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 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 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.

Nuclear pre-mRNA processing in plants: distinct modes of 3'-splice-site selection in plants and animals

1988 ◽  
Vol 8 (5) ◽  
pp. 2042-2051
Author(s):  
K Wiebauer ◽  
J J Herrero ◽  
W Filipowicz
Keyword(s):  
Splice Site ◽  
Hela Cells ◽  
Site Selection ◽  
Human Growth ◽  
Mrna Processing ◽  
Beta Globin ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Consensus Sequences ◽  
Intron 2

The report that human growth hormone pre-mRNA is not processed in transgenic plant tissues (A. Barta, K. Sommergruber, D. Thompson, K. Hartmuth, M.A. Matzke, and A.J.M. Matzke, Plant Mol. Biol. 6:347-357, 1986) has suggested that differences in mRNA splicing processes exist between plants and animals. To gain more information about the specificity of plant pre-mRNA processing, we have compared the splicing of the soybean leghemoglobin pre-mRNA with that of the human beta-globin pre-mRNA in transfected plant (Orychophragmus violaceus and Nicotiana tabacum) protoplasts and mammalian (HeLa) cells. Of the three introns of leghemoglobin pre-mRNA, only intron 2 was correctly and efficiently processed in HeLa cells. The 5' splice sites of the remaining two introns were faithfully recognized, but correct processing of the 3' sites took place only rarely (intron 1) or not at all (intron 3); cryptic 3' splice sites were used instead. While the first intron in human beta-globin pre-mRNA was not spliced in transfected plant protoplasts, intron 2 processing occurred at a low level, indicating that some mammalian introns can be recognized by the plant intron-splicing machinery. However, excision of intron 2 proved to be incorrect, involving the authentic 5' splice site and a cryptic 3' splice site. Our results indicate that the mechanism of 3'-splice-site selection during intron excision differs between plants and animals. This conclusion is supported by analysis of the 3'-splice-site consensus sequences in animal and plant introns which revealed that polypyrimidine tracts, characteristic of animal introns, are not present in plant pre-mRNAs. It is proposed that an elevated AU content of plant introns is important for their processing.

Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites

1984 ◽  
Vol 4 (5) ◽  
pp. 966-972
Author(s):  
C Montell ◽  
E F Fisher ◽  
M H Caruthers ◽  
A J Berk
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Early Phase ◽  
Late Phase ◽  
Productive Infection ◽  
Splice Sites ◽  
Mrna Synthesis ◽  
Adenovirus E1b ◽  
Cryptic Splice Sites ◽  
Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

A novel protein factor is required for use of distal alternative 5' splice sites in vitro

1991 ◽  
Vol 11 (12) ◽  
pp. 5945-5953
Author(s):  
J E Harper ◽  
J L Manley
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Deae Cellulose ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Small Nuclear Ribonucleoprotein

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.

scholarly journals U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.

1993 ◽  
Vol 13 (5) ◽  
pp. 2666-2676 ◽  
Author(s):  
J B Cohen ◽  
S D Broz ◽  
A D Levinson
Keyword(s):  
Splice Site ◽  
Mammalian Cells ◽  
Mrna Processing ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Splice Sites ◽  
Gene Complementation ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Mutant Genes

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.

scholarly journals Herpes simplex virus ICP27 regulates alternative pre-mRNA polyadenylation and splicing in a sequence-dependent manner

2016 ◽  
Vol 113 (43) ◽  
pp. 12256-12261 ◽  
Author(s):  
Shuang Tang ◽  
Amita Patel ◽  
Philip R. Krause
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Splice Site ◽  
Alternative Polyadenylation ◽  
Common Mechanism ◽  
Specific Gene ◽  
Dependent Manner ◽  
Splice Sites ◽  
Cellular Genes ◽  
Simplex Virus

The herpes simplex virus (HSV) infected cell culture polypeptide 27 (ICP27) protein is essential for virus infection of cells. Recent studies suggested that ICP27 inhibits splicing in a gene-specific manner via an unknown mechanism. Here, RNA-sequencing revealed that ICP27 not only inhibits splicing of certain introns in <1% of cellular genes, but also can promote use of alternative 5′ splice sites. In addition, ICP27 induced expression of pre-mRNAs prematurely cleaved and polyadenylated from cryptic polyadenylation signals (PAS) located in intron 1 or 2 of ∼1% of cellular genes. These previously undescribed prematurely cleaved and polyadenylated pre-mRNAs, some of which contain novel ORFs, were typically intronless, <2 Kb in length, expressed early during viral infection, and efficiently exported to cytoplasm. Sequence analysis revealed that ICP27-targeted genes are GC-rich (as are HSV genes), contain cytosine-rich sequences near the 5′ splice site, and have suboptimal splice sites in the impacted intron, suggesting that a common mechanism is shared between ICP27-mediated alternative polyadenylation and splicing. Optimization of splice site sequences or mutation of nearby cytosines eliminated ICP27-mediated splicing inhibition, and introduction of C-rich sequences to an ICP27-insensitive splicing reporter conferred this phenotype, supporting the inference that specific gene sequences confer susceptibility to ICP27. Although HSV is the first virus and ICP27 is the first viral protein shown to activate cryptic PASs in introns, we suspect that other viruses and cellular genes also encode this function.

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1926-1935
Author(s):  
P J Mitchell ◽  
G Urlaub ◽  
L Chasin
Keyword(s):  
Amino Acid ◽  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Splice Sites ◽  
Ovary Cells ◽  
Splicing Mutations ◽  
Intron 4

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

scholarly journals hnRNP A1 Regulates Alternative Splicing of Tau Exon 10 by Targeting 3′ Splice Sites

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 936 ◽  
Author(s):  
Yongchao Liu ◽  
Donggun Kim ◽  
Namjeong Choi ◽  
Jagyeong Oh ◽  
Jiyeon Ha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Ontology ◽  
Alternative Splicing ◽  
Splice Site ◽  
Hnrnp A1 ◽  
Gene Ontology Analysis ◽  
Splice Sites ◽  
Neuronal Function ◽  
Brain Functions ◽  
Exon 10

The ratio control of 4R-Tau/3R-Tau by alternative splicing of Tau exon 10 is important for maintaining brain functions. In this study, we show that hnRNP A1 knockdown induces inclusion of endogenous Tau exon 10, conversely, overexpression of hnRNP A1 promotes exon 10 skipping of Tau. In addition, hnRNP A1 inhibits splicing of intron 9, but not intron 10. Furthermore, hnRNP A1 directly interacts with the 3′ splice site of exon 10 to regulate its functions in alternative splicing. Finally, gene ontology analysis demonstrates that hnRNP A1-induced splicing and gene expression targets a subset of genes with neuronal function.

scholarly journals Comprehensive identification of alternative back-splicing in human tissue transcriptomes

2020 ◽  
Vol 48 (4) ◽  
pp. 1779-1789 ◽  
Author(s):  
Peng Zhang ◽  
Xiao-Ou Zhang ◽  
Tingting Jiang ◽  
Lingling Cai ◽  
Xiao Huang ◽  
...  
Keyword(s):  
Splice Site ◽  
Human Tissue ◽  
Single Gene ◽  
Brain Regions ◽  
Intron Length ◽  
Circular Rnas ◽  
Human Tissues ◽  
Alu Elements ◽  
Depth Analysis ◽  
Brain Tissues

Abstract Circular RNAs (circRNAs) are covalently closed RNAs derived from back-splicing of genes across eukaryotes. Through alternative back-splicing (ABS), a single gene produces multiple circRNAs sharing the same back-splice site. Although many ABS events have recently been discovered, to what extent ABS involves in circRNA biogenesis and how it is regulated in different human tissues still remain elusive. Here, we reported an in-depth analysis of ABS events in 90 human tissue transcriptomes. We observed that ABS occurred for about 84% circRNAs. Interestingly, alternative 5′ back-splicing occurs more prevalently than alternative 3′ back-splicing, and both of them are tissue-specific, especially enriched in brain tissues. In addition, the patterns of ABS events in different brain regions are similar to each other and are more complex than the patterns in non-brain tissues. Finally, the intron length and abundance of Alu elements positively correlated with ABS event complexity, and the predominant circRNAs had longer flanking introns and more Alu elements than other circRNAs in the same ABS event. Together, our results represent a resource for circRNA research—we expanded the repertoire of ABS events of circRNAs in human tissue transcriptomes and provided insights into the complexity of circRNA biogenesis, expression, and regulation.

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