Functional studies of Arabidopsis mitochondrial splicing factors
Date Issued
2014
Date
2014
Author(s)
Hsu, Ya-Wen
Abstract
Mitochondria, the central power plant in all eukaryotic cells, provide a majority of energy for normal cellular maintenance and functions. The function of mitochondrial respiratory chain needs proper mitochondrial gene expression which is regulated by extensive post-transcriptional controls including 5’ and 3’ RNA processing, intron splicing, RNA editing, and controlled RNA stability. Of these post-transcriptional processes, intron splicing is an example of acquired nonprokaryotic traits in plant mitochondria and involves in the vast majority of nuclear-encoded proteins. Disruption of these proteins causes the splicing defects of mitochondrial genes and results in the defective plant growth and development such as male sterility, embryo lethality and growth retardation. In this dissertation, three proteins with unique roles in Arabidopsis mitochondrial intron splicing were studied. In the first part, the biological functions and molecular mechanism of Arabidopsis mTERF15 (mitochondrial transcription termination factor 15) in mitochondria are investigated using molecular genetics, cytological and biochemical approaches. The null homozygous mterf15 mutant was found to result in substantial retardation of both vegetative and reproductive development, which was fully complemented by the wild-type genomic sequence. Surprisingly, mitochondrion-localized mTERF15 lacks obvious DNA-binding activity but processes mitochondrial nad2 intron 3 splicing through its RNA-binding ability. Impairment of this splicing event not only disrupted mitochondrial structure but also abolished the activity of mitochondrial respiratory chain complex I. These effects are in agreement with the severe phenotype of the mterf15 homozygous mutant. This study suggests that Arabidopsis mTERF15 functions as a splicing factor for nad2 intron 3 splicing in mitochondria, which is essential for normal plant growth and development. The second part of this dissertation focuses on the function of WTF9 (What is This Factor 9) and its interacting proteins, HSP60s, in mitochondrial intron splicing. Using co-immunoprecipitation, we identified HSP60s as interacting proteins of WTF9. HSP60s are recognized as molecular chaperones to assist protein folding in both eukaryotic and prokaryotic cells. However, accumulating evidence suggests HSP60s also participate in other biological functions such as RNA metabolism and protection. As noncanonical function of HSP60s, we found HSP60s interact with 48 nucleotides of ccmFC intron as WTF9. The seedling phenotype of hsp60-3a-1hsp60-3b-1 mutants with small stature suggests the importance of HSP60s in plant growth and development. Also, the splicing efficiency of rpl2 and ccmFC were reduced in hsp60-3a-1hsp60-3b-1 double mutants. These observations are similar to the findings in wtf9 mutants and suggest that HSP60s are involved in mitochondrial RNA splicing of rpl2 and ccmFC introns.
Subjects
Arabidopsis
mitochondria
intron
splicing factor
Type
thesis