RNA Silencing Induced by an Artificial Sequence That Prevents Proper Transcription Termination in Rice

Abstract

Posttranscriptional gene silencing (PTGS) is a sequence-specific mRNA degradation caused by small RNA, such as microRNA (miRNA) and small interfering RNA (siRNA). miRNAs are generated from MIRNA loci, whereas siRNAs originate from various sources of double-stranded RNA. In this study, an artificial RNA silencing inducible sequence (RSIS) was identified in rice (Oryza sativa). This sequence causes PTGS of 5' or 3' flanking-sequence-containing genes. Interestingly, two target genes can be simultaneously suppressed by linking a unique target sequence to either the 5' or 3' end of RSIS. Multiple gene suppression can be also achieved though a single transformation event by incorporating the multisite gateway system. Moreover, RSIS-mediated PTGS occurs in nuclei. Deep sequencing of small RNAs reveals that siRNAs are produced from RSIS-expressing cassettes and transitive siRNAs are produced from endogenous target genes. Furthermore, siRNAs are typically generated from untranscribed transgene terminator regions. The read-through transcripts from the RSIS-expression cassette were consistently observed, and most of these sequences were not polyadenylated. Collectively, this data indicates that RSIS inhibits proper transcription termination. The resulting transcripts are not polyadenylated. These transcripts containing RSIS become templates for double-stranded RNA synthesis in nuclei. This is followed by siRNA production and target degradation of target genes.