A guide to design and optimization of reporter assays for 3′ untranslated region mediated regulation of mammalian messenger RNAs

Etten, Jamie Van; Schagat, Trista; Goldstrohm, Aaron C.

doi:10.1016/j.ymeth.2013.04.020

Cited by 21 publications

(29 citation statements)

References 14 publications

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“…A relative response ratio (RRR) was calculated from RnLuc/FFLuc signals for each sample. Percent repression values were calculated as previously described (Van Etten et al, 2013; Weidmann and Goldstrohm, 2012). The pIZ-Halo-tag vector served as the negative control for Halo-NZC Nos constructs.…”

Section: Methodsmentioning

confidence: 99%

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

Weidmann

Qiu

Arvola

et al. 2016

eLife

169

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Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.DOI: http://dx.doi.org/10.7554/eLife.17096.001

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Section: Methodsmentioning

confidence: 99%

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

Weidmann

Qiu

Arvola

et al. 2016

eLife

169

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“…Firefly luciferase activity was normalized to Renilla luciferase activity to account for transfection efficiency. Analysis of data was performed as previously reported (Van Etten et al, 2013) and expressed as mean ± SEM of percent repression from 3 independent experiments using a two-sample equal variance t-test with GraphPad Prism 5.…”

Section: Methodsmentioning

confidence: 99%

Expression of microRNAs in human post-mortem amyotrophic lateral sclerosis spinal cords provides insight into disease mechanisms

Figueroa‐Romero

Hur

Lunn

et al. 2016

Molecular and Cellular Neuroscience

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Amyotrophic lateral sclerosis is a late-onset and terminal neurodegenerative disease. The majority of cases are sporadic with unknown causes and only a small number of cases are genetically linked. Recent evidence suggests that post-transcriptional regulation and epigenetic mechanisms, such as microRNAs, underlie the onset and progression of neurodegenerative disorders; therefore, altered microRNA expression may result in the dysregulation of key genes and biological pathways that contribute to the development of sporadic amyotrophic lateral sclerosis. Using systems biology analyses on postmortem human spinal cord tissue, we identified dysregulated mature microRNAs and their potential targets previously implicated in functional process and pathways associated with the pathogenesis of ALS. Furthermore, we report a global reduction of mature microRNAs, alterations in microRNA processing, and support for a role of the nucleotide binding protein, TAR DNA binding protein 43, in regulating sporadic amyotrophic lateral sclerosis-associated microRNAs, thereby offering a potential underlying mechanism for sporadic amyotrophic lateral sclerosis.

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“…Melt curve was generated with range 65-95°C at increments of 0.5°C. Data were analyzed using Pfaffl method (60), normalizing to the Rpl32 mRNA, with fold change calculated relative to the NTC negative control as previously described (36,61).…”

Section: Reverse Transcription and Quantitative Polymerase Chain Reacmentioning

confidence: 99%

Unique repression domains of Pumilio utilize deadenylation and decapping factors to accelerate destruction of target mRNAs

Arvola

Chang

Buytendorp

et al. 2019

Preprint

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Pumilio is an RNA-binding protein that represses a network of mRNAs to control embryogenesis, stem cell fate, fertility, and neurological functions in Drosophila. We sought to identify the mechanism of Pumilio-mediated repression and find that it accelerates degradation of target mRNAs, mediated by three N-terminal Repression Domains (RDs), which are unique to Pumilio orthologs. We show that the repressive activities of the Pumilio RDs depend on specific subunits of the Ccr4-Not (CNOT) deadenylase complex. Depletion of Pop2, Not1, Not2, or Not3 subunits alleviates Pumilio RD-mediated repression of protein expression and mRNA decay, whereas depletion of other CNOT components had little or no effect. Moreover, the catalytic activity of Pop2 deadenylase is important for Pumilio RD activity. Further, we show that the Pumilio RDs directly bind to the CNOT complex. We also report that the decapping enzyme, Dcp2, participates in repression by the N-terminus of Pumilio. These results support a model wherein Pumilio utilizes CNOT deadenylase and decapping complexes to accelerate destruction of target mRNAs. Because the N-terminal RDs are conserved in mammalian Pumilio orthologs, the results of this work broadly enhance our understanding of Pumilio function and roles in diseases including cancer, neurodegeneration, and epilepsy.

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A guide to design and optimization of reporter assays for 3′ untranslated region mediated regulation of mammalian messenger RNAs

Cited by 21 publications

References 14 publications

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

Expression of microRNAs in human post-mortem amyotrophic lateral sclerosis spinal cords provides insight into disease mechanisms

Unique repression domains of Pumilio utilize deadenylation and decapping factors to accelerate destruction of target mRNAs

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