<i>RPL30</i> regulation of splicing reveals distinct roles for Cbp80 in U1 and U2 snRNP cotranscriptional recruitment

Bragulat, Mireia; Meyer, Markus; Macías, Sara; Camats, Maria; Labrador, Mireia; Vilardell, Josep

doi:10.1261/rna.2366310

Cited by 10 publications

(8 citation statements)

References 40 publications

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“…Recently, we described how the cap-binding protein Cbc1 is involved in the rapid reprogramming of translation under osmotic stress [33]. Additionally, Cbc1 has been described to be required for the proper splicing of some RP genes [37]. To check whether Hog1 and/or Cbc1 play a role in the drop of RP pre-mRNAs under osmotic stress, we calculated the PMi for several RP genes in mutants hog1 and cbc1 .…”

Section: Resultsmentioning

confidence: 99%

Nonsense-Mediated mRNA Decay Controls the Changes in Yeast Ribosomal Protein Pre-mRNAs Levels upon Osmotic Stress

et al. 2013

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The expression of ribosomal protein (RP) genes requires a substantial part of cellular transcription, processing and translation resources. Thus, the RP expression must be tightly regulated in response to conditions that compromise cell survival. In Saccharomyces cerevisiae cells, regulation of the RP gene expression at the transcriptional, mature mRNA stability and translational levels during the response to osmotic stress has been reported. Reprogramming global protein synthesis upon osmotic shock includes the movement of ribosomes from RP transcripts to stress-induced mRNAs. Using tiling arrays, we show that osmotic stress yields a drop in the levels of RP pre-mRNAs in S. cerevisiae cells. An analysis of the tiling array data, together with transcription rates data, shows a poor correlation, indicating that the drop in the RP pre-mRNA levels is not merely a result of the lowered RP transcription rates. A kinetic study using quantitative RT-PCR confirmed the decrease in the levels of several RP-unspliced transcripts during the first 15 minutes of osmotic stress, which seems independent of MAP kinase Hog1. Moreover, we found that the mutations in the components of the nonsense-mediated mRNA decay (NMD), Upf1, Upf2, Upf3 or in exonuclease Xrn1, eliminate the osmotic stress-induced drop in RP pre-mRNAs. Altogether, our results indicate that the degradation of yeast RP unspliced transcripts by NMD increases during osmotic stress, and suggest that this might be another mechanism to control RP synthesis during the stress response.

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Section: Resultsmentioning

confidence: 99%

Nonsense-Mediated mRNA Decay Controls the Changes in Yeast Ribosomal Protein Pre-mRNAs Levels upon Osmotic Stress

et al. 2013

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“…Interestingly, Cbp80, the large subunit of the cap-binding complex in yeast (yCBC), was originally identified as Gcr3 in a screen for mutants with reduced glycolytic enzyme expression (Uemura and Jigami, 1992). The yCBC has also been implicated in transcriptional regulation (Hossain et al, 2013; Lahudkar et al, 2011), splicing fidelity, particularly at the 5′SS (Hossain et al, 2009), and the coordination of splicing and transcription (Bragulat et al, 2010; Gornemann et al, 2005). Intriguingly, we have found that CBC expression also changes in response to glucose availability (data not shown), suggesting that the CBC might play a critical role in regulating Gcr1 expression in response to glucose, a model we are currently testing.…”

Section: Discussionmentioning

confidence: 99%

Posttranscriptional Regulation of Gcr1 Expression and Activity Is Crucial for Metabolic Adjustment in Response to Glucose Availability

et al. 2016

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Summary The transcription factor Gcr1 controls expression of over 75% of the genes in actively growing yeast. Yet, despite its widespread effects, regulation of Gcr1 itself remains poorly understood. Here we show that posttranscriptional Gcr1 regulation is nutrient-dependent. Moreover, GCR1 RNA contains a long, highly conserved intron, which allows the cell to generate multiple RNA and protein isoforms whose levels change upon glucose depletion. Intriguingly, an isoform generated by intron retention is exported from the nucleus, and its translation is initiated from a conserved, intronic translation start site. Expression of gene products from both the spliced and unspliced RNAs is essential, as cells expressing only one isoform cannot adjust their metabolic program in response to environmental changes. Finally, we show that the Gcr1 proteins form dimers, providing an elegant mechanism by which this one gene, through its regulation, can perform the repertoire of transcriptional activities necessary for fine-tuned environmental response.

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“…The expressed protein contained an amino-terminal HA tag. SpeI and XhoI sites were introduced into the CBP80 pMalE clone ( 33 ) and then CBP80 was cloned into the equivalent sites of the p415-PL plasmid. The PL10 gene in the pRS315 plasmid with a GDH promoter and PGK terminator was as previously described ( 18 ).…”

Section: Methodsmentioning

confidence: 99%

“…The sec63 domain of BRR2 was cloned and expressed as previously described ( 42 ). The MalE-Cbp80 protein was expressed and purified as described elsewhere ( 33 ). The amino-terminal GST-tagged eIF4G1 fragments (1-596, 542-952, 882-952) were constructed by cloning the PCR-generated gene fragments into the BamHI-SmaI sites of pGEX-2T (GE Healthcare) and the expressed proteins were purified on glutathione Sepharose columns eluted with 10 mM reduced glutathione, 20 mM Tris–HCl, pH 7.5, 100 mM NaCl and 2 mM dithiothreitol (DTT).…”

Section: Methodsmentioning

confidence: 99%

The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus

Senissar

Saux

Belgareh‐Touzé

et al. 2014

Nucleic Acids Research

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The DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5′ 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrometry analyses, genetic experiments, sucrose gradients, in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs.

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RPL30 regulation of splicing reveals distinct roles for Cbp80 in U1 and U2 snRNP cotranscriptional recruitment

Cited by 10 publications

References 40 publications

Nonsense-Mediated mRNA Decay Controls the Changes in Yeast Ribosomal Protein Pre-mRNAs Levels upon Osmotic Stress

Nonsense-Mediated mRNA Decay Controls the Changes in Yeast Ribosomal Protein Pre-mRNAs Levels upon Osmotic Stress

Posttranscriptional Regulation of Gcr1 Expression and Activity Is Crucial for Metabolic Adjustment in Response to Glucose Availability

The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus

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