scholarly journals The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0257503
Author(s):  
Jessie A. C. Altieri ◽  
Klemens J. Hertel
Keyword(s):  
Degradation Kinetics ◽  
Mrna Splicing ◽  
Rrna Synthesis ◽  
Metabolic Labeling ◽  
Rna Turnover ◽  
Splice Sites ◽  
Synthesis And Processing ◽  
Splicing Efficiency ◽  
Extended Exposure ◽  
T7 Polymerase

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50μM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0–100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3’ and the 5’ splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 μM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

scholarly journals The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes

2021 ◽  
Author(s):  
Jessie A. C. Altieri ◽  
Klemens J. Hertel
Keyword(s):  
Degradation Kinetics ◽  
Mrna Splicing ◽  
Rrna Synthesis ◽  
Metabolic Labeling ◽  
Rna Turnover ◽  
Splice Sites ◽  
Synthesis And Processing ◽  
Splicing Efficiency ◽  
Extended Exposure ◽  
T7 Polymerase

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50µM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3’ and the 5’ splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 µM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

scholarly journals Harnessing the Nucleolar DNA Damage Response in Cancer Therapy

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1156
Author(s):  
Jiachen Xuan ◽  
Kezia Gitareja ◽  
Natalie Brajanovski ◽  
Elaine Sanij
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cancer Therapy ◽  
Dna Damage Response ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Pol I ◽  
Clinical Investigations ◽  
Damage Response ◽  
Synthesis And Processing

The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. They serve as the site of rRNA synthesis and processing, and ribosome assembly. There are 400–600 copies of rRNA genes (rDNA) in human cells and their highly repetitive and transcribed nature poses a challenge for DNA repair and replication machineries. It is only in the last 7 years that the DNA damage response and processes of DNA repair at the rDNA repeats have been recognized to be unique and distinct from the classic response to DNA damage in the nucleoplasm. In the last decade, the nucleolus has also emerged as a central hub for coordinating responses to stress via sequestering tumor suppressors, DNA repair and cell cycle factors until they are required for their functional role in the nucleoplasm. In this review, we focus on features of the rDNA repeats that make them highly vulnerable to DNA damage and the mechanisms by which rDNA damage is repaired. We highlight the molecular consequences of rDNA damage including activation of the nucleolar DNA damage response, which is emerging as a unique response that can be exploited in anti-cancer therapy. In this review, we focus on CX-5461, a novel inhibitor of Pol I transcription that induces the nucleolar DNA damage response and is showing increasing promise in clinical investigations.

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

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.

scholarly journals Assessment of branch point prediction tools to predict physiological branch points and their alteration by variants

2019 ◽  
Author(s):  
Raphael Leman ◽  
Hélène Tubeuf ◽  
Sabine Raad ◽  
Isabelle Tournier ◽  
Céline Derambure ◽  
...  
Keyword(s):  
Branch Point ◽  
Mrna Splicing ◽  
Large Set ◽  
Branch Points ◽  
Splice Sites ◽  
Prediction Tools ◽  
Mature Mrna ◽  
Bioinformatics Tools ◽  
The Impact

Abstract Background: Branch points (BPs) map within short motifs upstream of acceptor splice sites (3’ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3’ss. Results: We used a large set of constitutive and alternative human 3’ss collected from Ensembl (n = 264,787 3’ss) and from in-house RNAseq experiments (n = 51,986 3’ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3’ss (99.48 % and 65.84 % accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17 %. Conclusions: Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3’ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area. Keywords: Branch Point, Prediction, RNA, Benchmark, HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR, RNABPS, Variants

Mechanisms for selecting 5′ splice sites in mammalian pre-mRNA splicing

1994 ◽  
Vol 10 (3) ◽  
pp. 100-106 ◽  
Author(s):  
David S. Horowitz ◽  
Adrian R. Krainer
Keyword(s):  
Mrna Splicing ◽  
Splice Sites

scholarly journals Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons

2014 ◽  
Vol 42 (14) ◽  
pp. 8895-8904 ◽  
Author(s):  
Karol Szafranski ◽  
Claudia Fritsch ◽  
Frank Schumann ◽  
Lisa Siebel ◽  
Rileen Sinha ◽  
...  
Keyword(s):  
Physiological State ◽  
Mrna Splicing ◽  
Splice Sites

scholarly journals A common variant inMTHFD1Lis associated with neural tube defects and mRNA splicing efficiency

2009 ◽  
Vol 30 (12) ◽  
pp. 1650-1656 ◽  
Author(s):  
Anne Parle-McDermott ◽  
Faith Pangilinan ◽  
Kirsty K. O'Brien ◽  
James L. Mills ◽  
Alan M. Magee ◽  
...  
Keyword(s):  
Neural Tube ◽  
Neural Tube Defects ◽  
Mrna Splicing ◽  
Common Variant ◽  
Splicing Efficiency

scholarly journals Dhr1p, a Putative DEAH-Box RNA Helicase, Is Associated with the Box C+D snoRNP U3

2000 ◽  
Vol 20 (19) ◽  
pp. 7238-7246 ◽  
Author(s):  
Alan Colley ◽  
Jean D. Beggs ◽  
David Tollervey ◽  
Denis L. J. Lafontaine
Keyword(s):  
18S Rrna ◽  
Mrna Splicing ◽  
Rrna Synthesis ◽  
Detectable Effect ◽  
Rna Helicases ◽  
Structural Reorganization ◽  
Rrna Cleavage ◽  
Processing Steps

ABSTRACT Putative RNA helicases are involved in most aspects of gene expression. All previously characterized members of the DEAH-box family of putative RNA helicases are involved in pre-mRNA splicing. Here we report the analysis of two novel DEAH-box RNA helicases, Dhr1p and Dhr2p, that were found to be predominantly nucleolar. Both genes are essential for viability, and MET-regulated alleles were therefore created. Depletion of Dhr1p or Dhr2p had no detectable effect on pre-mRNA splicing in vivo or in vitro. Both Dhr1p and Dhr2p were, however, required for 18S rRNA synthesis. Depletion of Dhr2p inhibited pre-rRNA cleavage at sites A0, A1, and A2, while Dhr1p depletion inhibited cleavage at sites A1 and A2. No coprecipitation of snoRNAs was detected with ProtA-Dhr2p, but Dhr1p-ProtA was stably associated with the U3 snoRNA. Depletion of Dhr1p inhibited processing steps that require base pairing of U3 to the 5′ end of the 18S rRNA. We speculate that Dhr1p is targeted to the preribosomal particles by the U3-18S rRNA interaction and is required for the structural reorganization of the rRNA during formation of the central pseudoknot.

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