Mitochondrial Targeting of Catalytic RNAs

Abstract

Genetic transformation of mitochondria in multicellular eukaryotes has remained inaccessible, hindering fundamental investigations and applications to gene therapy or biotechnology. In this context, we have developed a strategy to target nuclear transgene-encoded RNAs into mitochondria in plants. We describe here mitochondrial targeting of trans-cleaving ribozymes destined to knockdown organelle RNAs for regulation studies and inverse genetics and biotechnological purposes. The design and functional assessment of chimeric RNAs combining the ribozyme and the mitochondrial shuttle are detailed, followed by all procedures to prepare constructs for in vivo expression, generate stable plant transformants, and establish target RNA knockdown in mitochondria.

Participation of non-coding RNAs in plant organellar biogenesis

“…Gómez & Pallás (2010a, b) had shown that a ncRNA with defined secondary structure is able to mediate functional foreign mRNA targeting into plastids. Mitochondrial biogenesis and metabolism can be also influenced by the expression of artificial miRNAs against mRNAs encoding important mitochondrial enzymes (Haas et al, 2008;Stoll et al, 2014) or chimeric ribozymes for directed knockdown of mtRNA, leading to a genetic manipulation of plant mitochondria (Val et al, 2011;Mileshina et al, 2015). How such transplastomic/transmitochondrial manipulations could affect plant organellar stress response under the field conditions?…”