TY - JOUR
T1 - Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity
AU - Gao, Zongliang
AU - van der Velden, Yme Ubeles
AU - Fan, Minghui
AU - van der Linden, Cynthia Alyssa
AU - Vink, Monique
AU - Herrera-Carrillo, Elena
AU - Berkhout, Ben
N1 - Publisher Copyright: © 2021 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.
PY - 2021/1/1
Y1 - 2021/1/1
N2 - RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both).
AB - RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both).
UR - http://www.scopus.com/inward/record.url?scp=85102940590&partnerID=8YFLogxK
U2 - https://doi.org/10.1074/jbc.RA120.015386
DO - https://doi.org/10.1074/jbc.RA120.015386
M3 - Article
C2 - 33154168
SN - 0021-9258
VL - 296
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
M1 - 100026
ER -