Concatemeric Broccoli reduces mRNA stability and induces aggregates

Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.

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Concatemeric Broccoli reduces mRNA stability, aggregates and induces p-body formation

10.1101/2020.12.07.414458 ◽

2020 ◽

Author(s):

Marco R. Rink ◽

Marisa A.P. Baptista ◽

Thomas Hennig ◽

Adam W. Whisnant ◽

Natalia Wolf ◽

...

Keyword(s):

Nuclear Export ◽

Substrate Binding ◽

Rna Stability ◽

Time Lapse ◽

Negative Effects ◽

Imaging Protocol ◽

P Bodies ◽

Copy Numbers ◽

Substrate Binding Sites ◽

Time Lapse Imaging

AbstractFluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation, a nuclear export defect and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Overexpression of Broccoli-tagged mRNA led to the formation of p-bodies. However, Broccoli RNAs did not localize to these sites. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.

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Faculty Opinions recommendation of Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1046453.496422 ◽

2006 ◽

Author(s):

Amnon Horovitz

Keyword(s):

Binding Sites ◽

Substrate Binding ◽

Substrate Binding Sites

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Faculty Opinions recommendation of Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718076922.793482351 ◽

2013 ◽

Author(s):

Robert Vandenberg

Keyword(s):

Dopamine Transporter ◽

Binding Sites ◽

Substrate Binding ◽

Multiple Substrate ◽

Substrate Binding Sites

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Covalent modification of substrate-binding sites of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-ADP-glucose-incorporated peptides.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36052-0 ◽

1986 ◽

Vol 261 (3) ◽

pp. 1058-1064

Author(s):

Y M Lee ◽

J Preiss

Keyword(s):

Escherichia Coli ◽

Structural Characterization ◽

Binding Sites ◽

Substrate Binding ◽

Covalent Modification ◽

Substrate Binding Sites

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Substrate-binding sites in acetylcholinesterase

Trends in Pharmacological Sciences ◽

10.1016/0165-6147(91)90621-x ◽

1991 ◽

Vol 12 ◽

pp. 422-426 ◽

Cited By ~ 26

Author(s):

Ferdinand Hucko ◽

Jaak Järv ◽

Christoph Weise

Keyword(s):

Binding Sites ◽

Substrate Binding ◽

Substrate Binding Sites

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Opposite stereospecificity of two tropinone reductases is conferred by the substrate-binding sites.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)32627-3 ◽

1994 ◽

Vol 269 (16) ◽

pp. 11695-11698 ◽

Cited By ~ 1

Author(s):

K. Nakajima ◽

T. Hashimoto ◽

Y. Yamada

Keyword(s):

Binding Sites ◽

Substrate Binding ◽

Substrate Binding Sites

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Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites

AJP Renal Physiology ◽

10.1152/ajprenal.00133.2004 ◽

2005 ◽

Vol 288 (2) ◽

pp. F327-F333 ◽

Cited By ~ 147

Author(s):

Rémon A. M. H. Van Aubel ◽

Pascal H. E. Smeets ◽

Jeroen J. M. W. van den Heuvel ◽

Frans G. M. Russel

Keyword(s):

Binding Sites ◽

Efflux Pump ◽

Substrate Binding ◽

Organic Anion ◽

Apical Cell ◽

Cell Uptake ◽

Hek293 Cells ◽

Hill Coefficient ◽

Anion Transporter ◽

Substrate Binding Sites

The end product of human purine metabolism is urate, which is produced primarily in the liver and excreted by the kidney through a well-defined basolateral blood-to-cell uptake step. However, the apical cell-to-urine efflux mechanism is as yet unidentified. Here, we show that the renal apical organic anion efflux transporter human multidrug resistance protein 4 (MRP4), but not apical MRP2, mediates ATP-dependent urate transport via a positive cooperative mechanism ( Km of 1.5 ± 0.3 mM, Vmax of 47 ± 7 pmol·mg−1·min−1, and Hill coefficient of 1.7 ± 0.2). In HEK293 cells overexpressing MRP4, intracellular urate levels were lower than in control cells. Urate inhibited methotrexate transport (IC50 of 235 ± 8 μM) by MRP4, did not affect cAMP transport, whereas cGMP transport was stimulated. Urate shifted cGMP transport by MRP4 from positive cooperativity ( Km and Vmax value of 180 ± 20 μM and 58 ± 4 pmol·mg−1·min−1, respectively, Hill coefficient of 1.4 ± 0.1) to single binding site kinetics ( Km and Vmax value of 2.2 ± 0.9 mM and 280 ± 50 pmol·mg−1·min−1, respectively). Finally, MRP4 could transport urate simultaneously with cAMP or cGMP. We conclude that human MRP4 is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites. We propose MRP4 as a candidate transporter for urinary urate excretion and suggest that MRP4 may also mediate hepatic export of urate into the circulation, because of its basolateral expression in the liver.

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