The biochemistry of RNA metabolism studied in situ

Nickerson, Jeffrey A.

doi:10.4161/rna.6.1.7563

Cited by 6 publications

(11 citation statements)

References 28 publications

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“…To study the effect of stress granule-associated mutant-FUS on the dynamic properties of stress granules, we employed fluorescence recovery after photobleaching (FRAP). FRAP measures the relative kinetics and affinities of protein binding within complexes, such as stress granules, over the time course of the experiment [44,45]. Because FRAP reports on binding events in live cells, fluorescently tagged-proteins were employed for these experiments [32,42,43,46].…”

Section: Resultsmentioning

confidence: 99%

“…The fluorescence signal recovers as bleached molecules unbind from sites in the stress granules, un-bleached fluorescent molecules exchange back into the photobleached area and then bind. The fluorescence recovery time is limited by either diffusion, which is faster than the rates we report here, or by binding kinetics; thus, proteins that are tightly bound to other proteins or cellular structures exhibit relatively long half times of fluorescence recovery ( t 1/2 ) [44]. The population of fluorescent molecules that exchange with their bleached counterparts over the time course of the experiment comprise the “mobile fraction” [44].…”

Section: Resultsmentioning

confidence: 99%

“…The fluorescence recovery time is limited by either diffusion, which is faster than the rates we report here, or by binding kinetics; thus, proteins that are tightly bound to other proteins or cellular structures exhibit relatively long half times of fluorescence recovery ( t 1/2 ) [44]. The population of fluorescent molecules that exchange with their bleached counterparts over the time course of the experiment comprise the “mobile fraction” [44]. The “immobile fraction” is the population that is tightly bound and does not exchange over the time course of the experiment.…”

Section: Resultsmentioning

confidence: 99%

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Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics

Baron

Kaushansky

Ward

et al. 2013

Mol Neurodegeneration

126

115

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BackgroundAmyotrophic lateral sclerosis (ALS)-linked fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is concentrated within cytoplasmic stress granules under conditions of induced stress. Since only the mutants, but not the endogenous wild-type FUS, are associated with stress granules under most of the stress conditions reported to date, the relationship between FUS and stress granules represents a mutant-specific phenotype and thus may be of significance in mutant-induced pathogenesis. While the association of mutant-FUS with stress granules is well established, the effect of the mutant protein on stress granules has not been examined. Here we investigated the effect of mutant-FUS on stress granule formation and dynamics under conditions of oxidative stress.ResultsWe found that expression of mutant-FUS delays the assembly of stress granules. However, once stress granules containing mutant-FUS are formed, they are more dynamic, larger and more abundant compared to stress granules lacking FUS. Once stress is removed, stress granules disassemble more rapidly in cells expressing mutant-FUS. These effects directly correlate with the degree of mutant-FUS cytoplasmic localization, which is induced by mutations in the nuclear localization signal of the protein. We also determine that the RGG domains within FUS play a key role in its association to stress granules. While there has been speculation that arginine methylation within these RGG domains modulates the incorporation of FUS into stress granules, our results demonstrate that this post-translational modification is not involved.ConclusionsOur results indicate that mutant-FUS alters the dynamic properties of stress granules, which is consistent with a gain-of-toxic mechanism for mutant-FUS in stress granule assembly and cellular stress response.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics

Baron

Kaushansky

Ward

et al. 2013

Mol Neurodegeneration

126

115

View full text Add to dashboard Cite

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“…, 2006; Lele and Ingber, 2006; Kota et al. , 2008; Nickerson, 2009). To identify mRNA export factors whose assembly into export complexes is regulated by the PI signal transduction pathway, we screened enhanced green fluorescent protein (EGFP)-fusion proteins of known mRNA export factors for altered photobleach recovery kinetics after drug inhibition of the PI pathway in live HeLa cells.…”

Section: Resultsmentioning

confidence: 99%

“…These include the steps of equilibrium binding and diffusion (Lele et al. , 2004; Nickerson, 2009): 1) the unbinding of bleached molecules, 2) the unbinding of still-fluorescent molecules outside of the bleach zone, 3) the exchange of these two pools of molecules by diffusion, and 4) the binding of fluorescent molecules in the bleach zone. FRAP recovery for an mRNA transcript might also depend on the movement of a completed and processed mRNA transcript away from the transcription site, with recovery coming from unbleached molecules binding to a new mRNA being transcribed (Ben-Ari et al.…”

Section: Discussionmentioning

confidence: 99%

Regulation of mRNA export by the PI3 kinase/AKT signal transduction pathway

Quaresma

Sievert

Nickerson

2013

MBoC

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Live cell imaging of the cancer‐related transcription factor RUNX2 during mitotic progression

Pockwinse

Kota

Quaresma

et al. 2011

Journal Cellular Physiology

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The nuclear matrix bound transcription factor RUNX2 is a lineage-specific developmental regulator that is linked to cancer. We have previously shown that RUNX2 controls transcription of both RNA polymerase II genes and RNA polymerase I dependent ribosomal RNA genes. RUNX2 is epigenetically retained through mitosis on both classes of target genes in condensed chromosomes. We have used fluorescence recovery after photobleaching (FRAP) to measure the relative binding kinetics of EGFP-RUNX2 at transcription sites in the nucleus and nucleoli during interphase, as well as on mitotic chromosomes. RUNX2 becomes more strongly bound as cells go from interphase through prophase, with a doubling of the most tightly bound "immobile fraction". RUNX2 exchange then becomes much more facile during metaphase to telophase. During interphase the less tightly bound pool of RUNX2 exchanges more slowly at nucleoli than at subnuclear foci, and the non-exchanging immobile fraction is greater in nucleoli. These results are consistent with a model in which the molecular mechanism of RUNX2 binding is different at protein-coding and ribosomal RNA genes. The binding interactions of RUNX2 change as cells go through mitosis, with binding affinity increasing as chromosomes condense and then decreasing through subsequent mitotic phases. The increased residence of RUNX2 at mitotic chromosomes may reflect its epigenetic function in "bookmarking" of target genes in cancer cells.

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The biochemistry of RNA metabolism studied in situ

Cited by 6 publications

References 28 publications

Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics

Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics

Regulation of mRNA export by the PI3 kinase/AKT signal transduction pathway

Live cell imaging of the cancer‐related transcription factor RUNX2 during mitotic progression

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