scholarly journals The rRNA-processing function of the yeast U14 small nucleolar RNA can be rescued by a conserved RNA helicase-like protein.

1997 ◽  
Vol 17 (7) ◽  
pp. 4124-4132 ◽  
Author(s):  
W Q Liang ◽  
J A Clark ◽  
M J Fournier
Keyword(s):  
Mutational Analysis ◽  
Rna Helicase ◽  
Suppressor Gene ◽  
Rrna Processing ◽  
Base Pairing ◽  
Small Nucleolar Rna ◽  
Stem Loop ◽  
Dead Box ◽  
Extragenic Suppressor ◽  
Nucleolar Rna

The phylogenetically conserved U14 small nucleolar RNA is required for processing of rRNA, and this function involves base pairing with conserved complementary sequences in 18S RNA. With a view to identifying other important U14 interactions, a stem-loop domain required for activity of Saccharomyces cerevisiae U14 RNAs (the Y domain) was first subjected to detailed mutational analysis. The mapping results showed that most nucleotides of the Y domain can be replaced without affecting function, except for loop nucleotides conserved among five different yeast species. Defective variants were then used to identify both intragenic and extragenic suppressor mutations. All of the intragenic mutations mapped within six nucleotides of the primary mutation, suggesting that suppression involves a change in conformation and that the loop element is involved in an essential intermolecular interaction rather than intramolecular base pairing. A high-copy extragenic suppressor gene, designated DBP4 (DEAD box protein 4), encodes an essential, putative RNA helicase of the DEAD-DEXH box family. Suppression by DBP4 (initially CA4 [T.-H. Chang, J. Arenas, and J. Abelson, Proc. Natl. Acad. Sci. USA 87:1571-1575, 1990]) restores the level of 18S rRNA and is specific for the Y domain but is not allele specific. DBP4 is predicted to function either in assembly of the U14 small nucleolar RNP or, more likely, in its interaction with other components of the rRNA processing apparatus. Mediating the interaction of U14 with precursor 18S RNA is an especially attractive possibility.

scholarly journals Base Pairing between U3 Small Nucleolar RNA and the 5′ End of 18S rRNA Is Required for Pre-rRNA Processing

1999 ◽  
Vol 19 (9) ◽  
pp. 6012-6019 ◽  
Author(s):  
Kishor Sharma ◽  
David Tollervey
Keyword(s):  
18S Rrna ◽  
Homologous Region ◽  
Compensatory Mutation ◽  
Loop Structure ◽  
Rrna Processing ◽  
Base Pairing ◽  
Small Nucleolar Rna ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Nucleolar Rna

ABSTRACT The loop of a stem structure close to the 5′ end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA). Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A1, the 5′ end of the 18S rRNA, and at site A2, located 1.9 kb away in internal transcribed spacer 1. This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing. The U3–pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA. In theEscherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5′ external transcribed spacer (5′ ETS). Cleavage at site A0in the yeast 5′ ETS strictly requires base pairing between U3 and a sequence within the 5′ ETS. In contrast, the U3-18S interaction is not required for A0 cleavage. U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA. The nucleotide at the site of A1 cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA. However, in contrast to the efficiency of cleavage, the position of A1 cleavage is not dependent on the U3-loop interaction. We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing.

scholarly journals Box H and Box ACA Are Nucleolar Localization Elements of U17 Small Nucleolar RNA

1999 ◽  
Vol 10 (11) ◽  
pp. 3877-3890 ◽  
Author(s):  
Thilo Sascha Lange ◽  
Michael Ezrokhi ◽  
Francesco Amaldi ◽  
Susan A. Gerbi
Keyword(s):  
Adverse Effect ◽  
Fluorescence Microscopy ◽  
Nucleolar Localization ◽  
Rrna Processing ◽  
Base Pairing ◽  
Small Nucleolar Rna ◽  
Family Function ◽  
Tail Structure ◽  
Localization Elements ◽  
Nucleolar Rna

The nucleolar localization elements (NoLEs) of U17 small nucleolar RNA (snoRNA), which is essential for rRNA processing and belongs to the box H/ACA snoRNA family, were analyzed by fluorescence microscopy. Injection of mutant U17 transcripts into Xenopus laevisoocyte nuclei revealed that deletion of stems 1, 2, and 4 of U17 snoRNA reduced but did not prevent nucleolar localization. The deletion of stem 3 had no adverse effect. Therefore, the hairpins of the hairpin–hinge–hairpin–tail structure formed by these stems are not absolutely critical for nucleolar localization of U17, nor are sequences within stems 1, 3, and 4, which may tether U17 to the rRNA precursor by base pairing. In contrast, box H and box ACA are major NoLEs; their combined substitution or deletion abolished nucleolar localization of U17 snoRNA. Mutation of just box H or just the box ACA region alone did not fully abolish the nucleolar localization of U17. This indicates that the NoLEs of the box H/ACA snoRNA family function differently from the bipartite NoLEs (conserved boxes C and D) of box C/D snoRNAs, where mutation of either box alone prevents nucleolar localization.

scholarly journals The DEAD-Box RNA Helicase AtRH7/PRH75 Participates in Pre-rRNA Processing, Plant Development and Cold Tolerance in Arabidopsis

2015 ◽  
Vol 57 (1) ◽  
pp. 174-191 ◽  
Author(s):  
Chun-Kai Huang ◽  
Yu-Lien Shen ◽  
Li-Fen Huang ◽  
Shaw-Jye Wu ◽  
Chin-Hui Yeh ◽  
...  
Keyword(s):  
Cold Tolerance ◽  
Plant Development ◽  
Rna Helicase ◽  
Processing Plant ◽  
Rrna Processing ◽  
Dead Box ◽  
The Dead

scholarly journals Rrp47p Is an Exosome-Associated Protein Required for the 3′ Processing of Stable RNAs

2003 ◽  
Vol 23 (19) ◽  
pp. 6982-6992 ◽  
Author(s):  
Philip Mitchell ◽  
Elisabeth Petfalski ◽  
Rym Houalla ◽  
Alexandre Podtelejnikov ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Mrna Decay ◽  
Nuclear Protein ◽  
Rrna Processing ◽  
Small Nucleolar Rna ◽  
Whole Cell ◽  
Cell Lysates ◽  
Small Nuclear Rnas ◽  
Nuclear Exosome ◽  
Nucleolar Rna

ABSTRACT Related exosome complexes of 3′→5′ exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg2+-labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 3′→5′ exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3′ processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47-Δ strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3′-extended nuclear pre-mRNAs or the cytoplasmic 3′→5′ mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs.

scholarly journals Yeast snR30 is a small nucleolar RNA required for 18S rRNA synthesis.

1993 ◽  
Vol 13 (4) ◽  
pp. 2469-2477 ◽  
Author(s):  
J P Morrissey ◽  
D Tollervey
Keyword(s):  
18S Rrna ◽  
Progressive Increase ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Small Nucleolar Rna ◽  
Conditional Allele ◽  
Cell Doubling Time ◽  
Associated Proteins ◽  
Nucleolar Rna

Subnuclear fractionation and coprecipitation by antibodies against the nucleolar protein NOP1 demonstrate that the essential Saccharomyces cerevisiae RNA snR30 is localized to the nucleolus. By using aminomethyl trimethyl-psoralen, snR30 can be cross-linked in vivo to 35S pre-rRNA. To determine whether snR30 has a role in rRNA processing, a conditional allele was constructed by replacing the authentic SNR30 promoter with the GAL10 promoter. Repression of snR30 synthesis results in a rapid depletion of snR30 and a progressive increase in cell doubling time. rRNA processing is disrupted during the depletion of snR30; mature 18S rRNA and its 20S precursor underaccumulate, and an aberrant 23S pre-rRNA intermediate can be detected. Initial results indicate that this 23S pre-rRNA is the same as the species detected on depletion of the small nucleolar RNA-associated proteins NOP1 and GAR1 and in an snr10 mutant strain. It was found that the 3' end of 23S pre-rRNA is located in the 3' region of ITS1 between cleavage sites A2 and B1 and not, as previously suggested, at the B1 site, snR30 is the fourth small nucleolar RNA shown to play a role in rRNA processing.

scholarly journals Nucleolar Localization Elements of Xenopus laevis U3 Small Nucleolar RNA

1998 ◽  
Vol 9 (10) ◽  
pp. 2973-2985 ◽  
Author(s):  
Thilo Sascha Lange ◽  
Michael Ezrokhi ◽  
Anton V. Borovjagin ◽  
Rafael Rivera-León ◽  
Melanie T. North ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Hinge Region ◽  
Nucleolar Localization ◽  
Rrna Processing ◽  
Small Nucleolar Rna ◽  
Critical Elements ◽  
U3 Snorna ◽  
External Transcribed Spacer ◽  
Localization Elements ◽  
Nucleolar Rna

The Nucleolar Localization Elements (NoLEs) of Xenopus laevis U3 small nucleolar RNA (snoRNA) have been defined. Fluorescein-labeled wild-type U3 snoRNA injected intoXenopus oocyte nuclei localized specifically to nucleoli as shown by fluorescence microscopy. Injection of mutated U3 snoRNA revealed that the 5′ region containing Boxes A and A′, known to be important for rRNA processing, is not essential for nucleolar localization. Nucleolar localization of U3 snoRNA was independent of the presence and nature of the 5′ cap and the terminal stem. In contrast, Boxes C and D, common to the Box C/D snoRNA family, are critical elements for U3 localization. Mutation of the hinge region, Box B, or Box C′ led to reduced U3 nucleolar localization. Results of competition experiments suggested that Boxes C and D act in a cooperative manner. It is proposed that Box B facilitates U3 snoRNA nucleolar localization by the primary NoLEs (Boxes C and D), with the hinge region of U3 subsequently base pairing to the external transcribed spacer of pre-rRNA, thus positioning U3 snoRNA for its roles in rRNA processing.

scholarly journals Cyclin-dependent Kinase 9 Links RNA Polymerase II Transcription to Processing of Ribosomal RNA

2013 ◽  
Vol 288 (29) ◽  
pp. 21173-21183 ◽  
Author(s):  
Kaspar Burger ◽  
Bastian Mühl ◽  
Michaela Rohrmoser ◽  
Britta Coordes ◽  
Martin Heidemann ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ribosomal Rna ◽  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Rrna Processing ◽  
Cyclin Dependent Kinase ◽  
Small Nucleolar Rna ◽  
Nucleolar Rna ◽  
Rnapii Transcription

Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3′ extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3′ processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3′ rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.

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