| Autor: |
McKinley LN; Depatment of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, United States.; Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, United States., Kern RG; Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, United States.; Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, United States., Assmann SM; Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, United States.; Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802, United States., Bevilacqua PC; Depatment of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, United States.; Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, United States.; Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, United States. |
| Abstrakt: |
Small nucleolytic ribozymes are RNAs that cleave their own phosphodiester backbone. While proteinaceous enzymes are regulated by a variety of known mechanisms, methods of regulation for ribozymes remain unclear. Twister is one ribozyme class for which many structural and catalytic properties have been elucidated. However, few studies have analyzed the activity of twister ribozymes in the context of a native flanking sequence, even though ribozymes as transcribed in nature do not exist in isolation. Interactions between the ribozyme and its neighboring sequences can induce conformational changes that inhibit self-cleavage, providing a regulatory mechanism that could naturally determine ribozyme activity in vivo and in synthetic applications. To date, eight twister ribozymes have been identified within the staple crop rice ( Oryza sativa ). Herein, we select several twister ribozymes from rice and show that they are differentially regulated by their flanking sequence using published RNA-seq data sets, structure probing, and cotranscriptional cleavage assays. We found that the Osa 1-2 ribozyme does not interact with its flanking sequences. However, sequences flanking the Osa 1-3 and Osa 1-8 ribozymes form inactive conformations, referred to here as "ribozymogens", that attenuate ribozyme self-cleavage activity. For the Osa 1-3 ribozyme, we show that activity can be rescued upon addition of a complementary antisense oligonucleotide, suggesting ribozymogens can be controlled via external signals. In all, our data provide a plausible mechanism wherein flanking sequence differentially regulates ribozyme activity in vivo . More broadly, the ability to regulate ribozyme behavior locally has potential applications in control of gene expression and synthetic biology. |
