The molecular mechanism of dsRNA processing by a bacterial Dicer.
Alanine
/ genetics
Amino Acid Sequence
/ genetics
Dimerization
Escherichia coli
/ enzymology
Gene Expression Regulation, Bacterial
Gene Silencing
Glutamic Acid
/ genetics
Mutant Proteins
/ chemistry
Mutation
RNA, Double-Stranded
/ genetics
RNA, Small Interfering
/ genetics
Ribonuclease III
/ chemistry
Sequence Analysis, DNA
Substrate Specificity
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
21 05 2019
21 05 2019
Historique:
accepted:
17
03
2019
revised:
01
03
2019
received:
10
10
2018
pubmed:
28
3
2019
medline:
26
11
2019
entrez:
28
3
2019
Statut:
ppublish
Résumé
Members of the ribonuclease (RNase) III family regulate gene expression by processing dsRNAs. It was previously shown that Escherichia coli (Ec) RNase III recognizes dsRNA with little sequence specificity and the cleavage products are mainly 11 nucleotides (nt) long. It was also shown that the mutation of a glutamate (EcE38) to an alanine promotes generation of siRNA-like products typically 22 nt long. To fully characterize substrate specificity and product size of RNase IIIs, we performed in vitro cleavage of dsRNAs by Ec and Aquifex aeolicus (Aa) enzymes and delineated their products by next-generation sequencing. Surprisingly, we found that both enzymes cleave dsRNA at preferred sites, among which a guanine nucleotide was enriched at a specific position (+3G). Based on sequence and structure analyses, we conclude that RNase IIIs recognize +3G via a conserved glutamine (EcQ165/AaQ161) side chain. Abolishing this interaction by mutating the glutamine to an alanine eliminates the observed +3G preference. Furthermore, we identified a second glutamate (EcE65/AaE64), which, when mutated to alanine, also enhances the production of siRNA-like products. Based on these findings, we created a bacterial Dicer that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.
Identifiants
pubmed: 30916338
pii: 5420528
doi: 10.1093/nar/gkz208
pmc: PMC6511835
doi:
Substances chimiques
Mutant Proteins
0
RNA, Double-Stranded
0
RNA, Small Interfering
0
Glutamic Acid
3KX376GY7L
Ribonuclease III
EC 3.1.26.3
Alanine
OF5P57N2ZX
Types de publication
Journal Article
Research Support, N.I.H., Intramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
4707-4720Informations de copyright
Published by Oxford University Press on behalf of Nucleic Acids Research 2019.
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