Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation

Aboulhouda, Soufiane; Santo, Rachael Di; Thérizols, Gabriel; Weinberg, David E.

doi:10.21769/bioprotoc.2573

Cited by 14 publications

(11 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Moreover, we analysed the translation status of Hif1a mRNA, using its association with ribosomes, because the translational activation of specific mRNAs occurred during macrophage activation 39 . Hif1a mRNA was present in large polysome fractions 40 in MYD88 L265P expressing cells compared with control cells (Fig. 4 e), suggesting MYD88 signals also activate Hif1a mRNA translational efficiency.…”

Section: Resultsmentioning

confidence: 88%

See 1 more Smart Citation

MYD88 signals induce tumour-initiating cell generation through the NF-κB-HIF-1α activation cascade

Tanimura

Nakazato

Tanaka

2021

Sci Rep

View full text Add to dashboard Cite

Tumour-promoting inflammation is a hallmark of cancer, and chronic inflammatory disease increases the risk of cancer. In this context, MYD88, a downstream signalling molecule of Toll-like receptors that initiates inflammatory signalling cascades, has a critical role in tumour development in mice and its gene mutation was found in human cancers. In inflammation-induced colon cancer, tumour suppressor p53 mutations have also been detected with high frequency as early events. However, the molecular mechanism of MYD88-induced cancer development is poorly understood. Here, we demonstrated that MYD88 induced the protein accumulation of the transcription factor HIF-1α through NF-κB in p53-deficient cells. HIF-1α accumulation was not caused by enhanced protein stability but by NF-κB-mediated transcriptional activation, the enhanced translation of HIF-1α and JNK activation. In contrast, MYD88-induced mRNA expressions of HIF-1α and HIF-1-target genes were attenuated in the presence of p53. Furthermore, constitutively active forms of MYD88 induced tumour-initiating cell (TIC) generation in p53-deficient cells, as determined by tumour xenografts in nude mice. TIC generating activity was diminished by the suppression of NF-κB or HIF-1α. These results indicate that MYD88 signals induce the generation of TICs through the NF-κB-HIF-1α activation cascade in p53-deficient cells and suggest this molecular mechanism underlies inflammation-induced cancer development.

show abstract

Section: Resultsmentioning

confidence: 88%

“…The polysomal fraction was isolated from cells as previously described 40 . Briefly, the cells were washed twice with PBS containing 100 μg/ml cycloheximide (CHX) and lysed with lysis buffer.…”

Section: Methodsmentioning

confidence: 99%

MYD88 signals induce tumour-initiating cell generation through the NF-κB-HIF-1α activation cascade

Tanimura

Nakazato

Tanaka

2021

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Polysome analyses by sucrose gradient fractionation are described in more detail at Bio-protocol (Aboulhouda et al, 2017). Yeast strains were grown to mid-log phase (OD 600 ~ 0.5) in YPD and harvested by vacuum filtration as previously described (Weinberg et al, 2016).…”

Section: Methodsmentioning

confidence: 99%

The fail-safe mechanism of post-transcriptional silencing of unspliced HAC1 mRNA

Santo

Aboulhouda

Weinberg

2016

eLife

Self Cite

View full text Add to dashboard Cite

HAC1 encodes a transcription factor that is the central effector of the unfolded protein response (UPR) in budding yeast. When the UPR is inactive, HAC1 mRNA is stored as an unspliced isoform in the cytoplasm and no Hac1 protein is detectable. Intron removal is both necessary and sufficient to relieve the post-transcriptional silencing of HAC1 mRNA, yet the precise mechanism by which the intron prevents Hac1 protein accumulation has remained elusive. Here, we show that a combination of inhibited translation initiation and accelerated protein degradation—both dependent on the intron—prevents the accumulation of Hac1 protein when the UPR is inactive. Functionally, both components of this fail-safe silencing mechanism are required to prevent ectopic production of Hac1 protein and concomitant activation of the UPR. Our results provide a mechanistic understanding of HAC1 regulation and reveal a novel strategy for complete post-transcriptional silencing of a cytoplasmic mRNA.DOI: http://dx.doi.org/10.7554/eLife.20069.001

show abstract

“…Testicular seminiferous tubules were dissected and collected according to previous literature with minor modifications(Aboulhouda et al, 2017; Karamysheva et al, 2018). Briefly after adding polysome extraction buffer (20mM Tris-HCl, pH 7.4, 100 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 0.5% NP-40, 1x protease inhibitor cocktail (EDTA-free), 200 μg/ml CHX, 200 units/mL of RNase inhibitor), the tubules were transferred to a small (0.5-1.0 mL) Dounce homogenizer to disrupt the tissues with seven to eight strokes of the glass pestle.…”

Section: Methodsmentioning

confidence: 99%

Mitofusin2 cooperates with Nuage-associated proteins and involves mRNA translational machinery in controlling mRNA fates during spermatogenesis

Wang

Zhang

et al. 2020

Preprint

View full text Add to dashboard Cite

Mitochondria play a critical role in spermatogenesis and regulated by several mitochondrial fusion proteins. Its interaction with other organelles forms several unique structures, including mitochondria-associated ER membrane (MAM) and a specific type of Nuage close to mitochondria. However, the importance of mitochondria functions and mitochondrial fusion proteins in its associated-structure formation and mRNA translation during spermatogenesis remain unclear. Here, we show that Mitofusin 2 (MFN2), a mitochondrial fusion GTPase protein, cooperates with Nuage-associated proteins, including MIWI, DDX4, TDRKH and GASZ and involves translational machinery to control the fates of gamete-specific mRNAs in spermatogenesis. Conditional mutation of Mfn2 in postnatal germ cells results in male sterility due to germ cell developmental defects characterized by disruption of mitochondrial morphology, abnormal MAMs structure, aberrant mRNA translational processes, and anomalous splicing events. Moreover, MFN2 interacts with MFN1, another mitochondrial fusion protein with high-sequence similar to MFN2, in testes to facilitate spermatogenesis. Mutation of Mfn1 and Mfn2 simultaneously in testes causes very severe infertile phenotypes. Importantly, we further show that MFN2 is enriched in polysome fractions in testes and interacts with MSY2, a germ cell-specific DNA/RNA-binding protein, and eukaryotic elongation factor 1 alpha (eEF1A) to control gamete-specific mRNA translational delay during spermatogenesis. Collectively, our findings demonstrate that MFN2 works with Nuage-associated proteins and involves translational secession to regulate gamete-specific mRNA fates. Our data reveal a novel molecular link among Mitofusins, Nuage-associated proteins, and mRNA translational processes in controlling male germ cell development.

show abstract

Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation

Cited by 14 publications

References 11 publications

MYD88 signals induce tumour-initiating cell generation through the NF-κB-HIF-1α activation cascade

MYD88 signals induce tumour-initiating cell generation through the NF-κB-HIF-1α activation cascade

The fail-safe mechanism of post-transcriptional silencing of unspliced HAC1 mRNA

Mitofusin2 cooperates with Nuage-associated proteins and involves mRNA translational machinery in controlling mRNA fates during spermatogenesis

Contact Info

Product

Resources

About