Identification of Novel Nuclear Localization Signals of Drosophila Myeloid Leukemia Factor

Abstract

ABSTRACT. Myeloid leukemia factor 1 (MLF1) was first identified as part of a leukemic fusion protein produced by a chromosomal translocation, and MLF family proteins are present in many animals. In mammalian cells, MLF1 has been described as mainly cytoplasmic, but in Drosophila, one of the dMLF isoforms (dMLFA) localized mainly in the nucleus while the other isoform (dMLFB), that appears to be produced by the alternative splicing, displays both nuclear and cytoplasmic localization. To investigate the difference in subcellular localization between MLF family members, we examined the subcellular localization of deletion mutants of dMLFA isoform. The analyses showed that the C-terminal 40 amino acid region of dMLFA is necessary and sufficient for nuclear localization. Based on amino acid sequences, we hypothesized that two nuclear localization signals (NLSs) are present within the region. Site-directed mutagenesis of critical residues within the two putative NLSs leads to loss of nuclear localization, suggesting that both NLS motifs are necessary for nuclear localization.

lncRNA Sensing of a Viral Suppressor of RNAi Activates Non-canonical Innate Immune Signaling in Drosophila

“…The fusion cDNAs encoding DG, DGm2, and DGm were constructed by overlap polymerase chain reaction (PCR) and inserted into the pMT/V5-His A vector. The cDNA encoding the two nuclear localization signals of Drosophila MLFA protein (SIHPHPYAANPRRQQRAVKHFHTEDEAASSYKAVKRGKKK) (Sugano and Yamaguchi, 2007) was synthesized and fused with cDNA of FHV B2 at the 3 0 terminus by overlap PCR, then the fused cDNA encoding B2-NLS was cloned into pMT/V5-His A vector. The primers are listed in Table S3.…”

Identification of Nuclear Localization Signals of Drosophila G9a Histone H3 Methyltransferase

“…For detailed analysis of the nuclear localization signal, oligonucleotides were synthesized to construct the plasmid pUAST-EGFPdG9aNLS1, pUAST-EGFP-dG9aNLS2, pUAST-EGFP-dG9aNLS3, pUAST-EGFP-dG9aNLS4. After annealing the following pairs of synthetic oligonucleotids, the double stranded ologonucleotides were cloned into the pUAST-EGFP vector (Sugano and Yamaguchi, 2008).…”

Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis

“…Only one MLF gene is present in Drosophila, whereas there are two MLF paralogs in vertebrates (21). As in mammals, MLF encodes for a nucleocytoplasmic shuttling protein with no recognizable structural domain apart from a 14-3-3 binding motif (22). However, its in vivo function remains unclear, given that mlf mutant flies are subviable and do not exhibit any obvious developmental defect (23).…”