TY - JOUR
T1 - Delineation of the Exact Transcription Termination Signal for Type 3 Polymerase III
AU - Gao, Zongliang
AU - Herrera-Carrillo, Elena
AU - Berkhout, Ben
PY - 2018
Y1 - 2018
N2 - 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.
AB - 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.
UR - https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85040082321&origin=inward
UR - https://www.ncbi.nlm.nih.gov/pubmed/29499947
U2 - https://doi.org/10.1016/j.omtn.2017.11.006
DO - https://doi.org/10.1016/j.omtn.2017.11.006
M3 - Article
C2 - 29499947
SN - 2162-2531
VL - 10
SP - 36
EP - 44
JO - Molecular Therapy. Nucleic Acids
JF - Molecular Therapy. Nucleic Acids
ER -