Nuclear RNA Exosome and Pervasive Transcription: Dual Sculptors of Genome Function

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.

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U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2666-2676.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2666-2676

Author(s):

J B Cohen ◽

S D Broz ◽

A D Levinson

Keyword(s):

Splice Site ◽

Mammalian Cells ◽

Mrna Processing ◽

Mrna Splicing ◽

Small Nuclear Rna ◽

Splice Sites ◽

Gene Complementation ◽

Small Nuclear Rnas ◽

Nuclear Rna ◽

Mutant Genes

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.

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A Nuclear Surveillance Pathway for mRNAs with Defective Polyadenylation

Molecular and Cellular Biology ◽

10.1128/mcb.25.22.9996-10004.2005 ◽

2005 ◽

Vol 25 (22) ◽

pp. 9996-10004 ◽

Cited By ~ 75

Author(s):

Laura Milligan ◽

Claire Torchet ◽

Christine Allmang ◽

Tracey Shipman ◽

David Tollervey

Keyword(s):

Rna Surveillance ◽

Major Function ◽

Mrna Stabilization ◽

Nuclear Rna ◽

Cleavage And Polyadenylation ◽

Polyadenylation Sites ◽

Partial Activity ◽

Mrna Polyadenylation ◽

Type Strains ◽

Exosome Complex

ABSTRACT The pap1-5 mutation in poly(A) polymerase causes rapid depletion of mRNAs at restrictive temperatures. Residual mRNAs are polyadenylated, indicating that Pap1-5p retains at least partial activity. In pap1-5 strains lacking Rrp6p, a nucleus-specific component of the exosome complex of 3′-5′ exonucleases, accumulation of poly(A)+ mRNA was largely restored and growth was improved. The catalytically inactive mutant Rrp6-1p did not increase growth of the pap1-5 strain and conferred much less mRNA stabilization than rrp6Δ. This may indicate that the major function of Rrp6p is in RNA surveillance. Inactivation of core exosome components, Rrp41p and Mtr3p, or the nuclear RNA helicase Mtr4p gave different phenotypes, with accumulation of deadenylated and 3′-truncated mRNAs. We speculate that slowed mRNA polyadenylation in the pap1-5 strain is detected by a surveillance activity of Rrp6p, triggering rapid deadenylation and exosome-mediated degradation. In wild-type strains, assembly of the cleavage and polyadenylation complex might be suboptimal at cryptic polyadenylation sites, causing slowed polyadenylation.

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Altered rRNA processing disrupts nuclear RNA homeostasis via competition for the poly(A)-binding protein Nab2

Nucleic Acids Research ◽

10.1093/nar/gkaa964 ◽

2020 ◽

Vol 48 (20) ◽

pp. 11675-11694

Author(s):

Lisbeth-Carolina Aguilar ◽

Biplab Paul ◽

Taylor Reiter ◽

Louis Gendron ◽

Arvind Arul Nambi Rajan ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Metabolism ◽

Rdna Transcription ◽

Cellular Processes ◽

Genome Wide ◽

Nuclear Rna ◽

Rna Exosome ◽

Nucleolar Rna

Abstract RNA-binding proteins (RBPs) are key mediators of RNA metabolism. Whereas some RBPs exhibit narrow transcript specificity, others function broadly across both coding and non-coding RNAs. Here, in Saccharomyces cerevisiae, we demonstrate that changes in RBP availability caused by disruptions to distinct cellular processes promote a common global breakdown in RNA metabolism and nuclear RNA homeostasis. Our data shows that stabilization of aberrant ribosomal RNA (rRNA) precursors in an enp1-1 mutant causes phenotypes similar to RNA exosome mutants due to nucleolar sequestration of the poly(A)-binding protein (PABP) Nab2. Decreased nuclear PABP availability is accompanied by genome-wide changes in RNA metabolism, including increased pervasive transcripts levels and snoRNA processing defects. These phenotypes are mitigated by overexpression of PABPs, inhibition of rDNA transcription, or alterations in TRAMP activity. Our results highlight the need for cells to maintain poly(A)-RNA levels in balance with PABPs and other RBPs with mutable substrate specificity across nucleoplasmic and nucleolar RNA processes.

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The regulation and functions of the nuclear RNA exosome complex

Nature Reviews Molecular Cell Biology ◽

10.1038/nrm.2015.15 ◽

2016 ◽

Vol 17 (4) ◽

pp. 227-239 ◽

Cited By ~ 169

Author(s):

Cornelia Kilchert ◽

Sina Wittmann ◽

Lidia Vasiljeva

Keyword(s):

Nuclear Rna ◽

Rna Exosome ◽

Exosome Complex

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Artificial Box C/D RNAs Affect Pre-mRNA Maturation in Human Cells

BioMed Research International ◽

10.1155/2013/656158 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 7

Author(s):

Grigoriy A. Stepanov ◽

Dmitry V. Semenov ◽

Anna V. Savelyeva ◽

Elena V. Kuligina ◽

Olga A. Koval ◽

...

Keyword(s):

Human Cells ◽

Dependent Manner ◽

Control Of Gene Expression ◽

Cellular Processes ◽

Small Nuclear Rnas ◽

Mrna Maturation ◽

Target Rna ◽

Dose Dependent ◽

Nucleolar Rnas

Box C/D small nucleolar RNAs (snoRNAs) are known to guide the2′-O-ribose methylation of nucleotides in eukaryotic ribosomal RNAs and small nuclear RNAs. Recently snoRNAs are predicted to regulate posttranscriptional modifications of pre-mRNA. To expand understanding of the role of snoRNAs in control of gene expression, in this study we tested the ability of artificial box C/D RNAs to affect the maturation of target pre-mRNA. We found that transfection of artificial box C/D snoRNA analogues directed toHSPA8pre-mRNAs into human cells induced suppression of the target mRNA expression in a time- and dose-dependent manner. The artificial box C/D RNA directed to the branch point adenosine of the second intron, as well as the analogue directed to the last nucleotide of the second exon of theHSPA8pre-mRNA caused the most prominent influence on the level ofHSPA8mRNAs. Neither box D nor the ability to direct2′-O-methylation of nucleotides in target RNA was essential for the knockdown activity of artificial snoRNAs. Inasmuch as artificial box C/D RNAs decreased viability of transfected human cells, we propose that natural snoRNAs as well as their artificial analogues can influence the maturation of complementary pre-mRNA and can be effective regulators of vital cellular processes.

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

Molecular and Cellular Biology ◽

10.1128/mcb.20.2.441-452.2000 ◽

2000 ◽

Vol 20 (2) ◽

pp. 441-452 ◽

Cited By ~ 228

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.

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Distinct and evolutionary conserved structural features of the human nuclear exosome complex

eLife ◽

10.7554/elife.38686 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 18

Author(s):

Piotr Gerlach ◽

Jan M Schuller ◽

Fabien Bonneau ◽

Jérôme Basquin ◽

Peter Reichelt ◽

...

Keyword(s):

Structural Features ◽

Core Complex ◽

The Core ◽

Biochemical Assays ◽

Nuclear Rna ◽

Nuclear Exosome ◽

Non Coding Rnas ◽

Rna Exosome ◽

Distinct Features ◽

Exosome Complex

The nuclear RNA exosome complex mediates the processing of structured RNAs and the decay of aberrant non-coding RNAs, an important function particularly in human cells. Most mechanistic studies to date have focused on the yeast system. Here, we reconstituted and studied the properties of a recombinant 14-subunit human nuclear exosome complex. In biochemical assays, the human exosome embeds a longer RNA channel than its yeast counterpart. The 3.8 Å resolution cryo-EM structure of the core complex bound to a single-stranded RNA reveals that the RNA channel path is formed by two distinct features of the hDIS3 exoribonuclease: an open conformation and a domain organization more similar to bacterial RNase II than to yeast Rrp44. The cryo-EM structure of the holo-complex shows how obligate nuclear cofactors position the hMTR4 helicase at the entrance of the core complex, suggesting a striking structural conservation from lower to higher eukaryotes.

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