scholarly journals Nucleolar Factors Direct the 2′-O-Ribose Methylation and Pseudouridylation of U6 Spliceosomal RNA

1999 ◽  
Vol 19 (10) ◽  
pp. 6906-6917 ◽  
Author(s):  
Philippe Ganot ◽  
Beáta E. Jády ◽  
Marie-Line Bortolin ◽  
Xavier Darzacq ◽  
Tamás Kiss
Keyword(s):  
Small Nuclear Rna ◽  
Guide Rna ◽  
Guide Rnas ◽  
Trafficking Pathway ◽  
5.8S Rrna ◽  
Nuclear Rna ◽  
Nucleolar Rna ◽  
Correct Target ◽  
Selection Of ◽  
U6 Rna

ABSTRACT The nucleolus has long been known as a functionally highly specialized subnuclear compartment where synthesis, posttranscriptional modification, and processing of cytoplasmic rRNAs take place. In this study, we demonstrate that the nucleolus contains all thetrans-acting factors that are responsible for the accurate and efficient synthesis of the eight 2′-O-methylated nucleotides and three pseudouridine residues carried by the mammalian U6 spliceosomal small nuclear RNA. Factors mediating the formation of pseudouridine residues in the U3 small nucleolar RNA are also present and functionally active in the nucleolus. For selection of the correct target nucleotides in the U6 and U3 RNAs, the nucleolar 2′-O-methylation and pseudouridylation factors rely on short sequences located around the target nucleotide to be modified. This observation further underscores a recently proposed role for small nucleolar guide RNAs in the 2′-O-methylation of the U6 spliceosomal RNA (K. T. Tycowski, Z.-H. You, P. J. Graham, and J. A. Steitz, Mol. Cell 2:629–638, 1998). We demonstrate that a novel 2′-O-methylated nucleotide can be generated in the yeast U6 RNA by use of an artificial 2′-O-methylation small nucleolar guide RNA. We also show that a short fragment of the 5.8S rRNA, when expressed as part of the human U6 RNA, is faithfully 2′-O-methylated and pseudouridylated. These results are most consistent with a trafficking pathway in which the U6 spliceosomal RNA cycles through the nucleolus to undergo nucleolar RNA-directed modifications.

scholarly journals Sensitivity to UV radiation of small nuclear RNA synthesis in mammalian cells.

1983 ◽  
Vol 3 (12) ◽  
pp. 2151-2155 ◽  
Author(s):  
G L Eliceiri ◽  
J H Smith
Keyword(s):  
Uv Radiation ◽  
Uv Irradiation ◽  
Mammalian Cells ◽  
Repair Process ◽  
Rrna Synthesis ◽  
Small Nuclear Rna ◽  
Pyrimidine Dimers ◽  
5.8S Rrna ◽  
Nuclear Rna ◽  
U5 Snrna

It was demonstrated previously that the synthesis of small nuclear RNA (snRNA) species U1 and U2 in human cells is very sensitive to UV radiation. In the present work, the UV sensitivity of U3, U4, and U5 snRNA synthesis is shown to be also high. The synthesis of U1, U2, U3, U4, and U5 snRNAs progressively decreased during the first 2 h after UV irradiation (this was not observed in polyadenylated RNA) and had not returned to normal rates 6 h after UV exposure. In contrast, the restoration of 5.8S rRNA synthesis began immediately after UV irradiation and was essentially complete 6 h later. A small fraction of U1 and U5 (and possibly U2 and U3) snRNA synthesis remained unaffected by high UV doses, when cell radiolabeling began 10 min after UV irradiation. The present data suggest that a factor other than the level of pyrimidine dimers in DNA (possibly, steps in the post-irradiation DNA repair process) plays an important role in the mechanism of UV-induced inhibition of U1-U5 snRNA synthesis.

scholarly journals Ares-GT: design of guide RNAs targeting multiple genes for CRISPR-Cas experiments

2020 ◽  
Author(s):  
Eugenio G. Minguet
Keyword(s):  
Reference Genome ◽  
Query Sequence ◽  
Gene Families ◽  
Guide Rna ◽  
Guide Rnas ◽  
Online Tools ◽  
Multiple Input ◽  
Design Guide ◽  
Command Line Tool ◽  
Selection Of

ABSTRACTMotivationThere is a lack of tools to design guide RNA for CRISPR genome editing of gene families and usually good candidate sgRNAs are tagged with low scores precisely because they match several locations in the genome, thus time-consuming manual evaluation of targets is required. Moreover, online tools are limited to a restricted list of reference genome and lack the flexibility to incorporate unpublished genomes or contemplate genomes of populations with allelic variants.ResultsTo address these issues, I have developed the ARES-GT, a local command line tool in Python software. ARES-GT allows the selection of candidate sgRNAs that match multiple input query sequences, in addition of candidate sgRNAs that specifically match each query sequence. It also contemplates the use of unmapped contigs apart from complete genomes thus allowing the use of any genome provided by user and being able to handle intraspecies allelic variability and individual polymorphisms.AvailabilityARES-GT is available at GitHub (https://github.com/eugomin/ARES-GT.git).

scholarly journals snR30/U17 Small Nucleolar Ribonucleoprotein: A Critical Player during Ribosome Biogenesis

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2195
Author(s):  
Timothy John Vos ◽  
Ute Kothe
Keyword(s):  
Ribosome Biogenesis ◽  
Current Knowledge ◽  
Small Subunit ◽  
Future Research ◽  
Unique Role ◽  
Ribosome Synthesis ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ssu Processome ◽  
Nucleolar Rna

The small nucleolar RNA snR30 (U17 in humans) plays a unique role during ribosome synthesis. Unlike most members of the H/ACA class of guide RNAs, the small nucleolar ribonucleoprotein (snoRNP) complex assembled on snR30 does not direct pseudouridylation of ribosomal RNA (rRNA), but instead snR30 is critical for 18S rRNA processing during formation of the small subunit (SSU) of the ribosome. Specifically, snR30 is essential for three pre-rRNA cleavages at the A0/01, A1/1, and A2/2a sites in yeast and humans, respectively. Accordingly, snR30 is the only essential H/ACA guide RNA in yeast. Here, we summarize our current knowledge about the interactions and functions of snR30, discuss what remains to be elucidated, and present two non-exclusive hypotheses on the possible molecular function of snR30 during ribosome biogenesis. First, snR30 might be responsible for recruiting other proteins including endonucleases to the SSU processome. Second, snR30 may contribute to the refolding of pre-rRNA into a required conformation that serves as a checkpoint during ribosome biogenesis facilitating pre-rRNA cleavage. In both scenarios, the snR30 snoRNP may have scaffolding and RNA chaperoning activity. In conclusion, the snR30 snoRNP is a crucial player with an unknown molecular mechanism during ribosome synthesis, posing many interesting future research questions.

Mammalian U6 small nuclear RNA undergoes 3' end modifications within the spliceosome

1993 ◽  
Vol 13 (3) ◽  
pp. 1641-1650
Author(s):  
J Tazi ◽  
T Forne ◽  
P Jeanteur ◽  
G Cathala ◽  
C Brunel
Keyword(s):  
Single Species ◽  
Small Nuclear Rna ◽  
U6 Snrna ◽  
Major Form ◽  
Nuclear Extracts ◽  
Nuclear Rna ◽  
Minor Form ◽  
Multiple Species ◽  
A Minor ◽  
Selection Of

Mammalian U6 small nuclear RNA (snRNA) is heterogeneous with respect to the number of 3' terminal U residues. The major form terminates with five U residues and a 2',3' cyclic phosphate. Because of the presence in HeLa cell nuclear extracts of a terminal uridylyl transferase, a minor form of U6 snRNA is elongated, producing multiple species containing up to 12 U residues. In this study we have used glycerol gradients to demonstrate that these U6 snRNA forms are assembled into U6 ribonucleoprotein (RNP), U4/U6 snRNPs, and U4/U5/U6 tri-snRNP complexes. Furthermore, glycerol gradients combined with affinity selection of biotinylated pre-mRNAs led us to show that elongated forms of U6 snRNAs enter the spliceosome and that some of these become shortened with time to a single species having the same characteristics as the major form of U6 snRNA present in mammalian nuclear extracts. We propose that this elongation-shortening process is related to the function of U6 snRNA in mammalian pre-mRNA splicing.

scholarly journals Mammalian U6 small nuclear RNA undergoes 3' end modifications within the spliceosome.

1993 ◽  
Vol 13 (3) ◽  
pp. 1641-1650 ◽  
Author(s):  
J Tazi ◽  
T Forne ◽  
P Jeanteur ◽  
G Cathala ◽  
C Brunel
Keyword(s):  
Single Species ◽  
Small Nuclear Rna ◽  
U6 Snrna ◽  
Major Form ◽  
Nuclear Extracts ◽  
Nuclear Rna ◽  
Minor Form ◽  
Multiple Species ◽  
A Minor ◽  
Selection Of

Mammalian U6 small nuclear RNA (snRNA) is heterogeneous with respect to the number of 3' terminal U residues. The major form terminates with five U residues and a 2',3' cyclic phosphate. Because of the presence in HeLa cell nuclear extracts of a terminal uridylyl transferase, a minor form of U6 snRNA is elongated, producing multiple species containing up to 12 U residues. In this study we have used glycerol gradients to demonstrate that these U6 snRNA forms are assembled into U6 ribonucleoprotein (RNP), U4/U6 snRNPs, and U4/U5/U6 tri-snRNP complexes. Furthermore, glycerol gradients combined with affinity selection of biotinylated pre-mRNAs led us to show that elongated forms of U6 snRNAs enter the spliceosome and that some of these become shortened with time to a single species having the same characteristics as the major form of U6 snRNA present in mammalian nuclear extracts. We propose that this elongation-shortening process is related to the function of U6 snRNA in mammalian pre-mRNA splicing.

scholarly journals Base pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar Ribonucleoproteins

2019 ◽  
Author(s):  
Erin Katelyn Kelly ◽  
Dominic Philip Czekay ◽  
Ute Kothe
Keyword(s):  
Messenger Rna ◽  
Ribosome Biogenesis ◽  
Small Nuclear Rna ◽  
Base Pairing ◽  
Base Pairs ◽  
Guide Rna ◽  
Guide Rnas ◽  
Pairing Strength ◽  
Pairing Interactions ◽  
Time Courses

AbstractH/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base-pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the Saccharomyces cerevisiae H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide – substrate RNA pairs.

scholarly journals A discrete 3' region of U6 small nuclear RNA modulates the phosphorylation cycle of the C1 heterogeneous nuclear ribonucleoprotein particle protein.

1996 ◽  
Vol 16 (3) ◽  
pp. 1241-1246 ◽  
Author(s):  
S H Mayrand ◽  
P A Fung ◽  
T Pederson
Keyword(s):  
Rnase H ◽  
Micrococcal Nuclease ◽  
Small Nuclear Rna ◽  
Ribonucleoprotein Particle ◽  
Nuclear Extracts ◽  
Nuclear Rna ◽  
Hnrnp Protein ◽  
Protein Dephosphorylation ◽  
Nuclear Ribonucleoprotein ◽  
U6 Rna

The C heterogeneous ribonucleoprotein particle (hnRNP) protein bind to nascent pre-mRNA and may participate in assembly of the early prespliceosome. Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nuclear extracts regulates its binding to pre-mRNA (S. H. Mayrand, P. Dwen, and T. Pederson, Proc. Natl. Acad. Sci. USA 90:7764-7768, 1993). We have now further investigated the phosphorylation cycle of the C1 hnRNP protein, with emphasis on its regulation. Pretreatment of nuclear extracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a dependence on RNA. Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2, and U4 small nuclear RNAs did not affect the phosphorylation of C1 hnRNP protein. However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dephosphorylation step of the C1 hnRNP protein phosphorylation cycle. This inhibition was as pronounced as that seen with the serine/threonine protein phosphatase inhibitor okadaic acid. C1 hnRNP protein dephosphorylation could be completely restored by the addition of intact U6 RNA. Add-back experiments with mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucleotides 85 to 92 of U6 RNA. These results illuminate a hitherto unanticipated function of U6 RNA: the modulation of a phosphorylation-dephosphorylation cycle of C1 hnRNP protein that influences the binding affinity of this protein for pre-mRNA. This newly revealed function of U6 RNA is likely to play a very early role in the prespliceosome assembly pathway, prior to U6 RNA's entry into the mature spliceosome's active center.

scholarly journals Yeast Exosome Mutants Accumulate 3′-Extended Polyadenylated Forms of U4 Small Nuclear RNA and Small Nucleolar RNAs

2000 ◽  
Vol 20 (2) ◽  
pp. 441-452 ◽  
Author(s):  
Ambro van Hoof ◽  
Pascal Lennertz ◽  
Roy Parker
Keyword(s):  
Small Nucleolar Rnas ◽  
Small Nuclear Rna ◽  
The Core ◽  
Rnase Mrp ◽  
Degradation Reactions ◽  
5.8S Rrna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Core Components ◽  
Nucleolar Rnas

ABSTRACT The exosome is a protein complex consisting of a variety of 3′-to-5′ exonucleases that functions both in 3′-to-5′ trimming of rRNA precursors and in 3′-to-5′ degradation of mRNA. To determine additional exosome functions, we examined the processing of a variety of RNAs, including tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), RNase P, RNase MRP, and SRP RNAs, and 5S rRNAs in mutants defective in either the core components of the exosome or in other proteins required for exosome function. These experiments led to three important conclusions. First, exosome mutants accumulate 3′-extended forms of the U4 snRNA and a wide variety of snoRNAs, including snoRNAs that are independently transcribed or intron derived. This finding suggests that the exosome functions in the 3′ end processing of these species. Second, in exosome mutants, transcripts for U4 snRNA and independently transcribed snoRNAs accumulate as 3′-extended polyadenylated species, suggesting that the exosome is required to process these 3′-extended transcripts. Third, processing of 5.8S rRNA, snRNA, and snoRNA by the exosome is affected by mutations of the nuclear proteins Rrp6p and Mtr4p, whereas mRNA degradation by the exosome required Ski2p and was not affected by mutations inRRP6 or MTR4. This finding suggests that the cytoplasmic and nuclear forms of the exosome represent two functionally different complexes involved in distinct 3′-to-5′ processing and degradation reactions.

scholarly journals GRIBCG: A software for selection of sgRNAs in the design of balancer chromosomes

2018 ◽  
Author(s):  
Brian B. Merritt ◽  
Lily S. Cheung
Keyword(s):  
Rational Design ◽  
Chromosomal Rearrangements ◽  
Fruit Fly ◽  
Model Organisms ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genetics Research ◽  
Multicellular Organisms ◽  
Balancer Chromosomes ◽  
Selection Of

AbstractBackgroundBalancer chromosomes are tools used by fruit fly geneticists to prevent meiotic recombination. Recently, CRISPR/Cas9 genome editing has been shown capable of generating inversions similar to the chromosomal rearrangements present in balancer chromosomes. Extending the benefits of balancer chromosomes to other multicellular organisms could significantly accelerate biomedical and plant genetics research.ResultsHere, we present GRIBCG (Guide RNA Identifier for Balancer Chromosome Generation), a tool for the rational design of balancer chromosomes. GRIBCG identifies single guide RNAs (sgRNAs) for use with Streptococcus pyogenes Cas9 (SpCas9). These sgRNAs would efficiently cut a chromosome multiple times while minimizing off-target cutting in the rest of the genome. We describe the performance of this tool on six model organisms and compare our results to two routinely used fruit fly balancer chromosomes.ConclusionGRIBCG is the first of its kind tool for the design of balancer chromosomes using CRISPR/Cas9. GRIBCG can accelerate genetics research by providing a fast, systematic and simple to use framework to induce chromosomal rearrangements.

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