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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...

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Autores principales: Gao, Zongliang, Herrera-Carrillo, Elena, Berkhout, Ben
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2017
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.
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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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