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Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects.

ABSTRACT: RNA interference (RNAi) is an evolutionarily conserved phenomenon of post-transcriptional gene silencing mediated by small interfering RNAs (siRNAs) generated from short hairpin RNAs (shRNAs) by Dicer cleavage. Here, we report that siRNA precursors can be divided into two categories with different processing mechanisms and silencing activities that are dependent on stem loop length. We designed an alternative siRNA precursor for triggering RNAi named single-stranded Argonaute2 (Ago2)-processed interfering RNA (saiRNA). saiRNA is composed of a 16-18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleavage ribozyme derived from hepatitis delta virus (HDV) to the 3’ end of the saiRNA dramatically improved its silencing activity. Unlike classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during its processing not only eliminated the passenger strand but also avoided the association of mature siRNA with non-nucleolytic Ago proteins, thereby further reducing the risk of off-target effects. Additionally, saiRNA exhibited less competition with the biogenesis of endogenous miRNAs. Therefore, HDV ribozyme-enhanced saiRNA provides a reliable tool for RNAi applications.

ORGANISM(S): Homo sapiens

PROVIDER: GSE68631 | GEO | 2015/06/01

SECONDARY ACCESSION(S): PRJNA283266

REPOSITORIES: GEO

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Project description:Small RNAs are the common denominator of various RNA silencing pathways that regulate gene expression and protect the genome against mobile repetitive DNA sequences, retroelements, and transposons. In Schizosaccharomyces pombe, small interfering RNAs (siRNAs) are required for the faithful propagation of heterochromatin that is found at peri-centromeric repeats. In contrast to repetitive DNA, protein-coding genes are refractory to siRNA-mediated heterochromatin formation, unless siRNAs are expressed in mutant cells. Different studies have shown that siRNAs become potent mediators of RNAi-mediated epigenetic gene silencing in S. pombe cells that are mutant for mlo3+, dss1+, mst2+, or genes encoding subunits of the Paf1 complex (Paf1C). In this study, we have combined chemical mutagenesis with whole-genome sequencing in a sensitized S. pombe reporter strain to obtain a more comprehensive list of putative suppressors of small-RNA-mediated epigenetic gene silencing. This revealed more than 20 novel silencing-enabling mutations in genes that are associated with RNA processing, regulation of transcription, or post-translational protein modification. Focusing on factors involved in pre-mRNA cleavage and polyadenylation, we for example show that single amino acid substitutions in Yth1, which is responsible for polyadenylation signal recognition, lead to nearly 100% effective de novo formation of silent heterochromatin. In S. pombe, small RNA-mediated silencing relies on the continuous amplification of the siRNA pool through a positive feedback loop. The deposited data shows the generation of secondary small RNAs over the reporter gene exclusively in the presence of RNAi-enabling mutations in the pre-mRNA cleavage and polyadenylation machinery. Furthermore, the RNA-seq data show that overall transcriptome levels are not markedly changing in the mutations themselves. Altogether, our work shows that epigenetic gene silencing can be enabled by the acquisition of a plethora of mutant alleles in fission yeast.

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Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects.

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