title: The influence of the 5΄-terminal nucleotide on AgoshRNA activity and biogenesis: importance of the polymerase III transcription initiation site.
abstract: Recent evidence indicates that shRNAs with a relatively short basepaired stem do not require Dicer processing, but instead are processed by the Argonaute 2 protein (Ago2). We named these molecules AgoshRNAs as both their processing and silencing function are mediated by Ago2. This alternative processing yields only a single RNA guide strand, which can avoid off-target effects induced by the passenger strand of regular shRNAs. It is important to understand this alternative processing route in mechanistic detail such that one can design improved RNA reagents. We verified that AgoshRNAs trigger site-specific cleavage of a complementary mRNA. Second, we document the importance of the identity of the 5΄-terminal nucleotide and its basepairing status for AgoshRNA activity. AgoshRNA activity is significantly reduced or even abrogated with C or U at the 5΄-terminal and is enhanced by introduction of a bottom mismatch and 5΄-terminal nucleotide A or G. The 5΄-terminal RNA nucleotide also represents the +1 position of the transcriptional promoter in the DNA, thus further complicating the analysis. Indeed, we report that +1 modification affects the transcriptional efficiency and accuracy of start site selection, with A or G as optimal nucleotide. These combined results allow us to propose general rules for the design and expression of potent AgoshRNA molecules.
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands.
authors.forename: Elena
authors.initials: E
authors.lastname: Herrera-Carrillo
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands.
authors.forename: Zong-Liang
authors.initials: ZL
authors.lastname: Gao
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands.
authors.forename: Alex
authors.initials: A
authors.lastname: Harwig
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands.
authors.forename: Matthias T
authors.initials: MT
authors.lastname: Heemskerk
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands.
authors.forename: Ben
authors.initials: B
authors.lastname: Berkhout
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000378:metabolism*; D020029:Base Pairing; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D057809:HEK293 Cells; D006801:Humans; D009711:Nucleotides; D011485:Protein Binding; D059367:RNA Cleavage; D012320:RNA Polymerase III / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000378:metabolism*; D061785:Transcription Initiation, Genetic; D014709:Vero Cells
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D009711:Nucleotides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012320:RNA Polymerase III