The parathyroid hormone mRNA 3'-untranslated region AU-rich element is an unstructured functional element.
Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally by hypocalcemia and hypophosphatemia. This regulation is dependent upon binding of protective trans-acting factors to a specific element in the PTH mRNA 3'-untranslated region (UTR). We have previously demonstrated that a 63-nucleotide (nt) AU-rich PTH mRNA element is sufficient to confer regulation of RNA stability by calcium and phosphate in an in vitro degradation assay (IVDA). The 63-nt element consists of a core 26-nt minimal binding sequence and flanking regions. We have now studied the functionality of this element in HEK293 cells using reporter genes and showed that it destabilizes mRNAs for green fluorescent protein (GFP) and growth hormone, similar to its effect in the IVDA. To understand how the cis-element functions as an instability element, we have analyzed its structure by RNase H, primer extension, and computer modeling. The results indicate that the PTH mRNA 3'-UTR and in particular the region of the cis-element are dominated by significant open regions with little folded base pairing. Mutation analysis of the 26-nt core element demonstrated the importance of defined nucleotides for protein-RNA binding. In the GFP reporter system, the same mutations that prevented binding were also ineffective in destabilizing GFP mRNA in HEK293 cells. This is the first study of an AU-rich element that relates function to structure. The PTH mRNA 3'-UTR cis-acting element is an open region that utilizes the distinct sequence pattern to determine mRNA stability by its interaction with trans-acting factors.
Minerva Center for Calcium and Bone Metabolism, Nephrology Services, Hadassah Hospital, The Hebrew University Medical School, P.O. Box 12000, Jerusalem, Israel 91120.
Rachel
R
Kilav
Osnat
O
Bell
Shu-Yun
SY
Le
Justin
J
Silver
Tally
T
Naveh-Many
D020413:3' Untranslated Regions / Q000737:chemistry* / Q000502:physiology; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010281:Parathyroid Hormone / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry
D020413:3' Untranslated Regions; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D010281:Parathyroid Hormone; D012333:RNA, Messenger