Post-transcriptional Silencing and Functional Characterization of theDrosophila melanogasterHomolog of HumanSurf1

Abstract

Mutations in Surf1, a human gene involved in the assembly of cytochrome c oxidase (COX), cause Leigh syndrome, the most common infantile mitochondrial encephalopathy, characterized by a specific COX deficiency. We report the generation and characterization of functional knockdown (KD) lines for Surf1 in Drosophila. KD was produced by post-transcriptional silencing employing a transgene encoding a dsRNA fragment of the Drosophila homolog of human Surf1, activated by the UAS transcriptional activator. Two alternative drivers, Actin5C-GAL4 or elav-GAL4, were used to induce silencing ubiquitously or in the CNS, respectively. Actin5C-GAL4 KD produced 100% egg-to-adult lethality. Most individuals died as larvae, which were sluggish and small. The few larvae reaching the pupal stage died as early imagos. Electron microscopy of larval muscles showed severely altered mitochondria. elav-GAL4-driven KD individuals developed to adulthood, although cephalic sections revealed low COX-specific activity. Behavioral and electrophysiological abnormalities were detected, including reduced photoresponsiveness in KD larvae using either driver, reduced locomotor speed in Actin5C-GAL4 KD larvae, and impaired optomotor response as well as abnormal electroretinograms in elav-GAL4 KD flies. These results indicate important functions for SURF1 specifically related to COX activity and suggest a crucial role of mitochondrial energy pathways in organogenesis and CNS development and function.

Mitochondrial Transcription Factor B2 Is Essential for Metabolic Function in Drosophila melanogaster Development

“…Accordingly, the total amount of ATP in RNAiB2 larvae was maintained at 70% of control larvae, such that the defect in mitochondrial synthetic capacity is largely compensated by glycolysis. Furthermore, we found that RNAiB2 larvae exhibit a substantial increase in the activity of citrate synthase and complex II activities, and in the steadystate level of hsp60, which are considered to be markers of mitochondrial mass (33). We interpret these findings to suggest that OXPHOS impairment due to a reduced amount of TFB2M induces a compensatory response that increases mitochondrial mass.…”

“…RNAi-dependent knockdown was induced using the da-GAL4 strain as a driver, which expresses the yeast transcriptional activator GAL4 under control of the daughterless enhancer. da-GAL4 expresses GAL4 throughout development and in most if not all tissues, and has been used extensively to overexpress constitutively transgenes containing UAS binding sites in Drosophila (33).…”

Fly cryptochrome and the visual system

“…The walking optomotor test was performed as in ref. 52 (setup 1 in SI Materials and Methods). Details of setups 1 and 2 are given in SI Materials and Methods.…”

Mutations in Cytochrome c Oxidase Subunit VIa Cause Neurodegeneration and Motor Dysfunction in Drosophila

“…Agostino et al (2003) have generated a Surf1 knockout mouse model of Leigh syndrome that lacks Surfeit-1, an enzyme involved in COX assembly. Similarly, a Surf1 knockdown model has been generated in Drosophila by post-transcriptional silencing using dsRNA (Zordan et al 2006). Studies comparing biochemical and physiological characteristics of various models of COXrelated mitochondrial encephalomyopathies, including levy mutants and Surf1 models in mice and Drosophila, can help in understanding the mechanisms underlying the effects of such disorders.…”