Selective profiling of ribosomes associated with yeast Upf proteins

Ganesan, Robin; Leszyk, John D.; Jacobson, Andrew D.

doi:10.1016/j.ymeth.2018.12.008

Cited by 6 publications

(12 citation statements)

References 52 publications

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“…Ribosome profiling and RNA-seq libraries were prepared as described previously in our library preparation protocol for immunoprecipitated ribosomes [ 58 ]. Briefly, 1 L of cells of OD 600 between 0.6 and 0.8 were harvested by rapid vacuum filtration and flash-frozen in liquid nitrogen in the presence of Footprinting Buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl 2 ) plus 1% TritonX-100, 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1X protease inhibitors.…”

Section: Methodsmentioning

confidence: 99%

Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

Mangkalaphiban

Ganesan

et al. 2021

PLoS Genet

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Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

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Section: Methodsmentioning

confidence: 99%

Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

Mangkalaphiban

Ganesan

et al. 2021

PLoS Genet

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“…Gel bands were cut into 1 mm by 1 mm pieces and placed in 1.5-ml Eppendorf tubes with 1 ml of water for 30 min. Samples were then prepared for mass spectrometry, and liquid chromatography–tandem mass spectrometry analysis was performed essentially as described ( 54 ). Data analysis was also performed as described except that raw data files were peak-processed with Proteome Discoverer (version 2.0, Thermo Fisher Scientific) before database searching with Mascot Server (version 2.4) against the Swissprot_Human database.…”

Section: Methodsmentioning

confidence: 99%

Identification of the hyaluronic acid pathway as a therapeutic target for facioscapulohumeral muscular dystrophy

et al. 2019

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Facioscapulohumeral muscular dystrophy (FSHD) is linked to epigenetic derepression of the germline/embryonic transcription factor DUX4 in skeletal muscle. However, the etiology of muscle pathology is not fully understood, as DUX4 misexpression is not tightly correlated with disease severity. Using a DUX4-inducible cell model, we show that multiple DUX4-induced molecular pathologies that have been observed in patient-derived disease models are mediated by the signaling molecule hyaluronic acid (HA), which accumulates following DUX4 induction. These pathologies include formation of RNA granules, FUS aggregation, DNA damage, caspase activation, and cell death. We also observe previously unidentified pathologies including mislocalization of mitochondria and the DUX4- and HA-binding protein C1QBP. These pathologies are prevented by 4-methylumbelliferone, an inhibitor of HA biosynthesis. Critically, 4-methylumbelliferone does not disrupt DUX4-C1QBP binding and has only a limited effect on DUX4 transcriptional activity, establishing that HA signaling has a central function in pathology and is a target for FSHD therapeutics.

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“…Ribosome purification. Yeast cultures were grown, whole cell yeast lysates were prepared and digested with RNaseI, and ribosomes were recovered as described previously 30 .…”

Section: Methodsmentioning

confidence: 99%

“…Immunopurification. FLAG-tagged protein-associated ribosomes were isolated by immunopurification using anti-FLAG M2 Affinity gel as described previously 30 . Eluate was collected by centrifugation and spun through Amicon Ultra 2ml 100K centrifugal filter units (Millipore) until the volume was <200μl.…”

Section: Methodsmentioning

confidence: 99%

“…Likewise, as additional variables, we also examined cells in which all three UPF genes were simultaneously overexpressed, cells that were or were not treated with cycloheximide (CHX) prior to harvesting, and cells expressing mutant forms of the UPF genes. Upf factor-associated ribosomes were immunopurified as described previously 30 and input (total) and immunopurified (IP) ribosomes were analyzed by western blotting. As shown in Extended Data Figure 2, immunopurification yielded ribosomes with strong signals for both ribosomal protein Tcm1 (Rpl3) and the respective FLAG-tagged Upf factor.…”

Section: Purification Of Flag-upf1- Flag-upf2- and Flag-upf3-associated Ribosomes Yields Stoichiometric Recovery Of Upf Factors With Ribomentioning

confidence: 99%

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Progressive polysomal association of Upf1, Upf2, and Upf3 as ribosomes traverse mRNA coding regions

Ganesan

Mangkalaphiban

Baker

et al. 2021

Preprint

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SUMMARYUpf1, Upf2, and Upf3 are the central regulators of nonsense-mediated mRNA decay (NMD), the eukaryotic mRNA quality control pathway generally triggered when a premature termination codon is recognized by the ribosome. The NMD-related functions of the Upf proteins likely commence while these factors are ribosome-associated, but little is known of the timing of their ribosome binding, their specificity for ribosomes translating NMD substrates, or the nature and role of any ribosome:Upf complexes. Here, we have elucidated details of the ribosome-associated steps of NMD. By combining yeast genetics with selective ribosome profiling and co-sedimentation analyses of polysomes with wild-type and mutant Upf proteins, our approaches have identified distinct states of ribosome:Upf association. All three Upf factors manifest progressive polysome association as mRNA translation proceeds, but these events appear to be preceded by formation of a Upf1:80S complex as mRNAs initiate translation. This complex is likely executing an early mRNA surveillance function.

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Selective profiling of ribosomes associated with yeast Upf proteins

Cited by 6 publications

References 52 publications

Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

Identification of the hyaluronic acid pathway as a therapeutic target for facioscapulohumeral muscular dystrophy

Progressive polysomal association of Upf1, Upf2, and Upf3 as ribosomes traverse mRNA coding regions

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