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Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly
The pairing of 5′ and 3′ splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs a...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233244/ https://www.ncbi.nlm.nih.gov/pubmed/25403181 http://dx.doi.org/10.1101/gad.248625.114 |
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author | Sharma, Shalini Wongpalee, Somsakul Pop Vashisht, Ajay Wohlschlegel, James A. Black, Douglas L. |
author_facet | Sharma, Shalini Wongpalee, Somsakul Pop Vashisht, Ajay Wohlschlegel, James A. Black, Douglas L. |
author_sort | Sharma, Shalini |
collection | PubMed |
description | The pairing of 5′ and 3′ splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem–loop 4 (SL4) of the U1 snRNA, which recognizes the 5′ splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 3′ splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 5′ and 3′ splice site complexes within the assembling spliceosome. |
format | Online Article Text |
id | pubmed-4233244 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-42332442015-05-15 Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly Sharma, Shalini Wongpalee, Somsakul Pop Vashisht, Ajay Wohlschlegel, James A. Black, Douglas L. Genes Dev Research Paper The pairing of 5′ and 3′ splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem–loop 4 (SL4) of the U1 snRNA, which recognizes the 5′ splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 3′ splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 5′ and 3′ splice site complexes within the assembling spliceosome. Cold Spring Harbor Laboratory Press 2014-11-15 /pmc/articles/PMC4233244/ /pubmed/25403181 http://dx.doi.org/10.1101/gad.248625.114 Text en © 2014 Sharma et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Research Paper Sharma, Shalini Wongpalee, Somsakul Pop Vashisht, Ajay Wohlschlegel, James A. Black, Douglas L. Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly |
title | Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly |
title_full | Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly |
title_fullStr | Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly |
title_full_unstemmed | Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly |
title_short | Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly |
title_sort | stem–loop 4 of u1 snrna is essential for splicing and interacts with the u2 snrnp-specific sf3a1 protein during spliceosome assembly |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233244/ https://www.ncbi.nlm.nih.gov/pubmed/25403181 http://dx.doi.org/10.1101/gad.248625.114 |
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