Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3’ UTR structures

Ward, Alex M.; Bidet, Katell; Yinglin, Ang; Ler, Siok Ghee; Hogue, Kelly; Blackstock, Walter; Gunaratne, Jayantha; García-Blanco, Mariano A.

doi:10.4161/rna.8.6.17836

Cited by 163 publications

(230 citation statements)

References 59 publications

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“…We thus concluded that HeLa cell nuclear extracts contain all of the necessary factors required to recapitulate exon 6 splicing in vitro, and used these extracts in all further chromatography experiments. To identify trans-acting protein factors that bind exon 6 and/or adjacent intronic regions we used tobramycin RNA affinity chromatography (Hartmuth et al 2004;Ward et al 2011;K Hartmuth, pers. comm.).…”

Section: Resultsmentioning

confidence: 99%

Cleavage and polyadenylation specificity factor 1 (CPSF1) regulates alternative splicing of interleukin 7 receptor (IL7R) exon 6

Evsyukova

Bradrick²,

Gregory

et al. 2012

RNA

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Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage, and its expression is crucial for the development and maintenance of T cells. Alternative splicing of IL7R exon 6 results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing. We identified multiple exonic and intronic cis-acting elements that impact inclusion of exon 6. Moreover, we utilized RNA affinity chromatography followed by mass spectrometry to identify trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein-binding candidates. A consensus polyadenylation signal AAUAAA is present in intron 6 of IL7R directly downstream from the 59 splice site. Mutations to this site and CPSF1 knockdown both resulted in an increase in exon 6 inclusion. We found no evidence that this site is used to produce cleaved and polyadenylated mRNAs, suggesting that CPSF1 interaction with intronic IL7R pre-mRNA interferes with spliceosome binding to the exon 6 59 splice site. Our results suggest that competing mRNA splicing and polyadenylation regulate exon 6 inclusion and consequently determine the ratios of soluble to membrane-bound IL7R. This may be relevant for both T cell ontogeny and function and development of multiple sclerosis.

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Section: Resultsmentioning

confidence: 99%

Cleavage and polyadenylation specificity factor 1 (CPSF1) regulates alternative splicing of interleukin 7 receptor (IL7R) exon 6

Evsyukova

Bradrick²,

Gregory

et al. 2012

RNA

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“…Infection with West Nile virus or hepatitis C virus leads to a progressive reduction in the number of P-body foci 24 -36 hpi and relocalization of P-body components to viral replication centers (15)(16)(17). The genomic RNA of dengue virus interacts with the P-body component Rck/p54 (DDX6) and is presumed to be important for viral replication (18).…”

mentioning

confidence: 99%

mRNA Decapping Enzyme 1a (Dcp1a)-induced Translational Arrest through Protein Kinase R (PKR) Activation Requires the N-terminal Enabled Vasodilator-stimulated Protein Homology 1 (EVH1) Domain

Dougherty

Reineke

Lloyd

2014

Journal of Biological Chemistry

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“…4,38,39 Liu et al reported that mRNAs that are targeted for translational repression by endogenous or exogenous miRNAs become concentrated in P-bodies in a miRNAdependent manner. 40 In this study, we have confirmed that downregulation of G3BP2 by miR-23b could decrease kidney fibrosis and alleviate proteinuria in DN.…”

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confidence: 99%

MicroRNA-23b Targets Ras GTPase-Activating Protein SH3 Domain-Binding Protein 2 to Alleviate Fibrosis and Albuminuria in Diabetic Nephropathy

Zhao¹,

Li²,

Liu³

et al. 2016

JASN

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Diabetic nephropathy (DN) is a frequent and severe complication of diabetes that is structurally characterized by glomerular basement membrane thickening, extracellular matrix accumulation, and destabilization of podocyte foot processes. MicroRNAs (miRNAs) are dysregulated in DN, but identification of the specific miRs involved remains incomplete. Here, we confirm that the peripheral blood from patients with diabetes and the kidneys of animals with type 1 or 2 diabetes have low levels of miR-23b compared with those of their nondiabetic counterparts. Furthermore, exposure to high glucose downregulated miR-23b in cultured kidney cells. In contrast, renal expression of Ras GTPase-activating protein SH3 domain-binding protein 2 (G3BP2), a putative miR-23b target, increased in DN. In vitro, overexpression of miR-23b decreased, and inhibition of miR-23b increased, G3BP2 expression levels. Bioinformatics analysis also revealed p53 binding sites in the miR-23b promoter; in vitro inhibition of p53 or the upstream p38 mitogen-activated protein kinase (p38MAPK) upregulated miR-23b expression in high-glucose conditions. In turn, inhibition of G3BP2 or overexpression of miR-23b downregulated p53 and p38MAPK expression in high-glucose conditions. In vivo, overexpression of miR23b or inhibition of p53 in db/db mice reversed hyperalbuminuria and kidney fibrosis, whereas miR-23b antagomir treatment promoted renal fibrosis and increased albuminuria in wild-type mice. These data suggest that hyperglycemia regulates pathogenic processes in DN through an miR-23b/G3BP2 feedback circuit involving p38MAPK and p53. In conclusion, these results reveal a role for miR-23b in DN and indicate a novel potential therapeutic target.

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Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3’ UTR structures

Cited by 163 publications

References 59 publications

Cleavage and polyadenylation specificity factor 1 (CPSF1) regulates alternative splicing of interleukin 7 receptor (IL7R) exon 6

Cleavage and polyadenylation specificity factor 1 (CPSF1) regulates alternative splicing of interleukin 7 receptor (IL7R) exon 6

mRNA Decapping Enzyme 1a (Dcp1a)-induced Translational Arrest through Protein Kinase R (PKR) Activation Requires the N-terminal Enabled Vasodilator-stimulated Protein Homology 1 (EVH1) Domain

MicroRNA-23b Targets Ras GTPase-Activating Protein SH3 Domain-Binding Protein 2 to Alleviate Fibrosis and Albuminuria in Diabetic Nephropathy

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