Analysis of pre-mRNA processing in transfected plant protoplasts

Goodall, Gregory J.; Wiebauer, Karin; Filipowicz, Witold

doi:10.1016/0076-6879(90)81117-d

Cited by 165 publications

(146 citation statements)

References 21 publications

Supporting

Mentioning

146

Contrasting

Order By: Relevance

“…RNase A/T1 protection analysis was essentially performed as described in Goodall et al+ (1990)+ The 32 P-labeled antisense probe was transcribed with T7 polymerase from plasmid pTL66 linearized with Eco57 I+ The probe was treated with RQ1-RNase-free DNase (Promega) and gel purified+ Nine micrograms of total RNA were mixed with ;40 cpm of probe in 30 mL of PIPES buffer (40 mM PIPES, pH 6+7, 400 mM NaCl, 1 mM EDTA)/50% formamide+ Annealing was performed overnight at 48 8C+ Digestion in RNase buffer (10 mM Tris HCl, pH 7+5, 300 mM NaCl, 1 mM EDTA) was with 7+5 U of RNase T1/1+5 mg RNase A (both purchased from Boehringer) for 30 min at 25 8C+ Protected products were recovered by phenol-chloroform extraction and separated on an 8% polyacrylamide gel+ A sequencing reaction was used as a ladder+ With the antisense U24 transcript used (212 nt), the protected fragment corresponding to the mature 39 end of U24 was detected at the expected length of 77 nt+ Plasmid pTL66 was constructed as follows: a U24 genomic fragment encompassing the 39 end of U24 was recovered by KpnI/ DraI digestion from plasmid pFH2 (a kind gift of Y+ Henry) and subcloned in pBluescript+…”

Section: Rnase A/t1 Mappingmentioning

confidence: 99%

Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability

Lafontaine

Tollervey

1999

RNA

151

159

View full text Add to dashboard Cite

Eukaryotic nucleoli contain a large family of box C+D small nucleolar RNA (snoRNA) species, all of which are associated with a common protein Nop1p/fibrillarin. Nop58p was identified in a screen for synthetic lethality with Nop1p and shown to be an essential nucleolar protein. Here we report that a Protein A-tagged version of Nop58p coprecipitates all tested box C+D snoRNAs and that genetic depletion of Nop58p leads to the loss of all tested box C+D snoRNAs. The box H+ACA class of snoRNAs are not coprecipitated with Nop58p, and are not codepleted. The yeast box C+D snoRNAs include two species, U3 and U14, that are required for the early cleavages in pre-rRNA processing. Consistent with this, Nop58p depletion leads to a strong inhibition of pre-rRNA processing and 18S rRNA synthesis. Unexpectedly, depletion of Nop58p leads to the accumulation of 39 extended forms of U3 and U24, showing that the protein is also involved in snoRNA synthesis. Nop58p is the second common component of the box C+D snoRNPs to be identified and the first to be shown to be required for the stability and for the synthesis of these snoRNAs.

show abstract

Section: Rnase A/t1 Mappingmentioning

confidence: 99%

Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability

Lafontaine

Tollervey

1999

RNA

151

159

View full text Add to dashboard Cite

show abstract

“…RNase A/T1 protection analysis was performed essentially as described (Goodall et al 1990). Anti-sense probes were synthesized in the presence of [a-32 P]-CTP using the Maxiscript kit (Ambion) and gel purified; 1-5 mg of total RNA (prepared with TRIzol, Invitrogen) was mixed with probe (50,000 cpm) for hybridization and digestion with 40 mg/mL RNase A (Roche) and 2 mg/mL RNase T1 (Calbiochem).…”

Section: Fluorescence Methods and Immunochemicalsmentioning

confidence: 99%

Dynamic association and localization of human H/ACA RNP proteins

Kittur¹,

Darzacq²,

Roy³

et al. 2006

RNA

View full text Add to dashboard Cite

Mammalian H/ACA RNPs are essential for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. To form mature RNA-protein complexes, one H/ACA RNA associates with four core proteins. In the cell, this process is assisted by at least one nuclear assembly factor, NAF1. Here we report several unanticipated dynamic aspects of H/ACA RNP proteins. First, when overexpressed, NAF1 delocalizes to the cytoplasm. However, its nucleocytoplasmic shuttling properties remain unaffected. These observations demonstrate a subtle equilibrium between NAF1 expression levels and the availability of NAF1 nuclear binding sites. Second, although NAF1 is excluded from mature RNPs in nucleoli and Cajal bodies, NAF1 associates with mature H/ACA RNA in cell lysates. This association occurs post-lysis because it is observed even when NAF1 and the H/ACA RNA are expressed in separate cells. This documents a protein-RNP association in cell lysates that is absent from intact cells. Third, in similar experiments, all H/ACA core proteins, except NAP57, exchange with their exogenous counterparts, portraying an unexpected dynamic picture of H/ACA RNPs. Finally, the irreversible association of only NAP57 with H/ACA RNA and the conundrum that only NAP57 is mutated in X-linked dyskeratosis congenita (even though most core proteins are required for maintaining H/ACA RNAs) may be more than a coincidence.

show abstract

“…RNase protection was performed as described by Goodall et al (1990). RNA probes were synthesized in vitro with T3 or T7 RNA polymerase, using [a-^^P]CTP (30 Ci/mmole) and linear ized plasmids as templates.…”

Section: Rnase A/t^ Mappingmentioning

confidence: 99%

“…RNAs from HeLa cells, transfected COS cells, and yeast cells were isolated by guanidinium thiocyanate/phenol-chloroform extraction as described by Goodall et al (1990) and Tollervey and Mattaj (1987), respectively. The hot phenol/SDS method (Steele et al 1965) was used for isolation of RNA from subcel lular fractions of HeLa cells, yeast cell extracts, and pellets of immunoprecipitation reactions.…”

Section: Isolation Of Rnamentioning

confidence: 99%

The family of box ACA small nucleolar RNAs is defined by an evolutionarily conserved secondary structure and ubiquitous sequence elements essential for RNA accumulation.

Ganot¹,

Caizergues‐Ferrer²,

Kiss³

1997

Genes Dev.

319

387

View full text Add to dashboard Cite

Eukaryotic cells contain a large number of small nucleolar RNAs (snoRNAs).A major family of snoRNAs features a consensus ACA motif positioned 3 nucleotides from the 3' end of the RNA. In this study we have characterized nine novel human ACA snoRNAs (U64-U72). Structural probing of U64 RNA followed by systematic computer modeling of all known box ACA snoRNAs revealed that this class of snoRNAs is defined by a phylogenetically conserved secondary structure. The ACA snoRNAs fold into two hairpin structures connected by a single-stranded hinge region and followed by a short 3' tail. In eukaryotic cells, maturation of rRNAs takes place in the nucleolus. The 18S, 5.8S, and 25/28S rRNAs are syn thesized as a large precursor RNA (pre-rRNA) that car ries long external and internal spacer sequences. Shortly after transcription, many nucleotides in the coding re gions of pre-rRNA are methylated at the 2'-0-hydroxyl position and numerous U residues are converted into pseudouridines (Maden 1990;Eichler and Craig 1995). The covalently modified pre-rRNA is than processed into mature rRNAs through a series of endo-and exonucleolytic cleavages (Eichler and Craig 1995; Venema and Tollervey 1995;Sollner-Webb et al. 1996).The nucleolus contains a large number of snoRNAs. More than 80 distinct small nucleolar RNA (snoRNA) sequences have been identified in vertebrate and yeast

show abstract

Analysis of pre-mRNA processing in transfected plant protoplasts

Cited by 165 publications

References 21 publications

Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability

Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability

Dynamic association and localization of human H/ACA RNP proteins

The family of box ACA small nucleolar RNAs is defined by an evolutionarily conserved secondary structure and ubiquitous sequence elements essential for RNA accumulation.

Contact Info

Product

Resources

About