date, most artificial cytoplasms have used various polymers as molecular crowding agents and a series of small molecules as cosolutes. Studies under suchin vivo-likeconditions are yielding fresh insights, such as cooperative folding of functional RNAs and increased activity of ribozymes. These observations are accounted for in part by molecular crowding effects and interactions with other molecules. In this review, we report milestones in RNA foldingin vitroandin vivoand discuss ongoing experimental and computational efforts to bridge the gap between these two conditions in order to understand how RNA folds in the cell.

scholarly journals Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators

2015 ◽  
Vol 112 (16) ◽  
pp. E1984-E1993 ◽  
Author(s):  
Christophe Penno ◽  
Virag Sharma ◽  
Arthur Coakley ◽  
Mary O’Connell Motherway ◽  
Douwe van Sinderen ◽  
...  
Keyword(s):  
Rna Structure ◽  
Ebola Virus ◽  
Data Interpretation ◽  
Loop Formation ◽  
Nascent Transcript ◽  
Stem Loop ◽  
The Stability ◽  
Virus Expression ◽  
Transcriptional Slippage

Escherichia coliand yeast DNA-dependent RNA polymerases are shown to mediate efficient nascent transcript stem loop formation-dependent RNA-DNA hybrid realignment. The realignment was discovered on the heteropolymeric sequence T5C5 and yields transcripts lacking a C residue within a corresponding U5C4. The sequence studied is derived from aRoseiflexusinsertion sequence (IS) element where the resulting transcriptional slippage is required for transposase synthesis. The stability of the RNA structure, the proximity of the stem loop to the slippage site, the length and composition of the slippage site motif, and the identity of its 3′ adjacent nucleotides (nt) are crucial for transcripts lacking a single C. In many respects, the RNA structure requirements for this slippage resemble those for hairpin-dependent transcription termination. In a purified in vitro system, the slippage efficiency ranges from 5% to 75% depending on the concentration ratios of the nucleotides specified by the slippage sequence and the 3′ nt context. The only previous proposal of stem loop mediated slippage, which was in Ebola virus expression, was based on incorrect data interpretation. We propose a mechanical slippage model involving the RNAP translocation state as the main motor in slippage directionality and efficiency. It is distinct from previously described models, including the one proposed for paramyxovirus, where following random movement efficiency is mainly dependent on the stability of the new realigned hybrid. In broadening the scope for utilization of transcription slippage for gene expression, the stimulatory structure provides parallels with programmed ribosomal frameshifting at the translation level.

scholarly journals The Emerging Impact of RNA Editing in Tumor Growth and Metastasis

2021 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Mehrdad Nasrollahzadehsabet ◽  
Javad Behroozi
Keyword(s):  
Tumor Growth ◽  
Rna Editing ◽  
General Mechanism ◽  
Metastatic Progression ◽  
Target Mrna ◽  
Cancer Pathogenesis ◽  
Wide Range ◽  
Cancer Initiation ◽  
And Function

Context: RNA editing is an essential modification that needs to develop normal cells and is involved in a wide range of biological processes. It can arise in both coding and non-coding sequences with different functional effects. Although the expansion of transcriptome diversity is the primary goal of RNA editing, dysregulation and aberrant editing may act as an essential contributor to cancer pathogenesis. Evidence Acquisition: The current review aimed to investigate the role of RNA editing in cancer initiation and progression. Science Direct and PubMed databases were reviewed from 2000 to 2020 and 2003 to 2020, respectively, using various combinations of "RNA editing" and "cancer" keywords. Results: The location of editing sites has different functional impacts on tumorigenesis. Nonsynonymous editing in antizyme inhibitor 1 (AZIN1) leads to a metastatic progression of colorectal and gastric cancer. Recoding editing events in bladder cancer-associated protein (BLCAP) is correlated with the progression of cervical carcinogenesis. Editing events located at 3′UTRs are also a general mechanism to promote tumor growth in different types of cancers. A significant number of editing events in microRNAs with a functional role in cancer are also reported. These editing sites could change the fate and function of microRNAs, either by preventing target mRNA recognition or by dysregulating an off-target mRNA. Conclusions: There are increasing shreds of evidence on the key role of RNA editing events in cancer initiation and progression.

scholarly journals Reverse Transcription of a Self-Primed Retrotransposon Requires an RNA Structure Similar to the U5-IR Stem-Loop of Retroviruses

1998 ◽  
Vol 18 (11) ◽  
pp. 6859-6869 ◽  
Author(s):  
Jia-Hwei Lin ◽  
Henry L. Levin
Keyword(s):  
Reverse Transcription ◽  
Rna Structure ◽  
Unique Feature ◽  
Gc Content ◽  
Specific Sequence ◽  
Stem Loop ◽  
Rous Sarcoma ◽  
Large Loop ◽  
Sarcoma Virus ◽  
And Function

ABSTRACT An inverted repeat (IR) within the U5 region of the Rous sarcoma virus (RSV) mRNA forms a structure composed of a 7-bp stem and a 5-nucleotide (nt) loop. This U5-IR structure has been shown to be required for the initiation of reverse transcription. The mRNA of Tf1, long terminal repeat-containing retrotransposon from fission yeast (Schizosaccharomyces pombe) contains nucleotides with the potential to form a U5-IR stem-loop that is strikingly similar to that of RSV. The putative U5-IR stem-loop of Tf1 consists of a 7-bp stem and a 25-nt loop. Results from mutagenesis studies indicate that the U5-IR stem-loop in the mRNA of Tf1 does form and that it is required for Tf1 transposition. Although the loop is required for transposition, we were surprised that the specific sequence of the nucleotides within the loop was unimportant for function. Additional investigation indicates that the loss of transposition activity due to a reduction in the loop size to 6 nt could be rescued by increasing the GC content of the stem. This result indicates that the large loop in the Tf1 mRNA relative to that of the RSV allows the formation of the relatively weak U5-IR stem. The levels of Tf1 proteins expressed and the amounts of Tf1 RNA packaged into the virus-like particles were not affected by mutations in the U5-IR structure. However, all of the mutations in the U5-IR structure that caused defects in transposition produced low amounts of reverse transcripts. A unique feature in the initiation of Tf1 reverse transcription is that, instead of a tRNA, the first 11 nt of the Tf1 mRNA serve as the minus-strand primer. Analysis of the 5′ end of Tf1 mRNA revealed that the mutations in the U5-IR stem-loop that resulted in defects in reverse transcription caused a reduction in the cleavage activity required to generate the Tf1 primer. Our results indicate that the U5-IR stems of Tf1 and RSV are conserved in size, position, and function.

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