[27] Preparation of high molecular weight RNA

Köhrer, Karl; Domdey, Horst

doi:10.1016/0076-6879(91)94030-g

Cited by 554 publications

(367 citation statements)

References 5 publications

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“…Total RNA was purified by standard hot-phenol extraction (Kohrer and Domdey, 1991). After precipitation RNA samples were re-suspended in DEPC-treated water and quantified by spectrophotometry.…”

Section: Methodsmentioning

confidence: 99%

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The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis

Kimmig

Diaz

Zheng

et al. 2012

eLife

134

162

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The unfolded protein response (UPR) monitors the protein folding capacity of the endoplasmic reticulum (ER). In all organisms analyzed to date, the UPR drives transcriptional programs that allow cells to cope with ER stress. The non-conventional splicing of Hac1 (yeasts) and XBP1 (metazoans) mRNA, encoding orthologous UPR transcription activators, is conserved and dependent on Ire1, an ER membrane-resident kinase/endoribonuclease. We found that the fission yeast Schizosaccharomyces pombe lacks both a Hac1/XBP1 ortholog and a UPR-dependent-transcriptional-program. Instead, Ire1 initiates the selective decay of a subset of ER-localized-mRNAs that is required to survive ER stress. We identified Bip1 mRNA, encoding a major ER-chaperone, as the sole mRNA cleaved upon Ire1 activation that escapes decay. Instead, truncation of its 3′ UTR, including loss of its polyA tail, stabilized Bip1 mRNA, resulting in increased Bip1 translation. Thus, S. pombe uses a universally conserved stress-sensing machinery in novel ways to maintain homeostasis in the ER.DOI: http://dx.doi.org/10.7554/eLife.00048.001

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Section: Methodsmentioning

confidence: 99%

“…Total RNA was prepared from cells by the hot acid phenol method (Kohrer and Domdey, 1991), and subsequent enrichment performed. PolyA + mRNA was purified by two sequential rounds of enrichment using oligo-dT DynaBeads (Invitrogen) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis

Kimmig

Diaz

Zheng

et al. 2012

eLife

134

162

View full text Add to dashboard Cite

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“…albicans was grown as described above. After washing the cells in ice-cold citrate buffer (50 mM trisodium citrate pH 6.4, 5% glucose), RNA was isolated by hot phenol extraction (Kohrer et al, 1991). RNAs were quantitiated at A260, and 30 mg aliquots were separated on formaldehyde gels.…”

Section: Rna Analysismentioning

confidence: 99%

Siderophore uptake by Candida albicans: effect of serum treatment and comparison with Saccharomyces cerevisiae

Lesuisse

Knight

Camadro

et al. 2002

Yeast

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Iron uptake systems often function as virulence factors in pathogenic organisms. Candida albicans is a fungal pathogen that infects immunocompromised hosts, such as AIDS patients or granulocytopenic bone marrow transplant recipients. Here we show that iron uptake from siderophores occurs in C. albicans and is mediated by one or more highaffinity transport systems. Iron carried on ferrioxamine B, triacethyl-fusarinine, ferrichrome, or ferricrocin was actively taken up via a high-affinity mechanism. The kinetic parameters of uptake were similar to those found in S. cerevisiae. Furthermore, for ferrichrome and ferrioxamine B, cellular uptake of fluorescent analogues was observed. In C. albicans, iron uptake from siderophores was regulated by iron availability, with iron deprivation inducing uptake. Serum exposure, which induces a morphogenic shift from yeast to filamentous forms known to be required for virulence, also resulted in induction of iron transport from ferrichrome-type siderophores. In a tup1/tup1 strain which grows constitutively in the filamentous form, iron transport was derepressed for all siderophores tested. The genes mediating uptake and utilization of iron from siderophores in C. albicans have not been identified; however, the transcript abundance for CaSIT1 was regulated in a manner consistent with the pattern of iron uptake from ferrichrome-type siderophores. Furthermore, CaSIT1 overexpression in S. cerevisiae resulted in inhibited siderophore iron uptake, suggesting that the expressed protein may interact with proteins of S. cerevisiae involved in iron uptake from siderophores. In summary, iron uptake from ferrichrome-type siderophores was induced in filamentous C. albicans, and a potential role of this iron acquisition system in pathogenicity should be considered.

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“…Effects of P3 mutations on RNase P enzymatic activity were determined by probing whole cellular RNA with a radiolabeled anti-tRNA Leu3 DNA oligonucleotide+ Total cellular RNA was isolated from temperature-sensitive clones grown at permissive (30 8C) or nonpermissive temperatures (37 8C) for 4 h (Kohrer & Domdey, 1991)+ Concentration of isolated RNA was determined by measuring UV absorbance at 260 nm and 6-mg sample quantities were run on denaturing polyacrylamide gels, transferred to Nytran membrane and probed using a radiolabeled DNA oligonucleotide complementary to Leu3 tRNA (59-32 P-CTTAGACCGCTCGGCCAAAC-39)+ A radiolabeled DNA oligonucleotide complementary to signal recognition particle RNA (SCR1 RNA, 59-32 P-GGCGTGCAAT CCGTGTCT-39) was also used to probe the blots to normalize hybridization signal+ RNA blot hybridization profiles were examined for changes characteristic of RNase P enzymatic activity defects (Lee et al+, 1991b)+ These changes include an accumulation of 59 unprocessed pre-tRNA, 59 unprocessed with mature 39 end, and 59 unprocessed with spliced intron tRNA species+ RNA blot hybridization of RNase P RNA Whole cellular RNA from temperature-sensitive mutants grown at permissive and nonpermissive conditions was probed with a radiolabeled anti-RPR1 DNA oligonucleotide+ The probe is complementary to nt 310-327 of S. cerevisiae RNase P RNA (59-GACGTCCTACGATTGCAC-39)+ Electrophoresis and hybridization were as previously described (Pagan-Ramos et al+, 1996a)+ Holoenzyme assembly defects result in an accumulation of precursor RNase P RNA as previously described (Pagan-Ramos et al+, 1996a)+ The same radiolabeled anti-SCR1 RNA DNA oligonucleotide used in the pre-tRNA Leu3 blots was used as a hybridization control+…”

Section: Rna Blot Hybridization Of Pre-trna Leu3mentioning

confidence: 99%

An essential protein-binding domain of nuclear RNase P RNA

Ziehler

Morris

Scott

et al. 2001

RNA

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Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and protein subunits. The RNA subunits of each enzyme share substantial secondary structural features, and most of the protein subunits are shared between the two. One of the conserved RNA subdomains, designated P3, has previously been shown to be required for nucleolar localization. Phylogenetic sequence analysis suggests that the P3 domain interacts with one of the proteins common to RNase P and RNase MRP, a conclusion strengthened by an earlier observation that the essential domain can be interchanged between the two enzymes. To examine possible functions of the P3 domain, four conserved nucleotides in the P3 domain of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a library of all possible sequence combinations at those positions. Selection of functional genes in vivo identified permissible variations, and viable clones that caused yeast to exhibit conditional growth phenotypes were tested for defects in RNase P RNA and tRNA biosynthesis. Under nonpermissive conditions, the mutants had reduced maturation of the RPR1 RNA precursor, an expected phenotype in cases where RNase P holoenzyme assembly is defective. This loss of RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation characteristic of RNase P defects. To test whether mutations at the conserved positions inhibited interactions with a particular protein, specific binding of the individual protein subunits to the RNA subunit was tested in yeast using the threehybrid system. Pop1p, the largest subunit shared by RNases P and MRP, bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was eliminated by mutations at the conserved P3 residues. These results indicate that Pop1p interacts with the P3 domain common to RNases P and MRP, and that this interaction is critical in the maturation of RNase P holoenzyme.

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[27] Preparation of high molecular weight RNA

Cited by 554 publications

References 5 publications

The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis

The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis

Siderophore uptake by Candida albicans: effect of serum treatment and comparison with Saccharomyces cerevisiae

An essential protein-binding domain of nuclear RNase P RNA

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