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Article ; Online: Purification of Circular RNAs Using Poly(A) Tailing Followed by RNase R Digestion.

Xiao, Mei-Sheng / Wilusz, Jeremy E

Methods in molecular biology (Clifton, N.J.)

2024 Volume 2765, Page(s) 3–19

Abstract: Thousands of eukaryotic protein-coding genes can be alternatively spliced to yield linear mRNAs and circular RNAs (circRNAs). Some circRNAs accumulate to higher levels than their cognate linear mRNAs, but the vast majority are expressed at low levels. ... ...

Abstract	Thousands of eukaryotic protein-coding genes can be alternatively spliced to yield linear mRNAs and circular RNAs (circRNAs). Some circRNAs accumulate to higher levels than their cognate linear mRNAs, but the vast majority are expressed at low levels. Hence, for most circRNAs, only a handful of sequencing reads, if any, that span the backsplicing junction are observed in standard RNA-seq libraries. It thus has become common to use the 3'-5' exonuclease ribonuclease R (RNase R) to selectively degrade linear RNAs when aiming to prove transcript circularity or biochemically enrich circRNAs. However, RNase R fails to degrade linear RNAs with structured 3' ends or internal G-quadruplex structures. To overcome these shortcomings, we describe an improved protocol for circRNA purification from total RNA that employs a poly(A) tailing step prior to RNase R digestion, which is performed in a Li
MeSH term(s)	RNA, Circular ; RNA, Messenger ; RNA/genetics ; Exonucleases ; Digestion ; Exoribonucleases
Chemical Substances	RNA, Circular ; ribonuclease R (EC 3.1.27.-) ; RNA, Messenger ; RNA (63231-63-0) ; Exonucleases (EC 3.1.-) ; Exoribonucleases (EC 3.1.-)
Language	English
Publishing date	2024-02-21
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-3678-7_1
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Article ; Online: Circle the Wagons: Circular RNAs Control Innate Immunity.

Wilusz, Jeremy E

Cell

2019 Volume 177, Issue 4, Page(s) 797–799

Abstract: Circular RNAs are generated at low levels from many protein-coding genes. Liu et al. now reveal that many of these transcripts bind and inhibit the double-stranded RNA (dsRNA)-dependent kinase PKR. Upon viral infection, circular RNAs are globally ... ...

Abstract	Circular RNAs are generated at low levels from many protein-coding genes. Liu et al. now reveal that many of these transcripts bind and inhibit the double-stranded RNA (dsRNA)-dependent kinase PKR. Upon viral infection, circular RNAs are globally degraded to release PKR, which becomes activated to aid in the immune response.
MeSH term(s)	Immunity, Innate ; RNA, Circular ; RNA, Double-Stranded ; eIF-2 Kinase
Chemical Substances	RNA, Circular ; RNA, Double-Stranded ; eIF-2 Kinase (EC 2.7.11.1)
Keywords	covid19
Language	English
Publishing date	2019-04-30
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Comment
ZDB-ID	187009-9
ISSN	1097-4172 ; 0092-8674
ISSN (online)	1097-4172
ISSN	0092-8674
DOI	10.1016/j.cell.2019.04.020
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Article ; Online: Methods for circular RNAs.

Chen, Ling-Ling / Wilusz, Jeremy E

Methods (San Diego, Calif.)

2021 Volume 196, Page(s) 1–2

MeSH term(s)	MicroRNAs ; RNA/genetics ; RNA, Circular
Chemical Substances	MicroRNAs ; RNA, Circular ; RNA (63231-63-0)
Language	English
Publishing date	2021-10-01
Publishing country	United States
Document type	Editorial
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2021.09.011
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Zs.A 3239: Show issues

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Article ; Online: A 360° view of circular RNAs: From biogenesis to functions.

Wilusz, Jeremy E

Wiley interdisciplinary reviews. RNA

2018 Volume 9, Issue 4, Page(s) e1478

Abstract: The first circular RNA (circRNA) was identified more than 40 years ago, but it was only recently appreciated that circRNAs are common outputs of many eukaryotic protein-coding genes. Some circRNAs accumulate to higher levels than their associated linear ... ...

Abstract	The first circular RNA (circRNA) was identified more than 40 years ago, but it was only recently appreciated that circRNAs are common outputs of many eukaryotic protein-coding genes. Some circRNAs accumulate to higher levels than their associated linear mRNAs, especially in the nervous system, and have clear regulatory functions that result in organismal phenotypes. The pre-mRNA splicing machinery generates circRNAs via backsplicing reactions, which are often facilitated by intronic repeat sequences that base pair to one another and bring the intervening splice sites into close proximity. When spliceosomal components are limiting, circRNAs can become the preferred gene output, and backsplicing reactions are further controlled by exon skipping events and the combinatorial action of RNA binding proteins. This allows circRNAs to be expressed in a tissue- and stage-specific manner. Once generated, circRNAs are highly stable transcripts that often accumulate in the cytoplasm. The functions of most circRNAs remain unknown, but some can regulate the activities of microRNAs or be translated to produce proteins. Circular RNAs can further interface with the immune system as well as control gene expression events in the nucleus, including alternative splicing decisions. Circular RNAs thus represent a large class of RNA molecules that are tightly regulated, and it is becoming increasingly clear that they likely impact many biological processes. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Evolution and Genomics > Computational Analyses of RNA.
MeSH term(s)	Alternative Splicing/genetics ; Animals ; Cell Nucleus/genetics ; Cell Nucleus/metabolism ; Humans ; RNA/biosynthesis ; RNA/genetics ; RNA/immunology ; RNA/metabolism ; RNA, Circular ; RNA-Binding Proteins/metabolism
Chemical Substances	RNA, Circular ; RNA-Binding Proteins ; RNA (63231-63-0)
Language	English
Publishing date	2018-04-14
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2634714-3
ISSN	1757-7012 ; 1757-7004
ISSN (online)	1757-7012
ISSN	1757-7004
DOI	10.1002/wrna.1478
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Zs.A 6953: Show issues

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Article: Circular STAG2 RNA Modulates Bladder Cancer Progression via miR-145-5p/TAGLN2 and Is Considered as a Biomarker for Recurrence.

Du, Chris / Waltzer, Wayne C / Wilusz, Jeremy E / Spaliviero, Massimiliano / Darras, Frank / Romanov, Victor

Cancers

2024 Volume 16, Issue 5

Abstract: The current study aimed to elucidate the regulatory mechanisms of the circRNA hsa_circ_0139697 (circSTAG2(16-25)) in BCa and to consider the opportunity of using circSTAG2(16-25) isolated from BCa patient urine as a marker for disease development ... ...

Abstract	The current study aimed to elucidate the regulatory mechanisms of the circRNA hsa_circ_0139697 (circSTAG2(16-25)) in BCa and to consider the opportunity of using circSTAG2(16-25) isolated from BCa patient urine as a marker for disease development prediction. The selection of this circRNA was determined by the special role of its parental gene STAG2 in BCa biology. The circRNA hsa_circ_0139697 was chosen from 25 STAG2 circRNAs due to its differential expression in the urine of BCa patients and healthy volunteers. Higher levels of circSTAG2(16-25) were detected in urine samples obtained from patients with recurrent tumors. A higher expression of circSTAG2(16-25) was also detected in more tumorigenic BCa cell lines. The overexpression of circSTAG2(16-25) in BCa cells induced the elevation of proliferation, motility, and invasion. To study the mechanisms of circSTAG2(16-25) activity, we confirmed that circSTAG2(16-25) can bind miR-145-5p in vitro as was predicted by bioinformatic search. miR-145-5p was shown to suppress some genes that promoted BCa progression. One of these genes, TAGLN2, encodes the protein Transgelin 2, which plays a role in BCa cell motility and invasion. Therefore, the possible mechanism of action of circSTAG2(16-25) could be sponging the tumor suppressor miR-145-5p, which results in activation of TAGLN2. In addition, circSTAG2(16-25) might be considered as a potential biomarker for recurrence prediction.
Language	English
Publishing date	2024-02-28
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers16050978
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Article ; Online: Erratum: Attenuation of Eukaryotic Protein-Coding Gene Expression via Premature Transcription Termination.

Tatomer, Deirdre C / Wilusz, Jeremy E

Cold Spring Harbor symposia on quantitative biology

2020

Language	English
Publishing date	2020-02-27
Publishing country	United States
Document type	Journal Article ; Published Erratum
ISSN	1943-4456 ; 0091-7451
ISSN (online)	1943-4456
ISSN	0091-7451
DOI	10.1101/sqb.2019.84.039784
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Article ; Online: CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells.

Ai, Yuxi / Liang, Dongming / Wilusz, Jeremy E

Nucleic acids research

2022 Volume 50, Issue 11, Page(s) e65

Abstract: CRISPR/Cas13 effectors have garnered increasing attention as easily customizable tools for detecting and depleting RNAs of interest. Near perfect complementarity between a target RNA and the Cas13-associated guide RNA is required for activation of Cas13 ... ...

Abstract	CRISPR/Cas13 effectors have garnered increasing attention as easily customizable tools for detecting and depleting RNAs of interest. Near perfect complementarity between a target RNA and the Cas13-associated guide RNA is required for activation of Cas13 ribonuclease activity. Nonetheless, the specificity of Cas13 effectors in eukaryotic cells has been debated as the Cas13 nuclease domains can be exposed on the enzyme surface, providing the potential for promiscuous cleavage of nearby RNAs (so-called collateral damage). Here, using co-transfection assays in Drosophila and human cells, we found that the off-target effects of RxCas13d, a commonly used Cas13 effector, can be as strong as the level of on-target RNA knockdown. The extent of off-target effects is positively correlated with target RNA expression levels, and collateral damage can be observed even after reducing RxCas13d/guide RNA levels. The PspCas13b effector showed improved specificity and, unlike RxCas13d, can be used to deplete a Drosophila circular RNA without affecting the expression of the associated linear RNA. PspCas13b nonetheless still can have off-target effects and we notably found that the extent of off-target effects for Cas13 effectors differs depending on the cell type and target RNA examined. In total, these results highlight the need for caution when designing and interpreting Cas13-based knockdown experiments.
MeSH term(s)	Animals ; CRISPR-Cas Systems ; Drosophila/genetics ; Eukaryotic Cells ; RNA/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics
Chemical Substances	RNA, Guide, CRISPR-Cas Systems ; RNA (63231-63-0)
Language	English
Publishing date	2022-03-11
Publishing country	England
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkac159
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Zs.A 1067: Show issues

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Article ; Online: Biogenesis and Regulatory Roles of Circular RNAs.

Yang, Li / Wilusz, Jeremy E / Chen, Ling-Ling

Annual review of cell and developmental biology

2022 Volume 38, Page(s) 263–289

Abstract: Covalently closed, single-stranded circular RNAs can be produced from viral RNA genomes as well as from the processing of cellular housekeeping noncoding RNAs and precursor messenger RNAs. Recent transcriptomic studies have surprisingly uncovered that ... ...

Abstract	Covalently closed, single-stranded circular RNAs can be produced from viral RNA genomes as well as from the processing of cellular housekeeping noncoding RNAs and precursor messenger RNAs. Recent transcriptomic studies have surprisingly uncovered that many protein-coding genes can be subjected to backsplicing, leading to widespread expression of a specific type of circular RNAs (circRNAs) in eukaryotic cells. Here, we discuss experimental strategies used to discover and characterize diverse circRNAs at both the genome and individual gene scales. We further highlight the current understanding of how circRNAs are generated and how the mature transcripts function. Some circRNAs act as noncoding RNAs to impact gene regulation by serving as decoys or competitors for microRNAs and proteins. Others form extensive networks of ribonucleoprotein complexes or encode functional peptides that are translated in response to certain cellular stresses. Overall, circRNAs have emerged as an important class of RNAmolecules in gene expression regulation that impact many physiological processes, including early development, immune responses, neurogenesis, and tumorigenesis.
MeSH term(s)	Gene Expression Regulation/genetics ; MicroRNAs/genetics ; MicroRNAs/metabolism ; RNA/genetics ; RNA/metabolism ; RNA, Circular/genetics ; RNA, Untranslated ; RNA, Viral ; Ribonucleoproteins/genetics ; Ribonucleoproteins/metabolism
Chemical Substances	MicroRNAs ; RNA, Circular ; RNA, Untranslated ; RNA, Viral ; Ribonucleoproteins ; RNA (63231-63-0)
Language	English
Publishing date	2022-05-24
Publishing country	United States
Document type	Journal Article ; Review ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1293750-2
ISSN	1530-8995 ; 1081-0706
ISSN (online)	1530-8995
ISSN	1081-0706
DOI	10.1146/annurev-cellbio-120420-125117
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Se 1003: Show issues

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Article ; Online: Circular RNAs: Unexpected outputs of many protein-coding genes.

Wilusz, Jeremy E

RNA biology

2017 Volume 14, Issue 8, Page(s) 1007–1017

Abstract: Pre-mRNAs from thousands of eukaryotic genes can be non-canonically spliced to generate circular RNAs, some of which accumulate to higher levels than their associated linear mRNA. Recent work has revealed widespread mechanisms that dictate whether the ... ...

Abstract	Pre-mRNAs from thousands of eukaryotic genes can be non-canonically spliced to generate circular RNAs, some of which accumulate to higher levels than their associated linear mRNA. Recent work has revealed widespread mechanisms that dictate whether the spliceosome generates a linear or circular RNA. For most genes, circular RNA biogenesis via backsplicing is far less efficient than canonical splicing, but circular RNAs can accumulate due to their long half-lives. Backsplicing is often initiated when complementary sequences from different introns base pair and bring the intervening splice sites close together. This process is further regulated by the combinatorial action of RNA binding proteins, which allow circular RNAs to be expressed in unique patterns. Some genes do not require complementary sequences to generate RNA circles and instead take advantage of exon skipping events. It is still unclear what most mature circular RNAs do, but future investigations into their functions will be facilitated by recently described methods to modulate circular RNA levels.
MeSH term(s)	Animals ; Base Pairing ; Drosophila melanogaster/genetics ; Drosophila melanogaster/metabolism ; Exons ; Humans ; Introns ; Nucleic Acid Conformation ; RNA/genetics ; RNA/metabolism ; RNA Precursors/genetics ; RNA Precursors/metabolism ; RNA Splicing ; RNA Stability ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA-Binding Proteins/genetics ; RNA-Binding Proteins/metabolism ; Spliceosomes/genetics ; Spliceosomes/metabolism
Chemical Substances	RNA Precursors ; RNA, Messenger ; RNA, circular ; RNA-Binding Proteins ; RNA (63231-63-0)
Language	English
Publishing date	2017--03
Publishing country	United States
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ISSN	1555-8584
ISSN (online)	1555-8584
DOI	10.1080/15476286.2016.1227905
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Article ; Online: Attenuation of Eukaryotic Protein-Coding Gene Expression via Premature Transcription Termination.

Tatomer, Deirdre C / Wilusz, Jeremy E

Cold Spring Harbor symposia on quantitative biology

2020 Volume 84, Page(s) 83–93

Abstract: A complex network of RNA transcripts is generated from eukaryotic genomes, many of which are processed in unexpected ways. Here, we highlight how premature transcription termination events at protein-coding gene loci can simultaneously lead to the ... ...

Abstract	A complex network of RNA transcripts is generated from eukaryotic genomes, many of which are processed in unexpected ways. Here, we highlight how premature transcription termination events at protein-coding gene loci can simultaneously lead to the generation of short RNAs and attenuate production of full-length mRNA transcripts. We recently showed that the Integrator (Int) complex can be selectively recruited to protein-coding gene loci, including
Language	English
Publishing date	2020-02-21
Publishing country	United States
Document type	Journal Article
ISSN	1943-4456 ; 0091-7451
ISSN (online)	1943-4456
ISSN	0091-7451
DOI	10.1101/sqb.2019.84.039644
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