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Delineation of the Exact Transcription Termination Signal for Type 3 Polymerase III
Type 3 Pol III promoters such as U6 are widely used for expression of small RNAs, including short hairpin RNA for RNAi applications and guide RNA in CRISPR genome-editing platforms. RNA polymerase III uses a T-stretch as termination signal, but the exact properties have not been thoroughly investiga...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society of Gene & Cell Therapy
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725217/ https://www.ncbi.nlm.nih.gov/pubmed/29499947 http://dx.doi.org/10.1016/j.omtn.2017.11.006 |
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author | Gao, Zongliang Herrera-Carrillo, Elena Berkhout, Ben |
author_facet | Gao, Zongliang Herrera-Carrillo, Elena Berkhout, Ben |
author_sort | Gao, Zongliang |
collection | PubMed |
description | Type 3 Pol III promoters such as U6 are widely used for expression of small RNAs, including short hairpin RNA for RNAi applications and guide RNA in CRISPR genome-editing platforms. RNA polymerase III uses a T-stretch as termination signal, but the exact properties have not been thoroughly investigated. Here, we systematically measured the in vivo termination efficiency and the actual site of termination for different T-stretch signals in three commonly used human Pol III promoters (U6, 7SK, and H1). Both the termination efficiency and the actual termination site depend on the T-stretch signal. The T4 signal acts as minimal terminator, but full termination efficiency is reached only with a T-stretch of ≥6. The termination site within the T-stretch is quite heterogeneous, and consequently small RNAs have a variable U-tail of 1–6 nucleotides. We further report that such variable U-tails can have a significant negative effect on the functionality of the crRNA effector of the CRISPR-AsCpf1 system. We next improved these crRNAs by insertion of the HDV ribozyme to avoid U-tails. This study provides detailed design guidelines for small RNA expression cassettes based on Pol III. |
format | Online Article Text |
id | pubmed-5725217 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | American Society of Gene & Cell Therapy |
record_format | MEDLINE/PubMed |
spelling | pubmed-57252172017-12-18 Delineation of the Exact Transcription Termination Signal for Type 3 Polymerase III Gao, Zongliang Herrera-Carrillo, Elena Berkhout, Ben Mol Ther Nucleic Acids Article Type 3 Pol III promoters such as U6 are widely used for expression of small RNAs, including short hairpin RNA for RNAi applications and guide RNA in CRISPR genome-editing platforms. RNA polymerase III uses a T-stretch as termination signal, but the exact properties have not been thoroughly investigated. Here, we systematically measured the in vivo termination efficiency and the actual site of termination for different T-stretch signals in three commonly used human Pol III promoters (U6, 7SK, and H1). Both the termination efficiency and the actual termination site depend on the T-stretch signal. The T4 signal acts as minimal terminator, but full termination efficiency is reached only with a T-stretch of ≥6. The termination site within the T-stretch is quite heterogeneous, and consequently small RNAs have a variable U-tail of 1–6 nucleotides. We further report that such variable U-tails can have a significant negative effect on the functionality of the crRNA effector of the CRISPR-AsCpf1 system. We next improved these crRNAs by insertion of the HDV ribozyme to avoid U-tails. This study provides detailed design guidelines for small RNA expression cassettes based on Pol III. American Society of Gene & Cell Therapy 2017-11-21 /pmc/articles/PMC5725217/ /pubmed/29499947 http://dx.doi.org/10.1016/j.omtn.2017.11.006 Text en © 2017 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Article Gao, Zongliang Herrera-Carrillo, Elena Berkhout, Ben Delineation of the Exact Transcription Termination Signal for Type 3 Polymerase III |
title | Delineation of the Exact Transcription Termination Signal for Type 3 Polymerase III |
title_full | Delineation of the Exact Transcription Termination Signal for Type 3 Polymerase III |
title_fullStr | Delineation of the Exact Transcription Termination Signal for Type 3 Polymerase III |
title_full_unstemmed | Delineation of the Exact Transcription Termination Signal for Type 3 Polymerase III |
title_short | Delineation of the Exact Transcription Termination Signal for Type 3 Polymerase III |
title_sort | delineation of the exact transcription termination signal for type 3 polymerase iii |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725217/ https://www.ncbi.nlm.nih.gov/pubmed/29499947 http://dx.doi.org/10.1016/j.omtn.2017.11.006 |
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