scholarly journals Quantitative prediction of variant effects on alternative splicing using endogenous pre-messenger RNA structure probing

2021 ◽  
Author(s):  
Jayashree Kumar ◽  
Lela Lackey ◽  
Justin M. Waldern ◽  
Abhishek Dey ◽  
David H. Mathews ◽  
...  
Keyword(s):  
Rna Structure ◽  
Messenger Rna ◽  
Regulatory Elements ◽  
Quantitative Prediction ◽  
Mrna Structure ◽  
Splicing Regulatory Elements ◽  
Alternatively Spliced ◽  
Endogenous Precursor ◽  
Precursor Mrna

AbstractSplicing is a highly regulated process that depends on numerous factors. It is particularly challenging to quantitatively predict how a mutation will affect precursor messenger RNA (mRNA) structure and the subsequent functional consequences. Here we use a novel Mutational Profiling (-MaP) methodology to obtain highly reproducible endogenous precursor and mature mRNA structural probing data in vivo. We use these data to estimate Boltzmann suboptimal ensembles, and predict the structural consequences of mutations on precursor mRNA structure. Together with a structural analysis of recent cryo-EM spliceosome structures at different stages of the splicing cycle, we determined that the footprint of the Bact complex on precursor mRNA is best able to predict splicing outcomes for exon 10 inclusion of the alternatively spliced MAPT gene. However, structure alone only achieves 74% accuracy. We therefore developed a β-regression weighting framework that incorporates splice site strength, structure and exonic/intronic splicing regulatory elements which together achieves 90% accuracy for 47 known and six newly discovered splice-altering variants. This combined experimental/computational framework represents a path forward for accurate prediction of splicing related disease-causing variants.

scholarly journals mRNA structure dynamics identifies the embryonic RNA regulome

2018 ◽  
Author(s):  
Jean-Denis Beaudoin ◽  
Eva Maria Novoa ◽  
Charles E Vejnar ◽  
Valeria Yartseva ◽  
Carter Takacs ◽  
...  
Keyword(s):  
Rna Structure ◽  
Regulatory Elements ◽  
Structure Dynamics ◽  
Mrna Structure ◽  
Maternal To Zygotic Transition ◽  
Gene Regulatory ◽  
Folded Structures ◽  
Affect Gene Expression

RNA folding plays a crucial role in RNA function. However, our knowledge of the global structure of the transcriptome is limited to steady-state conditions, hindering our understanding of how RNA structure dynamics influences gene function. Here, we have characterized mRNA structure dynamics during zebrafish development. We observe that on a global level, translation guides structure rather than structure guiding translation. We detect a decrease in structure in translated regions, and we identify the ribosome as a major remodeler of RNA structure in vivo. In contrast, we find that 3’-UTRs form highly folded structures in vivo, which can affect gene expression by modulating miRNA activity. Furthermore, we find that dynamic 3’-UTR structures encode RNA decay elements, including regulatory elements in nanog and cyclin A1, key maternal factors orchestrating the maternal-to-zygotic transition. These results reveal a central role of RNA structure dynamics in gene regulatory programs.

scholarly journals Gymnotic Delivery of LNA Mixmers Targeting Viral SREs Induces HIV-1 mRNA Degradation

2019 ◽  
Vol 20 (5) ◽  
pp. 1088 ◽  
Author(s):  
Frank Hillebrand ◽  
Philipp Ostermann ◽  
Lisa Müller ◽  
Daniel Degrandi ◽  
Steffen Erkelenz ◽  
...  
Keyword(s):  
Particle Production ◽  
Regulatory Elements ◽  
Rna Degradation ◽  
Locked Nucleic Acid ◽  
Target Sequence ◽  
Viral Particle Production ◽  
Splicing Regulatory Elements ◽  
Alternatively Spliced ◽  
Infected Cells ◽  
Hiv 1

Transcription of the HIV-1 provirus generates a viral pre-mRNA, which is alternatively spliced into more than 50 HIV-1 mRNAs encoding all viral proteins. Regulation of viral alternative splice site usage includes the presence of splicing regulatory elements (SREs) which can dramatically impact RNA expression and HIV-1 replication when mutated. Recently, we were able to show that two viral SREs, GI3-2 and ESEtat, are important players in the generation of viral vif, vpr and tat mRNAs. Furthermore, we demonstrated that masking these SREs by transfected locked nucleic acid (LNA) mixmers affect the viral splicing pattern and viral particle production. With regard to the development of future therapeutic LNA mixmer-based antiretroviral approaches, we delivered the GI3-2 and the ESEtat LNA mixmers “nakedly”, without the use of transfection reagents (gymnosis) into HIV-1 infected cells. Surprisingly, we observed that gymnotically-delivered LNA mixmers accumulated in the cytoplasm, and seemed to co-localize with GW bodies and induced degradation of mRNAs containing their LNA target sequence. The GI3-2 and the ESEtat LNA-mediated RNA degradation resulted in abrogation of viral replication in HIV-1 infected Jurkat and PM1 cells as well as in PBMCs.

scholarly journals Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavage in vivo

2019 ◽  
Author(s):  
Minglei Yang ◽  
Hugh C. Woolfenden ◽  
Yueying Zhang ◽  
Xiaofeng Fang ◽  
Qi Liu ◽  
...  
Keyword(s):  
Rna Structure ◽  
Target Site ◽  
Site Structure ◽  
Mrna Structure ◽  
Sequence Complementarity ◽  
Primary Determinant ◽  
Site Binding

ABSTRACTMicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-Induced Silencing Complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. Here, we investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, the unfolding of the target site structure is the primary determinant for miRISC activity in vivo. Notably, we found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent structure Motif (TAM), can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can regulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.

scholarly journals RNA base-pairing complexity in living cells visualized by correlated chemical probing

2019 ◽  
Vol 116 (49) ◽  
pp. 24574-24582 ◽  
Author(s):  
Anthony M. Mustoe ◽  
Nicole N. Lama ◽  
Patrick S. Irving ◽  
Samuel W. Olson ◽  
Kevin M. Weeks
Keyword(s):  
Single Molecule ◽  
Rna Structure ◽  
Messenger Rna ◽  
Dimethyl Sulfate ◽  
Detection Methods ◽  
Base Pairing ◽  
Chemical Probing ◽  
Pairing Interactions ◽  
In Cells

RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having deficiencies. Here we convert the common reagent dimethyl sulfate into a useful probe of all 4 RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base-pairing interactions in cells. PAIR-MaP has superior resolution compared to alternative experiments, can resolve multiple sets of pairing interactions for structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and 2 bacterial messenger RNA 5′ untranslated regions reveals functionally important and complex structures undetected by prior analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells.

scholarly journals Annotating RNA motifs in sequences and alignments

2014 ◽  
Author(s):  
Paul P Gardner ◽  
Hisham Eldai
Keyword(s):  
Rna Structure ◽  
Messenger Rna ◽  
Building Blocks ◽  
Regulatory Elements ◽  
Rna Motifs ◽  
Regulate Gene Expression ◽  
Translational Machinery ◽  
Cellular Life ◽  
Functional Characterisation ◽  
Initial Discovery

RNA performs a diverse array of important functions across all cellular life. These functions include important roles in translation, building translational machinery and maturing messenger RNA. More recent discoveries include the miRNAs and bacterial sRNAs that regulate gene expression, the thermosensors, riboswitches and other cis-regulatory elements that help prokaryotes sense their environment and eukaryotic piRNAs that suppress transposition. However, there can be a long period between the initial discovery of a RNA and determining its function. We present a bioinformatic approach to characterise RNA motifs, which are the central building blocks of RNA structure. These motifs can, in some instances, provide researchers with functional hypotheses for uncharacterised RNAs. Moreover, we introduce a new profile-based database of RNA motifs - RMfam - and illustrate its application for investigating the evolution and functional characterisation of RNA. All the data and scripts associated with this work is available from: https://github.com/ppgardne/RMfam

scholarly journals Crystal structure of an atypical cobalamin riboswitch reveals RNA structural adaptability as basis for promiscuous ligand binding

2020 ◽  
Author(s):  
Clarence W Chan ◽  
Alfonso Mondragón
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Translational Control ◽  
Rna Structure ◽  
Messenger Rna ◽  
Regulatory Elements ◽  
Structural Features ◽  
Functional Coupling ◽  
Expression Platform ◽  
Structural Adaptability

Abstract Cobalamin riboswitches encompass a structurally diverse group of cis-acting, gene regulatory elements found mostly in bacterial messenger RNA and are classified into subtypes based on secondary and tertiary characteristics. An unusual variant of the cobalamin riboswitch with predicted structural features was identified in Bacillus subtilis over a decade ago, but its structure and mechanisms of cobalamin selectivity and translational control have remained unsolved. We present the crystal structure of the aptamer domain of this atypical cobalamin riboswitch and a model for the complete riboswitch, including its expression platform domain. We demonstrate that this riboswitch binds to multiple cobalamin derivatives and correlate its promiscuous behavior to its structure and unique arrangement of peripheral elements. Comparative structural analyses between conventional cobalamin riboswitches and the B. subtilis cobalamin riboswitch reveal that the likely basis for this promiscuous ligand binding is intrinsic structural adaptability encoded in the RNA structure. It suggests that cobalamin selectivity might ultimately be viewed as existing on a spectrum of affinity for each derivative rather than as belonging to distinct types based on ligand specificities. Our work provides an interesting and notable example of functional coupling of ligand-sensing and adaptive folding by a structured RNA molecule.

scholarly journals Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavage in vivo

2020 ◽  
Vol 48 (15) ◽  
pp. 8767-8781 ◽  
Author(s):  
Minglei Yang ◽  
Hugh C Woolfenden ◽  
Yueying Zhang ◽  
Xiaofeng Fang ◽  
Qi Liu ◽  
...  
Keyword(s):  
Rna Structure ◽  
Target Site ◽  
Site Structure ◽  
Mrna Structure ◽  
In Silico Studies ◽  
Sequence Complementarity ◽  
Site Binding

Abstract MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-induced silencing complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. We investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, we found that the unfolding of the target site structure plays a key role in miRISC activity in vivo. We found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent nucleotide Motif, can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can modulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.

scholarly journals On the Way to Understanding the Interplay between the RNA Structure and Functions in Cells: A Genome-Wide Perspective

2020 ◽  
Vol 21 (18) ◽  
pp. 6770
Author(s):  
Angelika Andrzejewska ◽  
Małgorzata Zawadzka ◽  
Katarzyna Pachulska-Wieczorek
Keyword(s):  
Rna Structure ◽  
Gene Expression Regulation ◽  
Biological Activities ◽  
Current View ◽  
Cellular Processes ◽  
Mrna Structure ◽  
Genome Wide ◽  
A Genome ◽  
Wide Perspective

RNAs adopt specific structures in order to perform their biological activities. The structure of RNA is an important layer of gene expression regulation, and can impact a plethora of cellular processes, starting with transcription, RNA processing, and translation, and ending with RNA turnover. The development of high-throughput technologies has enabled a deeper insight into the sophisticated interplay between the structure of the cellular transcriptome and the living cells environment. In this review, we present the current view on the RNA structure in vivo resulting from the most recent transcriptome-wide studies in different organisms, including mammalians, yeast, plants, and bacteria. We focus on the relationship between the mRNA structure and translation, mRNA stability and degradation, protein binding, and RNA posttranscriptional modifications.

scholarly journals Wheat in vivo RNA structure landscape reveals a prevalent role of RNA structure in modulating translational subgenome expression asymmetry

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xiaofei Yang ◽  
Haopeng Yu ◽  
Wenqing Sun ◽  
Ling Ding ◽  
Ji Li ◽  
...  
Keyword(s):  
Rna Structure ◽  
Tetraploid Wheat ◽  
Gc Content ◽  
Strong Impact ◽  
Contributing Factors ◽  
Mrna Structure ◽  
Major Route ◽  
New Perspective ◽  
Polyploid Crops

Abstract Background Polyploidy, especially allopolyploidy, which entails merging divergent genomes via hybridization and whole-genome duplication (WGD), is a major route to speciation in plants. The duplication among the parental genomes (subgenomes) often leads to one subgenome becoming dominant over the other(s), resulting in subgenome asymmetry in gene content and expression. Polyploid wheats are allopolyploids with most genes present in two (tetraploid) or three (hexaploid) functional copies, which commonly show subgenome expression asymmetry. It is unknown whether a similar subgenome asymmetry exists during translation. We aim to address this key biological question and explore the major contributing factors to subgenome translation asymmetry. Results Here, we obtain the first tetraploid wheat translatome and reveal that subgenome expression asymmetry exists at the translational level. We further perform in vivo RNA structure profiling to obtain the wheat RNA structure landscape and find that mRNA structure has a strong impact on translation, independent of GC content. We discover a previously uncharacterized contribution of RNA structure in subgenome translation asymmetry. We identify 3564 single-nucleotide variations (SNVs) across the transcriptomes between the two tetraploid wheat subgenomes, which induce large RNA structure disparities. These SNVs are highly conserved within durum wheat cultivars but are divergent in both domesticated and wild emmer wheat. Conclusions We successfully determine both the translatome and in vivo RNA structurome in tetraploid wheat. We reveal that RNA structure serves as an important modulator of translational subgenome expression asymmetry in polyploids. Our work provides a new perspective for molecular breeding of major polyploid crops.

Sign in / Sign up

Export Citation Format

Share Document