scholarly journals Knockout of circRNAs by base editing back-splice sites of circularized exons

2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Xiang Gao ◽  
Xu-Kai Ma ◽  
Xiang Li ◽  
Guo-Wei Li ◽  
Chu-Xiao Liu ◽  
...  
Keyword(s):  
Efficient Method ◽  
Functional Study ◽  
Circular Rnas ◽  
Splice Sites ◽  
Base Editing

AbstractMany circular RNAs (circRNAs) are produced from back-splicing of exons of precursor mRNAs and are generally co-expressed with cognate linear RNAs. Methods for circRNA-specific knockout are lacking, largely due to sequence overlaps between forms. Here, we use base editors (BEs) for circRNA depletion. By targeting splice sites involved in both back-splicing and canonical splicing, BEs can repress circular and linear RNAs. Targeting sites predominantly for circRNA biogenesis, BEs could efficiently repress the production of circular but not linear RNAs. As hundreds of exons are predominantly back-spliced to produce circRNAs, this provides an efficient method to deplete circRNAs for functional study.

scholarly journals Knockout of circRNAs by base editing back-splice sites of circularized exons

2021 ◽  
Author(s):  
Xiang Gao ◽  
Xu-Kai Ma ◽  
Xiang Li ◽  
Guo-Wei Li ◽  
Chu-Xiao Liu ◽  
...  
Keyword(s):  
Splice Site ◽  
Efficient Method ◽  
Complete Sequence ◽  
Circular Rnas ◽  
Splice Sites ◽  
Base Editing ◽  
Function Study

A large number of circular RNAs (circRNAs) are produced from back-splicing of exon(s) of precursor mRNAs and generally co-expressed with their cognate linear RNAs from the same gene loci. Methods for circRNA-specific knockout are lacking, largely due to complete sequence-overlaps between circular and cognate linear RNAs. Here, we report to use base editors (BEs) for circRNA depletion. By targeting splice sites involved in both back-splicing and canonical splicing, BEs can repress both circular and linear RNAs expression, which confirms the requirement of canonical splice site signals for back-splice. Importantly, by targeting back-splice sites predominantly for circRNA biogenesis, BEs could efficiently repress the production of circular, but not linear cognate RNAs. As hundreds of exons were found to be predominantly back-spliced to produce circRNAs, this study provides an efficient method to deplete circRNAs for function study.

scholarly journals CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary cells

2020 ◽  
Author(s):  
Mitchell G. Kluesner ◽  
Walker S. Lahr ◽  
Cara-Lin Lonetree ◽  
Branden A. Smeester ◽  
Patricia N. Claudio-Vázquez ◽  
...  
Keyword(s):  
Splice Site ◽  
Stop Codon ◽  
Cytidine Deaminase ◽  
Gene Knockout ◽  
Premature Stop Codon ◽  
Splice Sites ◽  
Immune Synapse ◽  
Base Editing ◽  
Human T Cells ◽  
Site Targeting

ABSTRACTBase editors allow for precise nucleotide editing without the need for genotoxic double-stranded breaks. Prior work has used base editors to knockout genes by introducing premature stop codons or by disrupting conserved splice-sites, but no direct comparison exists between these methods. Additionally, while base editor mediated disruption of splice sites has been used to shift the functional isoform pool, its utility for gene knockout requires further validation. To address these needs, we developed the program SpliceR (z.umn.edu/spliceR) to design cytidine-deaminase base editor (CBE) and adenosine-deaminase base editor (ABE) splice-site targeting guides. We compared the splice-site targeting and premature stop codon introduction in a knockout screen against the TCR-CD3 immune synapse in primary human T-cells. Our data suggests that 1) the CBE, BE4 is more reliable than the ABE, ABE7.10 for splice-site targeting knockout and 2) for both CBEs and ABEs, splice-donor targeting is the most reliable approach for base editing induced knockout.

scholarly journals Systematic identification of circular RNAs and corresponding regulatory networks unveil their potential roles in the midgut ofApis cerana ceranaworkers

2019 ◽  
Author(s):  
Dafu Chen ◽  
Huazhi Chen ◽  
Yu Du ◽  
Sihai Geng ◽  
Cuiling Xiong ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Apis Cerana ◽  
Regulation Of Gene Expression ◽  
Functional Study ◽  
Circular Rnas ◽  
Kegg Pathways ◽  
Midgut Development ◽  
Go Terms

AbstractBackgroundCircular RNAs (circRNAs) are newly discovered noncoding RNAs (ncRNAs) that play key roles in various biological functions, such as the regulation of gene expression and alternative splicing. CircRNAs have been identified in some species, including western honeybees. However, the understanding of honeybee circRNA is still very limited, and to date, no study on eastern honeybee circRNA has been conducted. Here, the circRNAs in the midguts ofApis cerana ceranaworkers were identified and validated, and the regulatory networks were constructed. Differentially expressed circRNAs (DEcircRNAs) and the corresponding competitively endogenous RNA (ceRNA) networks in the development of the worker’s midgut were further investigated.ResultsHere, 7- and 10-day-oldA. c. ceranaworkers’ midguts (Ac1 and Ac2) were sequenced using RNA-seq, and a total of 9589 circRNAs were predicted using bioinformatics. These circRNAs were approximately 201-800 nt in length and could be classified into six types; the annotated exonic circRNAs were the most abundant. Additionally, five novelA. c. ceranacircRNAs were confirmed by PCR amplification and Sanger sequencing, indicating the authenticity ofA. c. ceranacircRNAs. Interestingly, novel_circ_003723, novel_circ_002714, novel_circ_002451 and novel_circ_001980 were the most highly expressed circRNAs in both Ac1 and Ac2, which is indicative of their key roles in the development of the midgut. Moreover, 55 DEcircRNAs were identified in the Ac1 vs Ac2 comparison group, including 34 upregulated and 21 downregulated circRNAs. Further investigation showed that the source genes of circRNAs were classified into 34 GO terms and were involved in 141 KEGG pathways. In addition, the source genes of DEcircRNAs were categorized into 10 GO terms and 15 KEGG pathways, which demonstrated that the corresponding DEcircRNAs may affect the growth, development, and material and energy metabolisms of the worker’s midgut by regulating the expression of the related source genes. Additionally, the circRNA-miRNA regulatory networks were constructed and analyzed, and the results demonstrated that 1060 circRNAs can bind to 74 miRNAs and that 71.51% of circRNAs can be linked to only one miRNA. Furthermore, the DEcircRNA-miRNA-mRNA networks were constructed and explored, and the results indicate that the 13 downregulated circRNAs can bind to eight miRNAs and to 29 target genes. In addition, the results indicate that the 16 upregulated circRNAs can bind to 9 miRNAs and to 29 target genes, demonstrating that DEcircRNAs are likely involved in the regulation of midgut development via ceRNA mechanisms. Moreover, the regulatory networks of miR-6001-y-targeted DEcircRNAs were analyzed, and the results showed that eight DEcircRNAs may affect the development ofA. c. ceranaworkers’ midguts by targeting miR-6001-y. Finally, four randomly selected DEcircRNAs were verified via RT-qPCR, confirming the reliability of our sequencing data.ConclusionThis is the first systematic investigation of circRNAs and their corresponding regulatory networks in eastern honeybees. The identified circRNAs from theA. c. ceranaworker’s midgut will enrich the known reservoir of honeybee ncRNAs. DEcircRNAs may play a comprehensive role during the development of the worker’s midgut via the regulation of source genes and the interaction with miRNAs by acting as ceRNAs. The eight DEcircRNAs that targeted miR-6001-y were likely to be vital for the development of the worker’s midgut. Our results provide a valuable resource for the future studies ofA. c. ceranacircRNA and lay a foundation to reveal the molecular mechanisms underlying the regulatory networks of circRNAs responsible for the worker’s midgut development; in addition, these findings facilitate a functional study on the key circRNAs involved in the developmental process.Graphical Abstract

scholarly journals Conserved long-range RNA structures associated with pre-mRNA processing of human protein-coding genes

2020 ◽  
Author(s):  
Svetlana Kalmykova ◽  
Marina Kalinina ◽  
Stepan Denisov ◽  
Alexey Mironov ◽  
Dmitry Skvortsov ◽  
...  
Keyword(s):  
Long Range ◽  
Rna Folding ◽  
Current Knowledge ◽  
False Positive Rate ◽  
Human Protein ◽  
Circular Rnas ◽  
Splice Sites ◽  
Rna Structures ◽  
Protein Coding ◽  
Protein Coding Genes

AbstractThe ability of nucleic acids to form double-stranded structures is essential for all living systems on Earth. While DNA employs it for genome replication, RNA molecules fold into complicated secondary and tertiary structures. Current knowledge on functional RNA structures in human protein-coding genes is focused on locally-occurring base pairs. However, chemical crosslinking and proximity ligation experiments have demonstrated that long-range RNA structures are highly abundant. Here, we present the most complete to-date catalog of conserved long-range RNA structures in the human transcriptome, which consists of 1.1 million pairs of conserved complementary regions (PCCRs). PCCRs tend to occur within introns proximally to splice sites, suppress intervening exons, circumscribe circular RNAs, and exert an obstructive effect on cryptic and inactive splice sites. The double-stranded structure of PCCRs is supported by a significant decrease of icSHAPE nucleotide accessibility, high abundance of A-to-I RNA editing sites, and frequent nearby occurrence of forked eCLIP peaks. Introns with PCCRs show a distinct splicing pattern in response to RNA Pol II slowdown suggesting that splicing is widely affected by co-transcriptional RNA folding. Additionally, transcript starts and ends are strongly enriched in regions between complementary parts of PCCRs, leading to an intriguing hypothesis that RNA folding coupled with splicing could mediate co-transcriptional suppression of premature cleavage and polyadenylation events. PCCR detection procedure is highly sensitive with respect to bona fide validated RNA structures at the expense of having a high false positive rate, which cannot be reduced without loss of sensitivity. The catalog of PCCRs is visualized through a UCSC Genome Browser track hub to facilitate further genome research on long-range RNA structures.

scholarly journals Screening for functional circular RNAs using the CRISPR-Cas13 system

2020 ◽  
Author(s):  
Siqi Li ◽  
Xiang Li ◽  
Wei Xue ◽  
Lin Zhang ◽  
Shi-Meng Cao ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Functional Study ◽  
Circular Rnas ◽  
Rna Targeting ◽  
Scale Levels ◽  
A Cell ◽  
Cell Type Specific

SummaryCircular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood due to inadequate methods, such as RNAi and genome engineering, in distinguishing overlapped exons in circRNAs from those in linear cognate mRNAs1,2. Here we report that the programable RNA-guided, RNA-targeting CRISPR-Cas13, RfxCas13d, effectively and specifically discriminates circRNAs from mRNAs, using guide (g)RNAs targeting sequences spanning the back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation in vitro and in vivo by preventing FAM120A mRNA from binding the translation inhibitor IGF2BP2 for efficient translation. Application of RfxCas13d/BSJ-gRNA screening has also uncovered circMan1a2 with regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR-RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels.

scholarly journals CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mitchell G. Kluesner ◽  
Walker S. Lahr ◽  
Cara-lin Lonetree ◽  
Branden A. Smeester ◽  
Xiaohong Qiu ◽  
...  
Keyword(s):  
Mhc Class I ◽  
Gene Disruption ◽  
Modular Design ◽  
Design Tool ◽  
Splice Sites ◽  
Robust Method ◽  
Immune Synapse ◽  
Base Editing ◽  
Immortalized Cells ◽  
Disruption Method

AbstractCRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop (pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR (http://z.umn.edu/spliceR) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.

Role of small nuclear RNAs in eukaryotic gene expression

2013 ◽  
Vol 54 ◽  
pp. 79-90 ◽  
Author(s):  
Saba Valadkhan ◽  
Lalith S. Gunawardane
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Stability ◽  
Splice Sites ◽  
Branch Site ◽  
Small Nuclear Rnas ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Rna Biogenesis

Eukaryotic cells contain small, highly abundant, nuclear-localized non-coding RNAs [snRNAs (small nuclear RNAs)] which play important roles in splicing of introns from primary genomic transcripts. Through a combination of RNA–RNA and RNA–protein interactions, two of the snRNPs, U1 and U2, recognize the splice sites and the branch site of introns. A complex remodelling of RNA–RNA and protein-based interactions follows, resulting in the assembly of catalytically competent spliceosomes, in which the snRNAs and their bound proteins play central roles. This process involves formation of extensive base-pairing interactions between U2 and U6, U6 and the 5′ splice site, and U5 and the exonic sequences immediately adjacent to the 5′ and 3′ splice sites. Thus RNA–RNA interactions involving U2, U5 and U6 help position the reacting groups of the first and second steps of splicing. In addition, U6 is also thought to participate in formation of the spliceosomal active site. Furthermore, emerging evidence suggests additional roles for snRNAs in regulation of various aspects of RNA biogenesis, from transcription to polyadenylation and RNA stability. These snRNP-mediated regulatory roles probably serve to ensure the co-ordination of the different processes involved in biogenesis of RNAs and point to the central importance of snRNAs in eukaryotic gene expression.

Identification, characterization and expression profiling of circular RNAs in the early cotton fiber developmental stages

Genomics ◽  
2021 ◽  
Vol 113 (1) ◽  
pp. 356-365
Author(s):  
HARON Salih ◽  
Xiao Wang ◽  
Baojun Chen ◽  
Yinhua Jia ◽  
Wenfang Gong ◽  
...  
Keyword(s):  
Cotton Fiber ◽  
Expression Profiling ◽  
Developmental Stages ◽  
Circular Rnas

Circular RNAs in Neuroblastoma

2018 ◽  
Author(s):  
C Danßmann ◽  
J Toedling ◽  
F Klironomos ◽  
A Winkler ◽  
F Hertwig ◽  
...  
Keyword(s):  
Circular Rnas

Acquired Haemophilia: Functional Study of Antibodies to Factor VIII

1981 ◽  
Vol 45 (03) ◽  
pp. 285-289 ◽  
Author(s):  
J P Allain ◽  
A Gaillandre ◽  
D Frommel
Keyword(s):  
Factor Viii ◽  
Functional Study ◽  
Factor Viii Inhibitor ◽  
Acquired Haemophilia ◽  
Viii Inhibitor ◽  
Kinetics Of ◽  
Antibody Function ◽  
Native Plasma

SummaryFactor VIII complex and its interaction with antibodies to factor VIII have been studied in 17 non-haemophilic patients with factor VIII inhibitor. Low VIII:C and high VIIIR.Ag levels were found in all patients. VIII:WF levels were 50% of those of VTIIRrAg, possibly related to an increase of poorly aggregated and electrophoretically fast moving VIIIR:Ag oligomers.Antibody function has been characterized by kinetics of VIII :C inactivation, saturability by normal plasma and the slope of the affinity curve. Two major patterns were observed:1) Antibodies from 6 patients behaved similarly to those from haemophiliacs by showing second order inhibition kinetics, easy saturability and steep affinity slope (> 1).2) Antibodies from other patients, usually with lower titres, inactivated VIII :C according to complex order kinetics, were not saturable, and had a less steep affinity slope (< 0.7). In native plasma, or after mixing with factor VIII concentrate, antibodies of the second group did not form immune complexes with the whole factor VIII molecular complex. However, dissociation procedures did release some antibodies from apparently low molecular weight complexes formed in vivo or in vitro. For appropriate management of non-haemophilic patients with factor VIII inhibitor, it is important to determine the functional properties of their antibodies to factor VIII.

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