Related Experiment Videos
Evidence for ribosomes involved in splicing of yeast mitochondrial transcripts
Abstract:
We have investigated the processing of transcripts of the split gene COB in yeast mitochondrial DNA from cells whose mitochondrial translation was blocked by chloramphenicol for several generations of cell growth. First analysis of transcripts by electrophoresis and RNA/DNA-hybridization clearly showed that cell growth in the presence of CAP leads to an inhibition of processing yielding an increasing amount of splicing intermediates of the COB transcript and decreasing amounts of the 18S mRNA coding for apocytochrome b. This observation is in accordance with the now widely favoured idea that mitochondrial proteins are involved in splicing of COB transcripts and that their reduction should hamper processing and - therefore - lead to an accumulation of pre-mRNAs. However, further information obtained by pulse-labeling of pre-mRNA in vivo in the presence of CAP for various times shows that even 30 minutes after addition of CAP a reduction of the processing rate is obtained. Based on these findings we conclude that maturation of mtRNAs is not only dependent on mitochondrial proteins, but also on a more direct interaction of the translation machinery and RNA processing whose nature is so far unknown.
Insights
Blocking mitochondrial translation with chloramphenicol (CAP) inhibits yeast COB transcript processing. This suggests RNA maturation depends on both mitochondrial proteins and direct translation machinery interactions.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Mitochondrial Gene Expression
Background:
- The COB gene in yeast mitochondria is a split gene requiring transcript processing for functional mRNA production.
- Mitochondrial gene expression involves complex RNA processing steps, including splicing.
- Previous hypotheses suggested mitochondrial proteins are essential for COB transcript splicing.
Purpose of the Study:
- To investigate the effect of inhibited mitochondrial translation on COB transcript processing in yeast.
- To determine the role of mitochondrial translation and proteins in RNA maturation.
- To elucidate the relationship between translation machinery and RNA processing.
Main Methods:
- Yeast cell cultures grown with chloramphenicol (CAP) to block mitochondrial translation.
- Analysis of COB transcripts using electrophoresis and RNA/DNA hybridization.
- Pulse-labeling of pre-mRNA in vivo to assess processing rates over time.
Main Results:
- Inhibition of mitochondrial translation by CAP led to accumulation of COB splicing intermediates.
- A decrease in mature 18S mRNA coding for apocytochrome b was observed.
- Pulse-labeling revealed a reduced processing rate of pre-mRNA within 30 minutes of CAP addition.
Conclusions:
- Mitochondrial RNA (mtRNA) maturation is dependent on mitochondrial proteins.
- A direct, yet unknown, interaction between the translation machinery and RNA processing is crucial.
- Inhibition of translation directly impacts RNA processing efficiency.