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Article ; Online: Identification of circRNA-Interacting Proteins by Affinity Pulldown.

Yang, Jen-Hao / Pandey, Poonam R / Gorospe, Myriam

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

2021 Volume 2372, Page(s) 193–202

Abstract: Circular RNAs (circRNAs) comprise a vast class of covalently closed transcripts, generated primarily via backsplicing. Most circRNAs arise from full or partial exons, but they can also arise from introns, and from combinations of introns and exons. While ...

Abstract	Circular RNAs (circRNAs) comprise a vast class of covalently closed transcripts, generated primarily via backsplicing. Most circRNAs arise from full or partial exons, but they can also arise from introns, and from combinations of introns and exons. While high-throughput RNA-sequencing analysis has identified tens of thousands of circRNAs expressed in different tissues and growth conditions, the function of circRNAs has only been described for a handful of them. As most circRNAs appear not to encode peptides, their function is presumed to be linked to their interaction with a range of molecules, particularly other nucleic acids (notably microRNAs) and proteins. A major impediment to identifying circRNA-associated molecules is a lack of suitable methodologies capable of analyzing specifically circRNAs and not their linear RNA counterparts with which they share most of their sequence. Here, we describe a flexible and robust method for identifying the proteins that associate with a given circRNA. The affinity pulldown assay is based on the use of a biotinylated antisense oligomer that recognizes the circRNA-specific junction sequence. Following pulldown using streptavidin beads, the proteins are eluted from the circRNP (circribonucleoprotein) complex and identified by mass spectroscopy; validation by Western blot analysis and other methods would then confirm the identity of the circRNA-associated proteins. We present a detailed step-by-step protocol, tips to optimize the analysis, troubleshooting suggestions, and assistance in interpreting the results. In sum, this protocol enables the discovery of proteins present in circRNPs, a critical effort toward elucidating circRNA function.
MeSH term(s)	Exons ; Introns ; MicroRNAs ; RNA/genetics ; RNA, Circular/genetics ; Sequence Analysis, RNA
Chemical Substances	MicroRNAs ; RNA, Circular ; RNA (63231-63-0)
Language	English
Publishing date	2021-08-20
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Intramural
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-1697-0_17
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Article ; Online: RNA-mediated immunotherapy regulating tumor immune microenvironment: next wave of cancer therapeutics.

Pandey, Poonam R / Young, Ken H / Kumar, Dhiraj / Jain, Neeraj

Molecular cancer

2022 Volume 21, Issue 1, Page(s) 58

Abstract: Accumulating research suggests that the tumor immune microenvironment (TIME) plays an essential role in regulation of tumor growth and metastasis. The cellular and molecular nature of the TIME influences cancer progression and metastasis by altering the ... ...

Abstract	Accumulating research suggests that the tumor immune microenvironment (TIME) plays an essential role in regulation of tumor growth and metastasis. The cellular and molecular nature of the TIME influences cancer progression and metastasis by altering the ratio of immune- suppressive versus cytotoxic responses in the vicinity of the tumor. Targeting or activating the TIME components show a promising therapeutic avenue to combat cancer. The success of immunotherapy is both astounding and unsatisfactory in the clinic. Advancements in RNA-based technology have improved understanding of the complexity and diversity of the TIME and its effects on therapy. TIME-related RNA or RNA regulators could be promising targets for anticancer immunotherapy. In this review, we discuss the available RNA-based cancer immunotherapies targeting the TIME. More importantly, we summarize the potential of various RNA-based therapeutics clinically available for cancer treatment. RNA-dependent targeting of the TIME, as monotherapy or combined with other evolving therapeutics, might be beneficial for cancer patients' treatment in the near future.
MeSH term(s)	Antineoplastic Agents/pharmacology ; Humans ; Immunotherapy ; Neoplasms/drug therapy ; Neoplasms/therapy ; RNA ; Tumor Microenvironment
Chemical Substances	Antineoplastic Agents ; RNA (63231-63-0)
Language	English
Publishing date	2022-02-21
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2091373-4
ISSN	1476-4598 ; 1476-4598
ISSN (online)	1476-4598
ISSN	1476-4598
DOI	10.1186/s12943-022-01528-6
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Article ; Online: LINC00162 regulates cell proliferation and apoptosis by sponging PAQR4-targeting miR-485-5p.

Lee, Woo Joo / Ji, Haein / Jeong, Seong Dong / Pandey, Poonam R / Gorospe, Myriam / Kim, Hyeon Ho

Journal of cellular physiology

2022 Volume 237, Issue 7, Page(s) 2943–2960

Abstract: Growing evidence indicates that long intergenic noncoding RNAs play an important role in cancer progression by affecting gene regulation at the transcriptional and posttranscriptional levels. Recent studies have shown that long intergenic noncoding RNA ... ...

Abstract	Growing evidence indicates that long intergenic noncoding RNAs play an important role in cancer progression by affecting gene regulation at the transcriptional and posttranscriptional levels. Recent studies have shown that long intergenic noncoding RNA functions as a competitive endogenous RNA, which can interact with and mitigate the function of microRNA. In this study, we investigated the molecular mechanism by which LINC00162 regulates cell proliferation and apoptotic cell death. By analyzing RNA sequencing data, LINC00162 was identified to be a target of heterogeneous nuclear ribonucleoprotein K (hnRNPK). HnRNPK positively regulated LINC00162 expression through p38 mitogen-activated protein kinase. Lowering the level of either hnRNPK or LINC00162 decreased proliferation and colony formation while it increased apoptotic cell death. Small RNA sequencing followed by the antisense oligonucleotide pulldown, revealed that LINC00162 interacts directly with miR-485-5p which exhibited tumor-suppressing effects by suppressing cell proliferation and colony formation, and increasing apoptotic cell death. Through the bioinformatic approaches, progestin and adipoQ receptor 4 (PAQR4) was selected as a common target of LINC00162 and miR-485-5p. miR-485-5p decreased the expression of PAQR4 by directly binding to the 3'-untranslated region of PAQR4 messenger RNA. Knockdown of hnRNPK and LINC00162 increased the level of functional miR-485-5p, indicating that LINC00162 may compete for miR-485-5p, thereby derepressing PAQR4 expression. Overexpression of either hnRNPK or LINC00162, or inhibition of miR-485-5p, protected cells against etoposide-induced apoptotic death. Our findings demonstrate that a regulatory paradigm implicating hnRNPK, LINC00162, miR-485-5p, and PAQR4 plays an important role in cell proliferation and apoptosis, and is a promising target for cancer therapeutics.
MeSH term(s)	3' Untranslated Regions/genetics ; Apoptosis ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; MicroRNAs/genetics ; MicroRNAs/metabolism ; Neoplasms/genetics ; RNA, Long Noncoding/genetics ; RNA, Long Noncoding/metabolism ; Receptors, Progesterone/metabolism
Chemical Substances	3' Untranslated Regions ; MIRN485 microRNA, human ; MicroRNAs ; RNA, Long Noncoding ; Receptors, Progesterone
Language	English
Publishing date	2022-05-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3116-1
ISSN	1097-4652 ; 0021-9541
ISSN (online)	1097-4652
ISSN	0021-9541
DOI	10.1002/jcp.30758
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Article ; Online: LncRNA Malat1 suppresses pyroptosis and T cell-mediated killing of incipient metastatic cells.

Kumar, Dhiraj / Gurrapu, Sreeharsha / Wang, Yan / Bae, Seong-Yeon / Pandey, Poonam R / Chen, Hong / Mondal, Jayanta / Han, Hyunho / Wu, Chang-Jiun / Karaiskos, Spyros / Yang, Fei / Sahin, Aysegul / Wistuba, Ignacio I / Gao, Jianjun / Tripathy, Debasish / Gao, Hua / Izar, Benjamin / Giancotti, Filippo G

Nature cancer

2024 Volume 5, Issue 2, Page(s) 262–282

Abstract: The contribution of antitumor immunity to metastatic dormancy is poorly understood. Here we show that the long noncoding RNA Malat1 is required for tumor initiation and metastatic reactivation in mouse models of breast cancer and other tumor types. ... ...

Abstract	The contribution of antitumor immunity to metastatic dormancy is poorly understood. Here we show that the long noncoding RNA Malat1 is required for tumor initiation and metastatic reactivation in mouse models of breast cancer and other tumor types. Malat1 localizes to nuclear speckles to couple transcription, splicing and mRNA maturation. In metastatic cells, Malat1 induces WNT ligands, autocrine loops to promote self-renewal and the expression of Serpin protease inhibitors. Through inhibition of caspase-1 and cathepsin G, SERPINB6B prevents gasdermin D-mediated induction of pyroptosis. In this way, SERPINB6B suppresses immunogenic cell death and confers evasion of T cell-mediated tumor lysis of incipient metastatic cells. On-target inhibition of Malat1 using therapeutic antisense nucleotides suppresses metastasis in a SERPINB6B-dependent manner. These results suggest that Malat1-induced expression of SERPINB6B can titrate pyroptosis and immune recognition at metastatic sites. Thus, Malat1 is at the nexus of tumor initiation, reactivation and immune evasion and represents a tractable and clinically relevant drug target.
MeSH term(s)	Animals ; Mice ; Cell Line, Tumor ; Pyroptosis ; RNA Splicing ; RNA, Long Noncoding/genetics ; T-Lymphocytes/metabolism
Chemical Substances	RNA, Long Noncoding ; Malat1 long non-coding RNA, mouse
Language	English
Publishing date	2024-01-09
Publishing country	England
Document type	Journal Article
ISSN	2662-1347
ISSN (online)	2662-1347
DOI	10.1038/s43018-023-00695-9
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Article ; Online: Translation of insulin granule proteins are regulated by PDI and PABP.

Sarwade, Rucha D / Khalique, Abdul / Kulkarni, Shardul D / Pandey, Poonam R / Gaikwad, Naina / Seshadri, Vasudevan

Biochemical and biophysical research communications

2020 Volume 526, Issue 3, Page(s) 618–625

Abstract: Glucose mediated insulin biosynthesis is tightly regulated and shared between insulin granule proteins such as its processing enzymes, prohormone convertases, PC1/3 and PC2. However, the molecular players involved in the co-ordinated translation remain ... ...

Abstract	Glucose mediated insulin biosynthesis is tightly regulated and shared between insulin granule proteins such as its processing enzymes, prohormone convertases, PC1/3 and PC2. However, the molecular players involved in the co-ordinated translation remain elusive. The trans-acting factors like PABP (Poly A Binding Protein) and PDI (Protein Disulphide Isomerize) binds to a conserved sequence in the 5'UTR of insulin mRNA and regulates its translation. Here, we demonstrate that 5'UTR of PC1/3 and PC2 also associate with PDI and PABP. We show that a' and RRM 3-4 domains of PDI and PABP respectively, are necessary for RNA binding activity to the 5'UTRs of insulin and its processing enzymes.
MeSH term(s)	5' Untranslated Regions ; Animals ; Cell Line ; Cytoplasmic Granules/genetics ; Cytoplasmic Granules/metabolism ; Insulin/genetics ; Insulin/metabolism ; Mice ; Poly(A)-Binding Proteins/genetics ; Poly(A)-Binding Proteins/metabolism ; Proprotein Convertase 1/genetics ; Proprotein Convertase 1/metabolism ; Proprotein Convertase 2/genetics ; Proprotein Convertase 2/metabolism ; Protein Biosynthesis ; Protein Disulfide-Isomerases/genetics ; Protein Disulfide-Isomerases/metabolism ; RNA, Messenger/genetics ; RNA, Messenger/metabolism
Chemical Substances	5' Untranslated Regions ; Insulin ; Poly(A)-Binding Proteins ; RNA, Messenger ; Proprotein Convertase 1 (EC 3.4.21.93) ; Proprotein Convertase 2 (EC 3.4.21.94) ; Protein Disulfide-Isomerases (EC 5.3.4.1)
Language	English
Publishing date	2020-04-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	205723-2
ISSN	1090-2104 ; 0006-291X ; 0006-291X
ISSN (online)	1090-2104 ; 0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2020.03.106
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Article ; Online: Interaction of HuDA and PABP at 5'UTR of mouse insulin2 regulates insulin biosynthesis.

Pandey, Poonam R / Sarwade, Rucha D / Khalique, Abdul / Seshadri, Vasudevan

PloS one

2018 Volume 13, Issue 3, Page(s) e0194482

Abstract: Understanding the regulation of insulin biosynthesis is important as it plays a central role in glucose metabolism. The mouse insulin gene2 (Ins2) has two splice variants; long (Ins2L) and short (Ins2S), that differ only in their 5'UTR sequence and Ins2S ...

Abstract	Understanding the regulation of insulin biosynthesis is important as it plays a central role in glucose metabolism. The mouse insulin gene2 (Ins2) has two splice variants; long (Ins2L) and short (Ins2S), that differ only in their 5'UTR sequence and Ins2S is the major transcript which translate more efficiently as compared to Ins2L. Here, we show that cellular factors bind preferentially to the Ins2L 5'UTR, and that PABP and HuD can bind to Ins2 splice variants and regulate its translation. In vitro binding assay with insulin 5'UTR and different HuD isoforms indicate that the 'N' terminal region of HuD is important for RNA binding and insulin translation repression. Using reporter assay we showed that specifically full-length HuD A isoform represses translation of reporter containing insulin 5'UTR. We further show that PABP and HuD interact with each other in RNA-dependent manner and this interaction is affected by glucose and PDI (5'UTR associated translation activator). These results suggest that PABP interacts with HuD in basal glucose conditions making translation inhibitory complex, however upon glucose stimulation this association is affected and PABP is acted upon by PDI resulting in stimulation of insulin translation. Together, our findings snapshot the mechanism of post-transcriptional regulation of insulin biosynthesis.
MeSH term(s)	5' Untranslated Regions ; Animals ; Cell Line ; ELAV-Like Protein 4/genetics ; ELAV-Like Protein 4/metabolism ; Insulin/biosynthesis ; Insulin/genetics ; Mice ; Peptide Chain Initiation, Translational ; Poly(A)-Binding Proteins/genetics ; Poly(A)-Binding Proteins/metabolism
Chemical Substances	5' Untranslated Regions ; ELAV-Like Protein 4 ; Elavl4 protein, mouse ; Ins2 protein, mouse ; Insulin ; Poly(A)-Binding Proteins
Language	English
Publishing date	2018
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0194482
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Article: RPAD (RNase R treatment, polyadenylation, and poly(A)+ RNA depletion) method to isolate highly pure circular RNA

Pandey, Poonam R / Rout, Pranita K / Das, Aniruddha / Gorospe, Myriam / Panda, Amaresh C

Methods. 2019 Feb. 15, v. 155

2019

Abstract: Recent developments in high-throughput RNA sequencing methods coupled with innovative bioinformatic tools have uncovered thousands of circular (circ)RNAs. CircRNAs have emerged as a vast and novel class of regulatory RNAs with potential to modulate gene ... ...

Abstract	Recent developments in high-throughput RNA sequencing methods coupled with innovative bioinformatic tools have uncovered thousands of circular (circ)RNAs. CircRNAs have emerged as a vast and novel class of regulatory RNAs with potential to modulate gene expression by acting as sponges for microRNAs (miRNAs) and RNA-binding proteins (RBPs). The biochemical enrichment of circRNAs by exoribonuclease treatment or by depletion of polyadenylated RNAs coupled with deep-sequencing is widely used for the systematic identification of circRNAs. Although these methods enrich circRNAs substantially, they do not eliminate efficiently non-polyadenylated and highly-structured RNAs. Here, we describe a method we termed RPAD, based on initial RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion. These joint interventions drastically depleted linear RNAs leading to isolation of highly pure circRNAs from total RNA pools. By facilitating the isolation of highly pure circRNAs, RPAD enables the elucidation of circRNA biogenesis, sequence, and function.
Keywords	RNA-binding proteins ; biogenesis ; bioinformatics ; gene expression ; high-throughput nucleotide sequencing ; microRNA ; ribonucleases
Language	English
Dates of publication	2019-0215
Size	p. 41-48.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2018.10.022
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Article: Translation of insulin granule proteins are regulated by PDI and PABP

Sarwade, Rucha D / Khalique, Abdul / Kulkarni, Shardul D / Pandey, Poonam R / Gaikwad, Naina / Seshadri, Vasudevan

Biochemical and biophysical research communications. 2020 June 04, v. 526, no. 3

2020

Abstract	Glucose mediated insulin biosynthesis is tightly regulated and shared between insulin granule proteins such as its processing enzymes, prohormone convertases, PC1/3 and PC2. However, the molecular players involved in the co-ordinated translation remain elusive. The trans-acting factors like PABP (Poly A Binding Protein) and PDI (Protein Disulphide Isomerize) binds to a conserved sequence in the 5′UTR of insulin mRNA and regulates its translation. Here, we demonstrate that 5′UTR of PC1/3 and PC2 also associate with PDI and PABP. We show that a’ and RRM 3–4 domains of PDI and PABP respectively, are necessary for RNA binding activity to the 5′UTRs of insulin and its processing enzymes.
Keywords	biosynthesis ; disulfides ; glucose ; insulin ; research
Language	English
Dates of publication	2020-0604
Size	p. 618-625.
Publishing place	Elsevier Inc.
Document type	Article
Note	NAL-AP-2-clean
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2020.03.106
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Article ; Online: Methods for analysis of circular RNAs.

Pandey, Poonam R / Munk, Rachel / Kundu, Gautam / De, Supriyo / Abdelmohsen, Kotb / Gorospe, Myriam

Wiley interdisciplinary reviews. RNA

2019 Volume 11, Issue 1, Page(s) e1566

Abstract: Eukaryotic cells express a myriad of circular RNAs (circRNAs), many of them displaying tissue-specific expression patterns. They arise from linear precursor RNAs in which 5' and 3' ends become covalently ligated. Given these features, biochemical and ... ...

Abstract	Eukaryotic cells express a myriad of circular RNAs (circRNAs), many of them displaying tissue-specific expression patterns. They arise from linear precursor RNAs in which 5' and 3' ends become covalently ligated. Given these features, biochemical and computational approaches traditionally used to study linear RNA must be adapted for analysis of circular RNAs. Such circRNA-specific methodologies are allowing the systematic identification of circRNAs and the analysis of their biological functions. Here, we review the resources and molecular methods currently utilized to quantify circRNAs, visualize their distribution, identify interacting partners, and elucidate their function. We discuss the challenges of analyzing circRNAs and propose alternative approaches for studying this unique class of transcripts. This article is characterized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Methods > RNA Analyses in vitro and In Silico RNA Methods > RNA Analyses in Cells.
MeSH term(s)	Animals ; Computational Biology ; Eukaryota/metabolism ; Humans ; Kinetics ; RNA, Circular/analysis ; RNA, Circular/metabolism
Chemical Substances	RNA, Circular
Language	English
Publishing date	2019-09-05
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Intramural ; Review
ZDB-ID	2634714-3
ISSN	1757-7012 ; 1757-7004
ISSN (online)	1757-7012
ISSN	1757-7004
DOI	10.1002/wrna.1566
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Article ; Online: LncRNA OIP5-AS1-directed miR-7 degradation promotes MYMX production during human myogenesis.

Yang, Jen-Hao / Chang, Ming-Wen / Tsitsipatis, Dimitrios / Yang, Xiaoling / Martindale, Jennifer L / Munk, Rachel / Cheng, Aiwu / Izydore, Elizabeth / Pandey, Poonam R / Piao, Yulan / Mazan-Mamczarz, Krystyna / De, Supriyo / Abdelmohsen, Kotb / Gorospe, Myriam

Nucleic acids research

2022 Volume 50, Issue 12, Page(s) 7115–7133

Abstract: Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) modulate gene expression programs in physiology and disease. Here, we report a noncoding RNA regulatory network that modulates myoblast fusion into multinucleated myotubes, a process that occurs during ...

Abstract	Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) modulate gene expression programs in physiology and disease. Here, we report a noncoding RNA regulatory network that modulates myoblast fusion into multinucleated myotubes, a process that occurs during muscle development and muscle regeneration after injury. In early stages of human myogenesis, the levels of lncRNA OIP5-AS1 increased, while the levels of miR-7 decreased. Moreover, OIP5-AS1 bound and induced miR-7 decay via target RNA-directed miRNA decay; accordingly, loss of OIP5-AS1 attenuated, while antagonizing miR-7 accelerated, myotube formation. We found that the OIP5-AS1-mediated miR-7 degradation promoted myoblast fusion, as it derepressed the miR-7 target MYMX mRNA, which encodes the fusogenic protein myomixer (MYMX). Remarkably, an oligonucleotide site blocker interfered with the OIP5-AS1-directed miR-7 degradation, allowing miR-7 to accumulate, lowering MYMX production and suppressing myotube formation. These results highlight a mechanism whereby lncRNA OIP5-AS1-mediated miR-7 decay promotes myotube formation by stimulating a myogenic fusion program.
MeSH term(s)	Humans ; RNA, Long Noncoding/genetics ; MicroRNAs/genetics ; Muscle Development/genetics
Chemical Substances	RNA, Long Noncoding ; MicroRNAs ; MIRN7 microRNA, human
Language	English
Publishing date	2022-06-23
Publishing country	England
Document type	Journal Article
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/gkac524
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