Polysome Analysis and RNA Purification from Sucrose Gradients

Mašek, Tomáš; Valášek, Leoš Shivaya; Pospíšek, Martin

doi:10.1007/978-1-59745-248-9_20

Cited by 75 publications

(58 citation statements)

References 45 publications

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“…Frac-seq reveals isoform-specific differences in polyribosome association Polyribosome profiling is widely used to estimate the translation status of mRNAs (Masek et al 2011). We hypothesized that alternative splicing not only expands the protein coding capacity of human genes, but also establishes patterns of isoform-specific translational control.…”

Section: Resultsmentioning

confidence: 99%

Frac-seq reveals isoform-specific recruitment to polyribosomes

et al. 2013

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Pre-mRNA splicing is required for the accurate expression of virtually all human protein coding genes. However, splicing also plays important roles in coordinating subsequent steps of pre-mRNA processing such as polyadenylation and mRNA export. Here, we test the hypothesis that nuclear pre-mRNA processing influences the polyribosome association of alternative mRNA isoforms. By comparing isoform ratios in cytoplasmic and polyribosomal extracts, we determined that the alternative products of ∼30% (597/1954) of mRNA processing events are differentially partitioned between these subcellular fractions. Many of the events exhibiting isoform-specific polyribosome association are highly conserved across mammalian genomes, underscoring their possible biological importance. We find that differences in polyribosome association may be explained, at least in part by the observation that alternative splicing alters the cis-regulatory landscape of mRNAs isoforms. For example, inclusion or exclusion of upstream open reading frames (uORFs) in the 5′UTR as well as Alu-elements and microRNA target sites in the 3′UTR have a strong influence on polyribosome association of alternative mRNA isoforms. Taken together, our data demonstrate for the first time the potential link between alternative splicing and translational control of the resultant mRNA isoforms.

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Section: Resultsmentioning

confidence: 99%

Frac-seq reveals isoform-specific recruitment to polyribosomes

et al. 2013

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“…Such a dramatic destabilization of 60S subunits was unexpected, as ribosomal subunits are known to remain stable and functional for extended periods of time under high ionic strength, such as 0.5-0.8 M KCl/ NaCl (Falvey and Staehelin 1970;Martin and Hartwell 1970;Zylber and Penman 1970). Also, standard salt concentrations used to dissociate 80S ribosomes into subunits for their purification are in the same high range (Mašek et al 2011). Our data strongly suggest that the m 5 C2278 and G2288m affect the equilibrium of conformations that helices H70 and H71 can assume.…”

Section: Discussionmentioning

confidence: 99%

“…Surprisingly, even a mild increase in the salt concentration (300 mM NaCl, 5 mM MgCl 2 ), well below the concentrations used for purification of ribosomal subunits (Mašek et al 2011), led not only to further destabilization of 80S monosomes but also to appearance of smaller sub-60S peaks in the strains lacking methylations, but not the wildtype strain (Fig. 4).…”

Section: Rcm1 (Ynl022c) Methylates the Cytosine 2278 In 25s Rrnamentioning

confidence: 94%

A cluster of methylations in the domain IV of 25S rRNA is required for ribosome stability

Gigova

Duggimpudi

Pollex

et al. 2014

RNA

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In all three domains of life ribosomal RNAs are extensively modified at functionally important sites of the ribosome. These modifications are believed to fine-tune the ribosome structure for optimal translation. However, the precise mechanistic effect of modifications on ribosome function remains largely unknown. Here we show that a cluster of methylated nucleotides in domain IV of 25S rRNA is critical for integrity of the large ribosomal subunit. We identified the elusive cytosine-5 methyltransferase for C2278 in yeast as Rcm1 and found that a combined loss of cytosine-5 methylation at C2278 and ribose methylation at G2288 caused dramatic ribosome instability, resulting in loss of 60S ribosomal subunits. Structural and biochemical analyses revealed that this instability was caused by changes in the structure of 25S rRNA and a consequent loss of multiple ribosomal proteins from the large ribosomal subunit. Our data demonstrate that individual RNA modifications can strongly affect structure of large ribonucleoprotein complexes.

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“…Association of mRNA with the polysomes is an established approach to assess translation (17). This strategy was used to explore the pattern of translation of secreted proteins during oocyte maturation in a genome-wide fashion.…”

Section: Translational Regulation Of Mrna Coding For Secreted Proteinsmentioning

confidence: 99%

Dynamic secretion during meiotic reentry integrates the function of the oocyte and cumulus cells

Çakmak

Franciosi

Zamah

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The differentiation of the female gamete into a developmentally competent oocyte relies on the protected environment of the ovarian follicle. The oocyte plays a key role in establishing this microenvironment by releasing paracrine factors that control the functions of surrounding somatic cells. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are secreted during follicle growth and play pivotal roles in this local regulation. The current view is that the function of these secreted factors declines in the periovulatory period when the oocyte reenters the meiotic cell cycle. Here, we provide evidence that oocyte reentry into meiosis is instead associated with a shift in the pattern of secretion with a new set of bioactive molecules synthesized before ovulation. Using interleukin 7 (IL7) as a prototypic secreted factor, we show that its secretion is dependent on activation of mRNA translation in synchrony with the cell cycle and that its translation is under the control of somatic cells. IL7 is part of a local feedback loop with the soma because it regulates cumulus cell replication. Similar conclusions are reached when IL7 secretion is measured in human follicular fluid during in vitro fertilization cycles. IL7 concentration in the follicular fluid correlates with the oocyte ability to reach the MII stage of maturation. These findings are consistent with the hypothesis that a new set of local factors is secreted by the oocyte during ovulation. These dynamic secretions are likely critical for promoting the final stages of maturation and oocyte developmental competence.oocyte maturation | mRNA translation | oocyte secreted factors | interleukin-7

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Polysome Analysis and RNA Purification from Sucrose Gradients

Cited by 75 publications

References 45 publications

Frac-seq reveals isoform-specific recruitment to polyribosomes

Frac-seq reveals isoform-specific recruitment to polyribosomes

A cluster of methylations in the domain IV of 25S rRNA is required for ribosome stability

Dynamic secretion during meiotic reentry integrates the function of the oocyte and cumulus cells

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