Polysome Fractionation for Transcriptome-Wide Studies of mRNA Translation

Ristau, Johannes; Watt, Kathleen; Oertlin, Christian; Larsson, Ola

doi:10.1007/978-1-0716-1920-9_14

Cited by 10 publications

(11 citation statements)

References 66 publications

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“…Thus, for stressed samples, we calculate a “ribosome association” score which is TPM rib. bound /TPM Total 94 . This metric is similar to “translation efficiency” scores calculated for ribosome profiling studies 50 .…”

Section: Methodsmentioning

confidence: 99%

Transcriptome-wide mRNA condensation precedes stress granule formation and excludes stress-induced transcripts

Glauninger,

Bard,

Wong Hickernell

et al. 2024

Preprint

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Stress-induced condensation of mRNA and proteins into stress granules is conserved across eukaryotes, yet the function, formation mechanisms, and relation to well-studied conserved transcriptional responses remain largely unresolved. Stress-induced exposure of ribosome-free mRNA following translational shutoff is thought to cause condensation by allowing new multivalent RNA-dependent interactions, with RNA length and associated interaction capacity driving increased condensation. Here we show that, in striking contrast, virtually all mRNA species condense in response to multiple unrelated stresses in budding yeast, length plays a minor role, and instead, stress-induced transcripts are preferentially excluded from condensates, enabling their selective translation. Using both endogenous genes and reporter constructs, we show that translation initiation blockade, rather than resulting ribosome-free RNA, causes condensation. These translation initiation-inhibited condensates (TIICs) are biochemically detectable even when stress granules, defined as microscopically visible foci, are absent or blocked. TIICs occur in unstressed yeast cells, and, during stress, grow before the appearance of visible stress granules. Stress-induced transcripts are excluded from TIICs primarily due to the timing of their expression, rather than their sequence features. Together, our results reveal a simple system by which cells redirect translational activity to newly synthesized transcripts during stress, with broad implications for cellular regulation in changing conditions.

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Section: Methodsmentioning

confidence: 99%

Transcriptome-wide mRNA condensation precedes stress granule formation and excludes stress-induced transcripts

Glauninger,

Bard,

Wong Hickernell

et al. 2024

Preprint

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“…A total of 1.8 million MCF7 cells were plated in a 15 cm dish and the following day transfected for 48 hours with control siRNA or an siRNA targeting dyskerin. After harvesting, cells were subjected to polysome fractionation as described previously ( 78 ). Fractions were collected as described in ( 78 ), and equal volumes from fractions corresponding to more than three ribosomes were pooled and the RNA extracted.…”

Section: Methodsmentioning

confidence: 99%

“…After harvesting, cells were subjected to polysome fractionation as described previously ( 78 ). Fractions were collected as described in ( 78 ), and equal volumes from fractions corresponding to more than three ribosomes were pooled and the RNA extracted. Equal amounts of RNA were used for reverse transcription and qPCR, as described above.…”

Section: Methodsmentioning

confidence: 99%

Control of protein synthesis through mRNA pseudouridylation by dyskerin

et al. 2023

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Posttranscriptional modifications of mRNA have emerged as regulators of gene expression. Although pseudouridylation is the most abundant, its biological role remains poorly understood. Here, we demonstrate that the pseudouridine synthase dyskerin associates with RNA polymerase II, binds to thousands of mRNAs, and is responsible for their pseudouridylation, an action that occurs in chromatin and does not appear to require a guide RNA with full complementarity. In cells lacking dyskerin, mRNA pseudouridylation is reduced, while at the same time, de novo protein synthesis is enhanced, indicating that this modification interferes with translation. Accordingly, mRNAs with fewer pseudouridines due to knockdown of dyskerin are translated more efficiently. Moreover, mRNA pseudouridylation is severely reduced in patients with dyskeratosis congenita caused by inherited mutations in the gene encoding dyskerin (i.e., DKC1 ). Our findings demonstrate that pseudouridylation by dyskerin modulates mRNA translatability, with important implications for both normal development and disease.

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“…S3a). This isogenic cell pair was then used to assess TSC1-associated changes in gene expression at multiple levels using polysome-profiling 42 .…”

Section: Tsc1 Loss Leads To Widespread Alterations Of Mrna Translatio...mentioning

confidence: 99%

“…Polysome fractionation of post-mortem Brodmann area 19 samples from ASD-affected donors (n=6) and matched controls (n=4) provided by NIH NeuroBiobank was performed using an optimized sucrose gradient, as previously described 42,48 . RNA sequencing libraries were generated using the Smart-seq2 protocol as described previously 48 .…”

Section: Polysome Fractionation Of Post-mortem Brain Samplesmentioning

confidence: 99%

Translatome analysis of Tuberous Sclerosis Complex-1 patient-derived neural progenitor cells reveal rapamycin-dependent and independent alterations

Aksoylu

Martín

Robert

et al. 2023

Preprint

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Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD). The hamartin-tuberin (TSC1-TSC2) protein complex inactivates mechanistic target of rapamycin complex 1 (mTORC1) signaling, leading to increased protein synthesis via inactivation of translational repressor eIF4E-binding proteins (4E-BPs). In TSC1-null neural progenitor cells (NPCs), we previously reported early ND phenotypic changes, including increased proliferation/altered neurite outgrowth, which were unaffected by mTORC1-inhibitor rapamycin. Here, using polysome-profiling to quantify translational efficiencies at a transcriptome-wide level, we observed numerous TSC1-dependent alterations in NPCs, largely recapitulated in post-mortem brains from ASD donors. Although rapamycin partially reversed TSC1-associated alterations, most neural activity/synaptic- or ASD-related genes remained insensitive but were inhibited by third-generation bi-steric, mTORC1-selective inhibitor RMC-6272, which also reversed altered ND phenotypes. Together these data reveal potential implications for treatment of TAND.

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Polysome Fractionation for Transcriptome-Wide Studies of mRNA Translation

Cited by 10 publications

References 66 publications

Transcriptome-wide mRNA condensation precedes stress granule formation and excludes stress-induced transcripts

Transcriptome-wide mRNA condensation precedes stress granule formation and excludes stress-induced transcripts

Control of protein synthesis through mRNA pseudouridylation by dyskerin

Translatome analysis of Tuberous Sclerosis Complex-1 patient-derived neural progenitor cells reveal rapamycin-dependent and independent alterations

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