Mechanism of translation control of the alternative Drosophila melanogaster Voltage Dependent Anion-selective Channel 1 mRNAs

The eukaryotic porin, also called the Voltage Dependent Anion-selective Channel (VDAC), is the main pore-forming protein of the outer mitochondrial membrane. In Drosophila melanogaster, a cluster of genes evolutionarily linked to VDAC is present on chromosome 2L. The main VDAC isoform, called VDAC1 (Porin1), is expressed from the first gene of the cluster. The porin1 gene produces two splice variants, 1A-VDAC and 1B-VDAC, with the same coding sequence but different 5′ untranslated regions (UTRs). Here, we studied the influence of the two 5′ UTRs, 1A-5′ UTR and 1B-5′ UTR, on transcription and translation of VDAC1 mRNAs. In porin-less yeast cells, transformation with a construct carrying 1A-VDAC results in the expression of the corresponding protein and in complementation of a defective cell phenotype, whereas the 1B-VDAC sequence actively represses VDAC expression. Identical results were obtained using constructs containing the two 5′ UTRs upstream of the GFP reporter. A short region of 15 nucleotides in the 1B-5′ UTR should be able to pair with an exposed helix of 18S ribosomal RNA (rRNA), and this interaction could be involved in the translational repression. Our data suggest that contacts between the 5′ UTR and 18S rRNA sequences could modulate the translation of Drosophila 1B-VDAC mRNA. The evolutionary significance of this finding is discussed.

luciferase gene. The mutant 1A(ins16-31)-VDAC was obtained using the "QuikChange II XL Site-Directed Mutagenesis Kit" (QIAGEN) on the pMK26-1A-Luciferase construct. The accuracy of each construct was confirmed by DNA sequencing.
Cloning and expression of GFP constructs in HeLa cells. 1A-, 1B-, 1B(Δ16-31)-, and 1A(ins16-31)-UTRs were amplified using primers carrying NheI and BamHI restriction site, respectively at the 5' end and 3' end. The PCR products were cloned upstream of EGFP gene in pEGFP-N1 vector. 5'UTR-GFP constructs were used for transfection of HeLa cells. The accuracy of constructs was confirmed by DNA sequencing.
They were next incubated at the indicated temperature for 3-6 days.

Yeast lysates preparation
Yeast cells were grown in 50 ml of YPD medium for 12 hours at 30 °C, under 200 rpm shaking. The cells were harvested by centrifugation for 5 minutes at 3000 x g, at 25 °C and then washed twice with sterilized water. The cellular pellet was resuspended in 400 µl of lysis buffer (100 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl) and lysed in the presence of 1 volume of glass beads 425-600 µm (Sigma) by vortexing in ice 10 times for 60 seconds with 60 seconds intervals. Glass beads were eliminated by centrifugation at 4.500 rpm for 5 minutes and the supernatant was centrifuged 20 minutes at 12000 rpm at 4 °C to obtain the final lysate.

Production of antibodies and immunoblotting
Anti-D. melanogaster mt-porin polyclonal antibodies were generated in rabbits by using purified D. melanogaster recombinant porin1/VDAC1 as the antigen 2 . Rabbits were were immunized three times, following standard protocols. 100 µg of purified porin were used in any booster. After three immunization cycles, blood was collected and the serum was obtained.

Mass spectrometry analysis and protein identification
Protein samples from RNA pull-down experiments were then resolved in 12% SOD-PAGE. After Blue Comassie staining, each protein band in each lane was excised from gel and cut in very thin slices. Proteins in gel slices were identified as in 4 .

Supplementary Tables
Primers for qRT-PCR  Table S1. List of the primer sequences used in this work.  Table S3. List of proteins interacting with high affinity to 1B 10-37 RNA oligo identified by Mass Spectrometry analysis. Proteins interacting with the 1B 74-95 RNA oligo were also identified by MS analysis with the aim to identify unspecific RBPs able to recognize any RNA molecule. These latter proteins were then subtracted from the whole list of proteins interacting with the 10-37 RNA oligo.