Preferential translation of Hsp83 in Leishmania requires a thermosensitive polypyrimidine-rich element in the 3' UTR and involves scanning of the 5' UTR.
Heat shock proteins (HSPs) provide a useful system for studying developmental patterns in the digenetic Leishmania parasites, since their expression is induced in the mammalian life form. Translation regulation plays a key role in control of protein coding genes in trypanosomatids, and is directed exclusively by elements in the 3' untranslated region (UTR). Using sequential deletions of the Leishmania Hsp83 3' UTR (888 nucleotides [nt]), we mapped a region of 150 nt that was required, but not sufficient for preferential translation of a reporter gene at mammalian-like temperatures, suggesting that changes in RNA structure could be involved. An advanced bioinformatics package for prediction of RNA folding (UNAfold) marked the regulatory region on a highly probable structural arm that includes a polypyrimidine tract (PPT). Mutagenesis of this PPT abrogated completely preferential translation of the fused reporter gene. Furthermore, temperature elevation caused the regulatory region to melt more extensively than the same region that lacked the PPT. We propose that at elevated temperatures the regulatory element in the 3' UTR is more accessible to mediators that promote its interaction with the basal translation components at the 5' end during mRNA circularization. Translation initiation of Hsp83 at all temperatures appears to proceed via scanning of the 5' UTR, since a hairpin structure abolishes expression of a fused reporter gene.
David
Maya
M
Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.
Gabdank
Idan
I
Ben-David
Miriam
M
Zilka
Alon
A
Orr
Irit
I
Barash
Danny
D
Shapira
Michal
M
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D017930:Genes, Reporter; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism*; D007891:Leishmania / Q000235:genetics* / Q000378:metabolism*; D007894:Leishmania mexicana / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D017931:DNA Primers; D006360:Heat-Shock Proteins; C490310:Hsp83 protein, protozoan; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan