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Literature DB >> 12384611

A double reporter assay for detecting changes in the ratio of spliced and unspliced mRNA in mammalian cells.

Md Talat Nasim¹, H M Chowdhury, I C Eperon.

Abstract

Current methods for measuring the efficiency of splicing in mammalian cells rely on either direct analysis of the RNA, which does not lend itself to rapid assays, or on single reporter functions that are subject to numerous intrinsic variables. If two protein activities are encoded within a single reading frame but on separate exons, with an intervening sequence containing termination codons, then the expression of the second activity is dependent on removal of the intervening sequence by pre-mRNA splicing. Thus, the ratio of the activities encoded by exon 2 to exon 1 reflects the ratio of expression from spliced mRNA to the total expression of spliced and unspliced RNA. This provides a rapid and convenient assay for the effects on splicing efficiency of trans-acting factors or of alterations in the sequences of the intron and surrounding exon sequences.

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Year: 2002 PMID： 12384611 PMCID： PMC137153 DOI： 10.1093/nar/gnf108

Source DB: PubMed Journal: Nucleic Acids Res ISSN： 0305-1048 Impact factor: 16.971

17 in total

1. Eukaryotic selenocysteine incorporation follows a nonprocessive mechanism that competes with translational termination.

Authors: M T Nasim; S Jaenecke; A Belduz; H Kollmus; L Flohé; J E McCarthy
Journal: J Biol Chem Date: 2000-05-19 Impact factor: 5.157

2. In vitro selection of exonic splicing enhancer sequences: identification of novel CD44 enhancers.

Authors: G Woerfel; A Bindereif
Journal: Nucleic Acids Res Date: 2001-08-01 Impact factor: 16.971

3. The splicing of U12-type introns can be a rate-limiting step in gene expression.

Authors: Abhijit A Patel; Matthew McCarthy; Joan A Steitz
Journal: EMBO J Date: 2002-07-15 Impact factor: 11.598

4. A role for exon sequences and splice-site proximity in splice-site selection.

Authors: R Reed; T Maniatis
Journal: Cell Date: 1986-08-29 Impact factor: 41.582

5. Hierarchy for 5' splice site preference determined in vivo.

Authors: A L Lear; L P Eperon; I M Wheatley; I C Eperon
Journal: J Mol Biol Date: 1990-01-05 Impact factor: 5.469

6. The pre-mRNA 5' cap determines whether U6 small nuclear RNA succeeds U1 small nuclear ribonucleoprotein particle at 5' splice sites.

Authors: L O'Mullane; I C Eperon
Journal: Mol Cell Biol Date: 1998-12 Impact factor: 4.272

7. The role of nucleotide sequences in splice site selection in eukaryotic pre-messenger RNA.

Authors: L P Eperon; J P Estibeiro; I C Eperon
Journal: Nature Date: 1986 Nov 20-26 Impact factor: 49.962

8. Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing.

Authors: K K Nelson; M R Green
Journal: Genes Dev Date: 1988-03 Impact factor: 11.361

9. Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm.

Authors: P Legrain; M Rosbash
Journal: Cell Date: 1989-05-19 Impact factor: 41.582

10. Serine-arginine (SR) protein-like factors that antagonize authentic SR proteins and regulate alternative splicing.

Authors: A E Cowper; J F Cáceres; A Mayeda; G R Screaton
Journal: J Biol Chem Date: 2001-10-29 Impact factor: 5.157

20 in total

1. ATPase/helicase activities of p68 RNA helicase are required for pre-mRNA splicing but not for assembly of the spliceosome.

Authors: Chunru Lin; Liuqing Yang; Jenny J Yang; Youliang Huang; Zhi-Ren Liu
Journal: Mol Cell Biol Date: 2005-09 Impact factor: 4.272

2. The RNA binding motif protein 15B (RBM15B/OTT3) is a functional competitor of serine-arginine (SR) proteins and antagonizes the positive effect of the CDK11p110-cyclin L2α complex on splicing.

Authors: Pascal Loyer; Adeline Busson; Janeen H Trembley; Judith Hyle; Jose Grenet; Wei Zhao; Catherine Ribault; Tristan Montier; Vincent J Kidd; Jill M Lahti
Journal: J Biol Chem Date: 2010-11-02 Impact factor: 5.157

A double reporter assay for detecting changes in the ratio of spliced and unspliced mRNA in mammalian cells.

1. Eukaryotic selenocysteine incorporation follows a nonprocessive mechanism that competes with translational termination.

2. In vitro selection of exonic splicing enhancer sequences: identification of novel CD44 enhancers.

3. The splicing of U12-type introns can be a rate-limiting step in gene expression.

4. A role for exon sequences and splice-site proximity in splice-site selection.

5. Hierarchy for 5' splice site preference determined in vivo.

6. The pre-mRNA 5' cap determines whether U6 small nuclear RNA succeeds U1 small nuclear ribonucleoprotein particle at 5' splice sites.

7. The role of nucleotide sequences in splice site selection in eukaryotic pre-messenger RNA.

8. Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing.

9. Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm.

10. Serine-arginine (SR) protein-like factors that antagonize authentic SR proteins and regulate alternative splicing.

1. ATPase/helicase activities of p68 RNA helicase are required for pre-mRNA splicing but not for assembly of the spliceosome.

2. The RNA binding motif protein 15B (RBM15B/OTT3) is a functional competitor of serine-arginine (SR) proteins and antagonizes the positive effect of the CDK11p110-cyclin L2α complex on splicing.

3. Synthetic mRNA splicing modulator compounds with in vivo antitumor activity.

4. A high-throughput assay to identify small-molecule modulators of alternative pre-mRNA splicing.

5. Mutation of a U2 snRNA gene causes global disruption of alternative splicing and neurodegeneration.

6. Activation of pre-mRNA splicing by human RNPS1 is regulated by CK2 phosphorylation.

7. CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11(p110).

8. Intragenic Enhancers Attenuate Host Gene Expression.

9. Nuclear ARVCF protein binds splicing factors and contributes to the regulation of alternative splicing.

10. Identification of an exonic splicing silencer in exon 6A of the human VEGF gene.