The Precursor of Differentiation Factor HLDF and Ribosomal Protein RPS21 Have a Common N-Terminal Sequence
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- Smirnova, E.V., Garkovenko, A.V., Rakitina, T.V. et al. Russian Journal of Bioorganic Chemistry (2004) 30: 114. doi:10.1023/B:RUBI.0000023095.43172.c8
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The mature differentiation factor HLDF, isolated from cultural medium, comprises 54 aa, whereas the open reading frame of mRNA encodes a 97-aa protein. We presumed that the protein translation begins from the first ATG codon, whose environment mostly meets the requirements for the initiation point. Two more ATG triplets are localized in positions 48–50 and 100–102 (numbering according to the structure of S21), i.e., in the area preceding the cDNA fragment that encodes the N-terminal fragment of the mature protein. The mRNAs of HLDF and S21 ribosomal protein have previously been shown to be highly homologous, and, therefore, their differences appear to be derived from two point deletions in the cDNA of the HLDF-encoding sequence (a G residue in position 112 and a C residue in position 224). As a result, the mature differentiation factor and RPS21 may be the products of translation from different open reading frames, the differentiation factor may be synthesized in the cell as a precursor, and its N-terminal sequence may be identical to that of RPS21. To test this hypothesis, we prepared recombinant RPS21 and the polyclonal antibodies to HLDF, full-size RPS21, and the C-terminal RPS21 peptide. Immunochemical staining by specially produced antibodies of native HL-60 cells and the same cells brought into apoptosis or differentiation confirmed that the precursor of the differentiation factor and the ribosomal S21 protein have a common N-terminal sequence and different cellular localizations. Neither an intron-containing gene nor a pseudogene with the nucleotide sequence corresponding to the HLDF cDNA was detected in the human genome or in the HL-60 cell line genome. On the basis of these facts, we propose a hypothesis of the molecular mechanism of the HLDF mRNA biosynthesis by means of posttranslational modifications of pre-mRNA of RPS21.