Dissociation of Ribosomes into Large and Small Subunits

Rivera, Maria C.; Maguire, Bruce A.; Lake, James A.

doi:10.1101/pdb.prot081372

Cited by 3 publications

(3 citation statements)

References 3 publications

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“…Puromycin is commonly used as a protein synthesis inhibitor and dissociates ribosomes from translating peptides (Blobel and Sabatini 1971;Rivera et al 2015). However, we did not see a change in the number of RPM puncta in the absence of emetine, a drug traditionally used to repress translation and prevent puromycin dissociation of nascent peptides (David and Yewdell 2015).…”

Section: Stalled Polysomes Can Be Labeled By Puromycin But Nascent Ccontrasting

confidence: 75%

Polysomes identified by live imaging of nascent peptides are stalled in hippocampal and cortical neurites

2019

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In neurons, mRNAs can be repressed postinitiation and assembled into granules enabling the transport and later, regulated reactivation of the paused mRNAs. It has been suggested that a large percentage of transcripts in neuronal processes are stored in these stalled polysomes. Given this, it is predicted that nascent peptides should be abundant in these granules. Nascent peptides can be visualized in real time by the SunTag system. Using this system, we observe nascent peptides in neuronal processes that are resistant to runoff with the initiation inhibitor homoharringtonin (HHT) and to release by puromycin, properties expected from RNA granules consisting of stalled polysomes. In contrast, nascent peptides in nonneuronal cells and neuronal cell bodies were not resistant to HHT or puromycin. Stalled polysomes can also be visualized after runoff with ribopuromycylation and the RNA granules imaged with ribopuromycylation were the same as those with SunTag visualized nascent peptides. Accordingly, the ribopuromycylated puncta in neuronal dendrites were also resistant to puromycin. Thus, the SunTag technique corroborates in situ evidence of stalled polysomes and will allow for the live examination of these translational structures as a mechanism for mRNA transport and regulated protein synthesis.

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Section: Stalled Polysomes Can Be Labeled By Puromycin But Nascent Ccontrasting

confidence: 75%

Polysomes identified by live imaging of nascent peptides are stalled in hippocampal and cortical neurites

2019

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“…To validate the hypothesis that rRNAs within split ribosomal subunits become susceptible to RNase I, we separated the ribosome into the 30S and 50S subunits 48 and then incubated with RNase I. As expected, rRNAs of subunits were more degraded by RNase I than that of 70S ribosomes (Fig.…”

Section: Rnase I Degrades Rrna Within Dissociated Ribosomal Subunits ...mentioning

confidence: 57%

‘Tuning’ of ribosome levels mediated by RNase I and hibernating ribosomes

Minami,

Tanzawa,

Yang

et al. 2024

Preprint

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Ribosomes consume vast energy to synthesize proteins, so controlling the ribosome abundance is a significant concern for cells. Ribonucleases mediate ribosome degradation in response to stresses, while some ribosomes deactivate translational activity and protect themselves from degradation, called ribosome hibernation. RNase T2 is an endoribonuclease found in almost all organisms, and they are thought to be involved in the degradation of ribosomal RNA. Although it was recently reported that the activity ofEscherichia coliRNase T2, called RNase I, depends on the environmental conditions, the regulation mechanism remains elusive. Here, we report how rRNA degradation by RNase I is regulated by hibernating ribosomes. Combining the biochemical, cryo-electron microscopy, and single-molecule analyses, we found that hibernating ribosome is an inhibitor by forming a complex with RNase I. Moreover, RNase I does not bind to the translating ribosome, so rRNA is protected. On the other hand, RNase I degrades the rRNA of each subunit dissociated from stalled ribosomes on aberrant mRNA bytrans-translation. Under stress conditions, and even in the actively growing phase, some ribosomes are stalling or pausing. Although such ribosomes were thought to be recycled after being rescued, our results add a new insight that they are not recycled but degraded. These findings have broad implications for understanding the regulation of ribosome levels, which is critical for cellular homeostasis and response to environmental stresses.

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“…Discard the supernatant. Resuspend the pellet in prokaryotic storage buffer or in a buffer suitable for further purification of subunits, 70S ribosomes, or polysomes (see Protocol: Purification of 70S Ribosomes [Rivera et al 2014a], Protocol: Purification of Polysomes [Rivera et al 2014b], and Protocol: Dissociation of Ribosomes into Large and Small Subunits [Rivera et al 2014c]). 14.…”

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confidence: 99%

Isolation of Ribosomes by Chromatography

Maguire

2015

Cold Spring Harb Protoc

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Mixed-mode chromatography on cysteine-SulfoLink resin efficiently separates ribosomes from cell lysates and is particularly effective at rapidly removing endogenous proteases and nucleases, resulting in ribosomes of improved purity, integrity, and activity. Binding occurs partly by anion exchange of the RNA of the ribosomes, so that cells must be lysed in a buffer of moderate ionic strength (conductivity no more than 20 mS for chromatography of bacterial ribosomes) without any highly charged additives (e.g., heparin, which is used to inhibit RNases in yeast). A robust protocol for Escherichia coli is given here as an example.

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Dissociation of Ribosomes into Large and Small Subunits

Cited by 3 publications

References 3 publications

Polysomes identified by live imaging of nascent peptides are stalled in hippocampal and cortical neurites

Polysomes identified by live imaging of nascent peptides are stalled in hippocampal and cortical neurites

‘Tuning’ of ribosome levels mediated by RNase I and hibernating ribosomes

Isolation of Ribosomes by Chromatography

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